Note: Descriptions are shown in the official language in which they were submitted.
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DNA SHUFFLING OF DIOXYGENASE GENES FOR PRODUCTION OF
INDUSTRIAL CHEMICALS
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority from and the benefit of U.S. Provisional
Application No. 60/148,850, filed August 12, 1999, pursuant to 35 USC 119(e)
and any
other applicable statute or rule.
This application is related to USSN 09/373,333, filed August 12, 1999,
"DNA Shuffling to Produce Herbicide Selective Crops", Subramanian,
Venkitswaran, et al.,
which is a non-provisional application of USSN 60/096,288, filed August 12,
1998, USSN
60/111,146, filed December 7, 1998, and USSN 60/112,746, filed December 17,
1998, and
related to International Application No. PCT/LTS99/18394, filed August 12,
1999, "DNA
Shuffling to Produce Herbicide Selective Crops."
This application is also related to USSN 09/373,928, filed August 12, 1999,
"DNA Shuffling of Monooxygenase for Production of Industrial Chemicals,"
Affholter,
Joseph A., et al., which is a non-provisional of USSN 60/096,271, filed August
12, 1998,
and USSN 60/130,810, filed April 23, 1999, and related to International
Application No.
PCT/US99/18424, filed August 12, 1999, "DNA Shuffling of Monooxygenase for
Production of Industrial Chemicals."
COPYRIGHT NOTICE PURSUANT TO 37 C.F.R. ~ 1.71(e)
A portion of the disclosure of this patent document contains material which
is subject to copyright protection. The copyright owner has no objection to
the facsimile
reproduction by anyone of the patent document or the patent disclosure, as it
appears in the
Patent and Trademark Office patent file or records but otherwise reserves all
copyright
rights whatsoever.
FIELD OF THE INVENTION
This invention pertains to the shuffling of nucleic acids to achieve or
enhance industrial production of chemicals by dioxygenase genes.
SUBSTITUTE SHEET (RULE 26)
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BACKGROUND OF THE INVENTION
Oxygen-containing organic chemicals such as organic acids, hydroxy
carboxylic acids, alcohols, hydroxyaryls (e.g., hydroxyaryl carboxylic acids,
alkylphenols,
etc.) and glycols are important classes of industrial chemicals. Typically,
these products are
generated by successive introduction of various chemical functional groups by
oxidation,
(trans)alkylation, reduction, desaturation and other reactions of inexpensive
raw materials
such as saturated and unsaturated hydrocarbons (alkanes, alkenes, etc) and
simple aromatic
compounds (benzene, ethyl benzene, cumene, naphthalene, styrene, toluene,
xylenes, etc).
Dioxygenases (DOs) such as the arene dioxygenases (ADOs) typically
catalyze limited oxidation of these basic chemical building blocks. While
potentially
interesting from an industrial standpoint, these enzymes typically do not
exhibit sufficient
turnover numbers and/or desired specificity or regioselectivity to make them
usable as
industrial catalysts.
Surprisingly, the present invention provides a general (broad utility) method
for providing dioxygenase enzymes with higher activity and desired selectivity
and
specificity of the catalyzed reactions, thereby solving the problems outlined
above, as well
as providing a variety of other features which will be apparent upon review.
SUMMARY OF THE INVENTION
In the present invention, nucleic acid shuffling is used to generate new or
improved dioxygenase ("DO") genes. These dioxygenase genes are used to provide
dioxygenase enzymes, especially for industrial processes. These new or
improved genes
have surprisingly superior properties as compared to naturally occurnng
dioxygenase genes.
In the methods for obtaining dioxygenase genes, a plurality of parental forms
(homologs) of a selected nucleic acid are recombined. The selected nucleic
acid is derived
either from one or more parental nucleic acids) which encodes a dioxygenase
enzyme, or a
fragment thereof, or from a parental nucleic acid which does not encode
dioxygenase, but
which is a candidate for nucleic acid shuffling to develop dioxygenase
activity. The
plurality of forms of the selected nucleic acid differ from each other in at
least one (and
typically two or more) nucleotides, and, upon recombination, provide a library
of
recombinant dioxygenase nucleic acids. The library can be an in vitro set of
molecules, or
present in cells, phage or the like. The library is screened to identify at
least one
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recombinant dioxygenase nucleic acid that exhibits distinct or improved
dioxygenase
activity compared to the parental nucleic acid or nucleic acids.
Many formats for libraries of nucleic acids are known in the art and each of
these formats is generally applicable to the libraries of the present
invention. For example,
basic texts generally disclosing library formats of use in this invention
include Sambrook et
al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene
Transfer and
Expression: A Laboratory Manual (1990); and Current Protocols in Molecular
Biology,
(Ausubel et al., eds., 1994)).
In a preferred embodiment, the starting DNA segments are first recombined
by any of the formats described herein to generate a diverse library of
recombinant DNA
segments. Such a library can vary widely in size from having fewer than 10 to
more than
105, 107, or 10~ members. In general, the starting segments and the
recombinant libraries
generated include full-length coding sequences and any essential regulatory
sequences, such
as a promoter and polyadenylation sequence, required for expression. However,
if this is
not the case, the recombinant DNA segments in the library can be inserted into
a common
vector providing the missing sequences before performing screening/selection.
Although a variety of methods are optionally used, the library is typically
generated by nucleic acid shuffling. One preferred method comprises initiating
a
polynucleotide amplification process on overlapping segments of a population
of variant
polynucleotides, e.g., allelic or species variants, at least one of which
variant
polynucleotides typically encodes a dioxygenase polypeptide. This
amplification is
typically carried out under conditions whereby one segment serves as a
template for
extension of another segment, to generate a population of recombinant
polynucleotides.
The recombinant polynucleotides are typically selected or screened for a
desired property,
e.g., improved dioxygenase activity. The overlapping segments are optionally
produced by
cleavage of the population of variant polynucleotides, e.g., by DNaseI
digestion.
Alternatively, the overlapping segments are produced by chemical synthesis or
by
amplification of the population of polynucleotides.
For example, shuffling optionally comprises recombining at least first and
second forms of a nucleic acid that encodes a dioxygenase polypeptide, or
fragment thereof,
wherein the first and second forms differ from each other in two or more
nucleotides. The
library of recombinant polynucleotides is then typically expressed to obtain a
library of
recombinant polypeptides. In some embodiments, the method further comprises
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recombining at least one recombinant polynucleotide that encodes a member of
the library
of recombinant polynucleotides that encodes a member of the library of
recombinant
dioxygenase polypeptides, which is the same or different from the first and
second forms, to
produce a further library of recombinant polynucleotides. The further library
of
recombinant polynucleotides is expressed to obtain a further library of
recombinant
dioxygenase polypeptides. These steps are optionally repeated until the
further library of
recombinant polynucleotides contains a desired number of different recombinant
polynucleotides.
In another embodiment, shuffling comprises hybridizing at least two sets of
nucleic acids, wherein a first set of nucleic acids comprises single-stranded
nucleic acid
templates and a second set of nucleic acids comprises at least one set of
nucleic acid
fragments. The method further comprises elongating, ligating, or both,
sequence gaps
between the hybridized nucleic acid fragments, to generate one or more
substantially full-
length chimeric nucleic acid sequences corresponding to the single-stranded
nucleic acid
templates. In other embodiments, the method optionally comprises denaturing
the one or
more substantially full-length chimeric nucleic acid sequences and the single-
stranded
nucleic acid templates; separating the substantially full-length chimeric
nucleic acid
sequences from the single-stranded nucleic acid templates; and fragmenting the
separated
substantially full-length chimeric nucleic acid sequences by nuclease
digestion or physical
fragmentation to provide chimeric nucleic acid fragments.
If the sequence recombination format employed is an in vivo format, the
library of recombinant DNA segments generated already exists in a cell, which
is usually
the cell type in which expression of the enzyme with altered substrate
specificity is desired.
If sequence recombination is performed in vitro, the recombinant library is
preferably
introduced into the desired cell type before screening/selection. The members
of the
recombinant library can be linked to an episome or virus before introduction
or can be
introduced directly. In some embodiments of the invention, the library is
amplified in a first
host, and is then recovered from that host and introduced to a second host
more amenable to
expression, selection, or screening, or any other desirable parameter.
The manner in which the library is introduced into the cell type depends on
the DNA-uptake characteristics of the cell type (e.g., having viral receptors,
being capable
of conjugation, or being naturally competent). If the cell type is not
susceptible to natural
and chemical-induced competence, but is susceptible to electroporation, one
preferably
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employs electroporation. If the cell type is not susceptible to
electroporation as well, one
can employ biolistics. The biolistic PDS-1000 Gene Gun (Biorad, Hercules,
Calif.) uses
helium pressure to accelerate DNA-coated gold or tungsten microcarriers toward
target
cells. The process is applicable to a wide range of tissues, including plants,
bacteria, fungi,
algae, intact animal tissues, tissue culture cells, and animal embryos. One
can employ
electronic pulse delivery, which is essentially a mild electroporation format
for live tissues
in animals and patients. Zhao, Advanced Drug Delivery Reviews 17:257-262
(1995). Novel
methods for making cells competent are described in co-pending application
U.S. patent
application Ser. No. 08/621,430, filed Mar. 25, 1996. After introduction of
the library of
recombinant DNA genes, the cells are optionally propagated to allow expression
of genes to
occur.
In selecting for dioxygenase activity, a candidate shuffled DNA can be tested
for encoded dioxygenase activity in essentially any synthetic process. Common
processes
that can be screened include, for example, dihydroxylation of an aromatic
ring, olefinic or
polyenic alkene ~-bond dihydroxylation, oxidation of methyl or methlylene
groups attached
to an aromatic ring, oxidation of methyl or methylene groups attached to a ~t-
bond which is
not a part of an aromatic system, sulfur heteroatom monooxygenation,
desaturation of
alkane groups attached to aromatic ring or non-aromatic ~-bonds, oxidative
elimination of
halide (F, Cl, Br, I), nitrite, ammonia from halogen, nitro or amino
substituted ~-bonds
(aromatic and/or olefinic), N- dealkylation and dearylation of alkylamino- and
arylamino-
substituted aromatic compounds, O-dealkylation of alkoxy- and aryloxy-
substituted
aromatic compounds, and conversion of an aromatic ring to a substituted
conjugated dime.
Similarly, instead of, or in addition to, testing for an increase in
dioxygenase
specific activity, it is also desirable to screen for shuffled nucleic acids
which produce
higher levels of dioxygenase nucleic acid or enhanced or reduced recombinant
dioxygenase
polypeptide expression or stability encoded by the recombinant dioxygenase
nucleic acid.
A variety of screening methods can be used to screen a library, depending on
the dioxygenase activity for which the library is selected. By way of example,
the library to
be screened can be present in a population of cells. The library is selected
by growing the
cells in or on a medium comprising the chemical or compound to be oxidized or
reduced
and selecting for a detected physical difference between the oxidized or
reduced form of the
chemical or compound and the non-oxidized or reduced form of the chemical or
compound,
either in the cell, or the extracellular medium.
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Iterative selection for dioxygenase nucleic acids is also a feature of the
invention. In these methods, a selected nucleic acid identified as encoding
dioxygenase
activity can be shuffled, either with the parental nucleic acids, or with
other nucleic acids
(e.g., mutated forms of the selected nucleic acid) to produce a second
shuffled library. The
second shuffled library is then selected for one or more form of dioxygenase
activity, which
can be the same or different than the dioxygenase activity previously
selected. This process
can be iteratively repeated as many times as desired, until a nucleic acid
with optimized
properties is obtained. If desired, any dioxygenase nucleic acid identified by
any of the
methods herein can be cloned and, optionally, expressed.
The invention also provides methods of increasing dioxygenase activity by
whole genome shuffling. In these methods, a plurality of genomic nucleic acids
are
shuffled in a cell (in whole cell shuffling, entire genomes are shuffled,
rather than specific
sequences). The resulting shuffled nucleic acids are selected for one or more
dioxygenase
traits. The genomic nucleic acids can be from a species or strain different
from the cell in
which dioxygenase activity is desired. Similarly, the shuffling reaction can
be performed in
cells using genomic DNA from the same or different species, or strains.
Strains or enzymes
exhibiting enhanced or modified DO activity can be identified.
The distinct or improved dioxygenase activity encoded by a nucleic acid
identified after shuffling can encode for one or more of altered properties
selected from a
variety of properties of practical interest.
Of particular relevance to the practical use of DOs in making industrial
chemicals are several types of improvements in catalytic properties and in the
chemistry of
the catalyzed reactions. Preferred catalytic properties of DOs that can be
altered in a variety
of combinations using nucleic acid shuffling are as follows:
1. Rate of reaction which can be catalyzed by enzyme.
Changing the rate of oxidative reactions (turnover numbers, Vmax or other
related parameters indicative of reaction rate) for a given substrate, or for
a set of substrates,
is often useful as wild-type dioxygenases accept substrates of varying
structure, but the
reaction is often so slow as to be impractical for preparative use.
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2. Specificit~of oxidation.
Altering the specificity of oxidation is preferred when the oxidation of a
compound of interest occurs in a mixture of structurally related compounds,
some of which
may also serve as DO substrates (e.g. isomers of xylene and other
alkylbenzenes). Evolving
DOs by nucleic acid shuffling for greater specificity towards a particular
compound of
interest provides a means for using these enzymes in, for example, the
reactive separation of
compound mixtures, thus allowing for downstream separation of undesired
substrates and
their alternative uses.
3. Regioselectivity of oxidation.
Often, DOs form multiple or alternative products from a substrate when
alternative substrate binding is possible and two or more sites for
introducing oxygen are
present. For the purpose of making industrial chemicals, high or otherwise
altered
selectivity of DO reaction is often preferred. Other properties roughly
falling into the
category of regioselectivity of reactions catalyzed by DOs include, for
example, absolute
configuration of chiral products and their enantiomeric purity (e.g., chiral
hydroxylation),
where chirality of products is possible.
4. Mode of action.
In respect to different substrates, or even to a single compound, DOs can
catalyze different reactions including, but not limited to:
a) monooxygenation of sulfur atoms in various thioethers;
b) O- and N-dealkylation of appropriately substituted arenes;
c) oxidative dehalogenations and denitrations of halogenated and nitrated
arenes;
d) monoxygenation (e.g., monohydoxylation) of "benzylic" (i.e., attached to
a benzene or other aromatic ring) carbon atoms, whether methylene or methyl
groups;
e) monooxygenation (e.g., monohydoxylation) of allylic (i.e., attached to a
non-aromatic ~c-bond) carbon atoms, whether methylene and methyl groups;
f) desaturation reactions of alkyl or cycloalkyl carbon fragments attached to
an aromatic ring (e.g. formation of styrene from ethylbenzene and indene from
indan);
For the purpose of using arene dioxygenases ("ADO"s) in making industrial
chemicals, it is often desired to control or change mode of action of wild-
type (natural)
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dioxygenases, e.g. dioxygenase reaction versus monooxygenase reaction, where
two or
more functional groups amenable to these reactions are present in the
substrate's structure.
Among other properties of interest are those which may not be directly
associated with the catalytic mechanism; however these physical and other
general
properties can make a profound impact on biocatalyst performance in a
practical setting.
These properties include, for example: an increase in the range of dioxygenase
substrates
which the distinct or improved polypeptide operates on, an increased
expression level of a
polypeptide encoded by the nucleic acid, a decrease in susceptibility of a
polypeptide
encoded by the nucleic acid to protease cleavage, a decrease in susceptibility
of a
polypeptide encoded by the nucleic acid to high or low pH levels, a decrease
in
susceptibility of the protein encoded by the nucleic acid to high or low
temperatures, an
optimization of nucleic acid codon usage for effective expression of ADO
polypeptides in a
particular host cell, a reduction in the sensitivity of the ADO polypeptides
and/or an
organism expressing the polypeptide to inactivation by organic solvents, a
decrease in ADO
inactivation or inhibition by substrates, products or reactive intermediates
arising from
ADO reaction or from other metabolic reactions of a host cell, and a decrease
in toxicity to
a host cell of a polypeptide encoded by the selected nucleic acid
The selected nucleic acids to be shuffled can be from any of a variety of
sources, including synthetic or cloned DNAs. Exemplary targets for
recombination include
nucleic acids encoding arene dioxygenases and the like. Typically, shuffled
nucleic acids
are cloned into expression vectors to achieve desired expression levels.
One feature of the invention is production of libraries and shuffling mixtures
for use in the methods as set forth above. For example, a phage display
library comprising
shuffled forms of a nucleic acid is provided. Similarly, a shuffling mixture
comprising at
least three homologous DNAs, each of which is derived from a nucleic acid
encoding a
polypeptide or polypeptide fragment is provided. These polypeptides can be,
for example,
arene dioxygenases, and the like.
Isolated nucleic acids identified by selection of the libraries in the methods
above are also a feature of the invention.
Also provided are biotransformative methods using the dioxygenases of the
invention for preparing diverse oxidized organic species. Representative
species include
vicinal diols, hydroxylated aromatic carboxylic acids, hydroxy alkylarene, a-
hydroxcarboxylic acids and the like. Methods for preparing adducts of oxidized
organic
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species are also provided. To aid in practicing the biotransformative methods
of the
invention, there are also provided improved polypeptides and host organisms
expressing
these polypeptides.
Additional objects and advantages of this invention will be apparent to those
of skill in the art from the detailed description that follows.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Schematic showing the initial dioxygenation of an arene ~-bond of
a trialkylbenzene to produce a diol using a dioxygenase and the subsequent
dehydration of
this diol to a hydroxy trialkylbenzene.
Figure 2. Schematic showing the conversion of a monohydroxy arene to a
dihydroxy arene using a dioxygenase.
Figure 3. Schematic showing esterification and de-esterification strategies
using transferases, esterases and chemical dehydration.
Figure 4. Schematic showing the initial dioxygenation of an arene ~-bond of
a dialkylbenzene to produce a diol using a dioxygenase and the subsequent
dehydration of
this diol to a hydroxy dialkylbenzene.
Figure 5. Schematic showing the oxygenation of an alkylarene to the
corresponding arene carboxylic acid, the subsequent oxidation of an arene ~-
bond to the
corresponding diol and the alkylation and/or acylation of the carboxylic acid
and/or
hydroxyl moieties of the diol.
Figure 6. Schematic of the selective oxidation of one alkyl group of a
dialkylarene to the corresponding arene carboxylic acid, the subsequent
oxidation of an
arene ~-bond to the corresponding diol, the dehydration of the diol and the
alkylation and/or
acylation of the carboxylic acid and/or hydroxyl moieties of the diol.
Figure 7. Schematic showing the oxygenation of an alkylarene to the
corresponding arenealkyl carboxylic acid, the subsequent oxidation of an arene
~-bond to
the corresponding diol and the alkylation and/or acylation of the carboxylic
acid and/or
hydroxyl moieties of the diol.
Figure 8. Schematic showing coumarin and coumarin derivatives.
Figure 9. Schematic showing synthetic routes to cinnamic acid, coumarin
and derivatives of these compounds.
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Figure 10. Schematic showing the oxygenation of a ~-bond of a polycyclic
arene to the corresponding diol, the opening of the diol functionalized ring
and the
conversion of the ring opened product into a lactone.
Figure 11. Structures of exemplary feedstock olefinic compounds and
structures of a-hydroxy carboxylic acids produced from these feedstocks by
dioxygenase
action.
Figure 12. Enzymatic reaction schemes for multistep biochemical
transformations of olefins to AHAs.
Figure 13. Enzymatic reaction schemes for converting free AHAs to ester
derivatives.
Figures 14A- 14R. Table of presently preferred substrates and
oxygenations.
Figure 15. Enzymatic reaction schemes for multistep biochemical
transformations of olefins to AHAs.
Figure 16. Schematic illustrating crossovers for clones obtained from
dioxygenase shuffling.
The absolute configuration of the chiral centers is not indicated in the above
Figures. The chiral centers of the chiral compounds can be R, S, or a mixture
of these
configurations.
DETAILED DESCRIPTION OF THE INVENTION
Abbreviations
"AHA" refers to an a-hydroxycarboxylic acid.
"HCA" refers to a hydroxylated aromatic carboxylic acid.
"ADO" refers to an arene dioxygenase.
"DO" refers to a dioxygenase.
Definitions
Unless clearly indicated to the contrary, the following definitions supplement
definitions of terms known in the art.
The term "shuffling" is used herein to indicate recombination between non-
identical sequences, in some embodiments shuffling may include crossover via
homologous
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recombination or via non-homologous recombination, such as via cre/lox and/or
flp/frt
systems. Shuffling can be carried out by employing a variety of different
formats, including
for example, in vitro and in vivo shuffling formats, in silico shuffling
formats, shuffling
formats that utilize either double-stranded or single-stranded templates,
primer based
shuffling formats, nucleic acid fragmentation-based shuffling formats, and
oligonucleotide-
mediated shuffling formats, all of which are based on recombination events
between non-
identical sequences and are described in more detail or referenced herein
below, as well as
other similar recombination-based formats.
A "recombinant" nucleic acid is a nucleic acid produced by recombination
between two or more nucleic acids, or any nucleic acid made by an in vitro or
artificial
process. The term "recombinant" when used with reference to a cell indicates
that the cell
includes (and optionally replicates) a heterologous nucleic acid, or expresses
a peptide or
protein encoded by a heterologous nucleic acid. Recombinant cells can contain
genes that
are not found within the native (non-recombinant) form of the cell.
Recombinant cells can
also contain genes found in the native form of the cell where the genes are
modified and re-
introduced into the cell by artificial means. The term also encompasses cells
that contain a
nucleic acid endogenous to the cell that has been artificially modified
without removing the
nucleic acid from the cell; such modifications include those obtained by gene
replacement,
site-specific mutation, and related techniques.
A "recombinant dioxygenase nucleic acid" is a recombinant nucleic acid
encoding a protein or RNA which confers dioxygenase activity to a cell when
the nucleic
acid is expressed in the cell.
A "plurality of forms" of a selected nucleic acid refers to a plurality of
homologs of the nucleic acid. The homologs can be from naturally occurnng
homologs
(e.g., two or more homologous genes) or by artificial synthesis of one or more
nucleic acids
having related sequences, or by modification of one or more nucleic acid to
produce related
nucleic acids. Nucleic acids are homologous when they are derived, naturally
or artificially,
from a common ancestor sequence. During natural evolution, this occurs when
two or more
descendent sequences diverge from a parent sequence over time, i.e., due to
mutation and
natural selection. Under artificial conditions, divergence occurs, e.g., in
one of two ways.
First, a given sequence can be artificially recombined with another sequence,
as occurs, e.g.,
during typical cloning, to produce a descendent nucleic acid. Alternatively, a
nucleic acid
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can be synthesized de novo, by synthesizing a nucleic acid which varies in
sequence from a
given parental nucleic acid sequence.
When there is no explicit knowledge about the ancestry of two nucleic acids,
homology is typically inferred by sequence comparison between two sequences.
Where two
S nucleic acid sequences show sequence similarity it is inferred that the two
nucleic acids
share a common ancestor. The precise level of sequence similarity required to
establish
homology varies in the art depending on a variety of factors. For purposes of
this
disclosure, two sequences are considered homologous where they share
sufficient sequence
identity to allow recombination to occur between two nucleic acid molecules.
Typically,
nucleic acids require regions of close similarity spaced roughly the same
distance apart to
permit recombination to occur.
The terms "identical" or percent "identity," in the context of two or more
nucleic acid or polypeptide sequences, refer to two or more sequences or
subsequences that
are the same or have a specified percentage of amino acid residues or
nucleotides that are
the same, when compared and aligned for maximum correspondence, as measured
using
one of the sequence comparison algorithms described below (or other algorithms
available
to persons of skill) or by visual inspection.
The phrase "substantially identical," in the context of two nucleic acids or
polypeptides (e.g., DNAs encoding a dioxygenase, or the amino acid sequence of
the
dioxygenase) refers to two or more sequences or subsequences that have at
least about 60%,
preferably 80%, most preferably 90-95% nucleotide or amino acid residue
identity, when
compared and aligned for maximum correspondence, as measured using one of the
following sequence comparison algorithms or by visual inspection. Such
"substantially
identical" sequences are typically considered to be homologous. Preferably,
the "substantial
identity" exists over a region of the sequences that is at least about 50
residues in length,
more preferably over a region of at least about 100 residues, and most
preferably the
sequences are substantially identical over at least about 150 residues, or
over the full length
of the two sequences to be compared.
For sequence comparison and homology determination, typically one
sequence acts as a reference sequence to which test sequences are compared.
When using a
sequence comparison algorithm, test and reference sequences are input into a
computer,
subsequence coordinates are designated, if necessary, and sequence algorithm
program
parameters are designated. The sequence comparison algorithm then calculates
the percent
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sequence identity for the test sequences) relative to the reference sequence,
based on the
designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by
the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482
(1981), by the
homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443
(1970), by
the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci.
USA
85:2444 (1988), by computerized implementations of these algorithms (GAP,
BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer
Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally,
Ausubel et
al., infra).
One example of an algorithm that is suitable for determining percent
sequence identity and sequence similarity is the BLAST algorithm, which is
described in
Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing
BLAST analyses
is publicly available through the National Center for Biotechnology
Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high
scor7ng
sequence pairs (HSPs) by identifying short words of length W in the query
sequence, which
either match or satisfy some positive-valued threshold score T when aligned
with a word of
the same length in a database sequence. T is referred to as the neighborhood
word score
threshold (Altschul et al., supra). These initial neighborhood word hits act
as seeds for
initiating searches to find longer HSPs containing them. The word hits are
then extended in
both directions along each sequence for as far as the cumulative alignment
score can be
increased. Cumulative scores are calculated using, for nucleotide sequences,
the parameters
M (reward score for a pair of matching residues; always > 0) and N (penalty
score for
mismatching residues; always < 0). For amino acid sequences, a scoring matrix
is used to
calculate the cumulative score. Extension of the word hits in each direction
are halted
when: the cumulative alignment score falls off by the quantity X from its
maximum
achieved value; the cumulative score goes to zero or below, due to the
accumulation of one
or more negative-scoring residue alignments; or the end of either sequence is
reached. The
BLAST algorithm parameters W, T, and X determine the sensitivity and speed of
the
alignment. The BLASTN program (for nucleotide sequences) uses as defaults a
wordlength
(W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a
comparison of both
strands. For amino acid sequences, the BLASTP program uses as defaults a
wordlength (W)
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of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff
&
Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
In addition to calculating percent sequence identity, the BLAST algorithm
also performs a statistical analysis of the similarity between two sequences
(see, e.g., Karlin
& Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of
similarity
provided by the BLAST algorithm is the smallest sum probability (P(N)), which
provides an
indication of the probability by which a match between two nucleotide or amino
acid
sequences would occur by chance. For example, a nucleic acid is considered
similar to a
reference sequence if the smallest sum probability in a comparison of the test
nucleic acid to
the reference nucleic acid is less than about 0.1, more preferably less than
about 0.01, and
most preferably less than about 0.001.
Another indication that two nucleic acid sequences are substantially
identical/homologous is that the two molecules hybridize to each other under
stringent
conditions. The phrase "hybridizing specifically to," refers to the binding,
duplexing, or
hybridizing of a molecule only to a particular nucleotide sequence under
stringent
conditions, including when that sequence is present in a complex mixture
(e.g., total
cellular) DNA or RNA. "Bind(s) substantially" refers to complementary
hybridization
between a probe nucleic acid and a target nucleic acid and embraces minor
mismatches that
can be accommodated by reducing the stringency of the hybridization media to
achieve the
desired detection of the target polynucleotide sequence.
"Stringent hybridization conditions" and "stringent hybridization wash
conditions" in the context of nucleic acid hybridization experiments such as
Southern and
northern hybridizations are sequence dependent, and are different under
different
environmental parameters. Longer sequences hybridize specifically at higher
temperatures.
An extensive guide to the hybridization of nucleic acids is found in Tijssen,
LABORATORY
TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY--HYBRIDIZATION WITH NUCLEIC
ACm PROBES, part I, chapter 2 "Overview of principles of hybridization and the
strategy of
nucleic acid probe assays," Elsevier, New York (1993). Generally, highly
stringent
hybridization and wash conditions are selected to be about 5 °C lower
than the thermal
melting point (Tm) for the specific sequence at a defined ionic strength and
pH. Typically,
under "stringent conditions" a probe will hybridize to its target subsequence,
but no to
unrelated sequences.
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The Tm is the temperature (under defined ionic strength and pH) at which
50% of the target sequence hybridizes to a perfectly matched probe. Very
stringent
conditions are selected to be equal to the Tm for a particular probe. An
example of stringent
hybridization conditions for hybridization of complementary nucleic acids
which have more
than 100 complementary residues on a filter in a Southern or northern blot is
50%
formamide with 1 mg of heparin at 42 °C, with the hybridization being
carried out
overnight. An example of highly stringent wash conditions is 0.15M NaCI at 72
°C for
about 15 minutes. An example of stringent wash conditions is a 0.2x SSC wash
at 65 °C for
minutes (see, Sambrook, infra., for a description of SSC buffer). Often, a
high
10 stringency wash is preceded by a low stringency wash to remove background
probe signal.
An example medium stringency wash for a duplex of, e.g., more than 100
nucleotides, is lx
SSC at 45°C for 15 minutes. An example low stringency wash for a duplex
of, e.g., more
than 100 nucleotides, is 4-6x SSC at 40 °C for 15 minutes. For short
probes (e.g., about 10
to 50 nucleotides), stringent conditions typically involve salt concentrations
of less than
15 about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or
other salts) at
pH 7.0 to 8.3, and the temperature is typically at least about 30 °C.
Stringent conditions can
also be achieved with the addition of destabilizing agents such as formamide.
In general, a
signal to noise ratio of 2x (or higher) than that observed for an unrelated
probe in the
particular hybridization assay indicates detection of a specific
hybridization. Nucleic acids
which do not hybridize to each other under stringent conditions are still
substantially
identical if the polypeptides which they encode are substantially identical.
This occurs, e.g.,
when a copy of a nucleic acid is created using the maximum codon degeneracy
permitted by
the genetic code.
A further indication that two nucleic acid sequences or polypeptides are
substantially identical/homologous is that the polypeptide encoded by the
first nucleic acid
is immunologically cross reactive with, or specifically binds to, the
polypeptide encoded by
the second nucleic acid. Thus, a polypeptide is typically substantially
identical to a second
polypeptide, for example, where the two peptides differ only by conservative
substitutions.
"Conservatively modified variations" of a particular polynucleotide sequence
refers to those polynucleotides that encode identical or essentially identical
amino acid
sequences, or where the polynucleotide does not encode an amino acid sequence,
to
essentially identical sequences. Because of the degeneracy of the genetic
code, a large
CA 02377669 2001-12-12
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number of functionally identical nucleic acids encode any given polypeptide.
For instance,
the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid
arginine.
Thus, at every position where an arginine is specified by a codon, the codon
can be altered
to any of the corresponding codons described without altering the encoded
polypeptide.
Such nucleic acid variations are "silent variations," which are one species of
"conservatively modified variations." Every polynucleotide sequence described
herein
which encodes a polypeptide also describes every possible silent variation,
except where
otherwise noted. One of skill will recognize that each codon in a nucleic acid
(except AUG,
which is ordinarily the only codon for methionine) can be modified to yield a
functionally
identical molecule by standard techniques. Accordingly, each "silent
variation" of a nucleic
acid which encodes a polypeptide is implicit in each described sequence.
Furthermore, one of skill will recognize that individual substitutions,
deletions or additions which alter, add or delete a single amino acid or a
small percentage of
amino acids (typically less than 5%, more typically less than 1%) in an
encoded sequence
are "conservatively modified variations" where the alterations result in the
substitution of an
amino acid with a chemically similar amino acid. Conservative substitution
tables
providing functionally similar amino acids are. well known in the art. The
following five
groups each contain amino acids that are conservative substitutions for one
another:
Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I);
Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing:
Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H);
Acidic:
Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q). See also,
Creighton,
PROTEINS, W.H. Freeman and Company (1984). In addition, individual
substitutions,
deletions or additions which alter, add or delete a single amino acid or a
small percentage of
amino acids in an encoded sequence are also "conservatively modified
variations."
Sequences that differ by conservative variations are generally homologous.
A "subsequence" refers to a sequence of nucleic acids or amino acids that
comprise a part of a longer sequence of nucleic acids or amino acids (e.g.,
polypeptide)
respectively.
The term "gene" is used broadly to refer to any segment of DNA associated
with expression of a given RNA or protein. Thus, genes include regions
encoding
expressed RNAs (which typically include polypeptide coding sequences) and,
often, the
regulatory sequences required for their expression. Genes can be obtained from
a variety of
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sources, including cloning from a source of interest or synthesizing from
known or
predicted sequence information, and may include sequences designed to have
desired
parameters.
The term "isolated", when applied to a nucleic acid or protein, denotes that
the nucleic acid or protein is essentially free of other cellular components
with which it is
associated in the natural state.
The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides
and polymers thereof in either single- or double-stranded form. Unless
specifically limited,
the term encompasses nucleic acids containing known analogues of natural
nucleotides
which have similar binding properties as the reference nucleic acid and are
metabolized in a
manner similar to naturally occurnng nucleotides. Unless otherwise indicated,
a particular
nucleic acid sequence also implicitly encompasses conservatively modified
variants thereof
(e.g. degenerate codon substitutions) and complementary sequences and as well
as the
sequence explicitly indicated. Specifically, degenerate codon substitutions
may be achieved
by generating sequences in which the third position of one or more selected
(or all) codons
is substituted with mixed-base and/or deoxyinosine residues (Batzer et al.,
Nucleic Acid
Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985);
Cassol et al.
(1992) ; Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)). The term
nucleic acid is
generic to the terms "gene", "DNA," "cDNA", "oligonucleotide," "RNA," "mRNA,"
"polynucleotide" and the like.
"Nucleic acid derived from a gene" refers to a nucleic acid for whose
synthesis the gene, or a subsequence thereof, has ultimately served as a
template. Thus, an
mRNA, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that
cDNA,
a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA,
etc., are all
derived from the gene and detection of such derived products is indicative of
the presence
and/or abundance of the original gene and/or gene transcript in a sample.
A nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic acid sequence. For instance, a promoter or
enhancer is
operably linked to a coding sequence if it increases the transcription of the
coding sequence.
A "recombinant expression cassette" or simply an "expression cassette" is a
nucleic acid construct, generated recombinantly or synthetically, with nucleic
acid elements
that are capable of effecting expression of a structural gene in hosts
compatible with such
sequences. Expression cassettes include at least promoters and optionally,
transcription
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termination signals. Typically, the recombinant expression cassette includes a
nucleic acid
to be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a
promoter.
Additional factors necessary or helpful in effecting expression may also be
used as
described herein. For example, an expression cassette can also include
nucleotide
sequences that encode a signal sequence that directs secretion of an expressed
protein from
the host cell. Transcription termination signals, enhancers, and other nucleic
acid sequences
that influence gene expression, can also be included in an expression
cassette.
"Alkyl" refers to straight- and branched-chain, saturated and unsaturated
hydrocarbons. "Lower alkyl", as used herein, refers to "alkyl" groups having
from about 1
to about 6 carbon atoms.
"Substituted alkyl" refers to alkyl as just described including one or more
functional groups such as lower alkyl, aryl, acyl, halogen (i.e., alkylhalos,
e.g., CF3),
hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, aryloxy, aryloxyalkyl,
mercapto,
both saturated and unsaturated cyclic hydrocarbons, heterocycles and the like.
These
groups may be attached to any carbon of the alkyl moiety.
The term "aryl" is used herein to refer to an aromatic substituent which may
be a single aromatic ring or multiple aromatic rings which are fused together,
linked
covalently, or linked to a common group such as a methylene or ethylene
moiety. The
common linking group may also be a carbonyl as in benzophenone. The aromatic
rings)
may include phenyl, napthyl, biphenyl, diphenylmethyl and benzophenone among
others.
The term "aryl" encompasses "arylalkyl."
The term "alkylarene" is used herein to refer to a subset of "aryl" in which
the aryl group is substituted with an alkyl group as defined herein.
"Substituted aryl" refers to aryl as just described including one or more
functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g. CF3),
hydroxy, amino,
alkoxy, alkylamino, acylamino, acyloxy, mercapto and both saturated and
unsaturated
cyclic hydrocarbons which are fused to the aromatic ring(s), linked covalently
or linked to a
common group such as a methylene or ethylene moiety. The linking group may
also be a
carbonyl such as in cyclohexyl phenyl ketone. The term "substituted aryl"
encompasses
"substituted arylalkyl."
The term "acyl" is used to describe a ketone substituent, -C(O)R, wherein
R is alkyl or substituted alkyl, aryl or substituted aryl as defined herein.
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The term "halogen" is used herein to refer to fluorine, bromine, chlorine and
iodine atoms.
The term "hydroxy" is used herein to refer to the group -OH.
The term "amino" is used to describe primary amines, R-NH2, wherein R is
alkyl or substituted alkyl, aryl or substituted aryl as defined herein.
The term "alkoxy" is used herein to refer to the -OR group, wherein R is a
lower alkyl, substituted lower alkyl, aryl, substituted aryl, arylalkyl or
substituted arylalkyl
wherein the alkyl, aryl, substituted aryl, arylalkyl and substituted arylalkyl
groups are as
described herein. Suitable alkoxy radicals include, for example, methoxy,
ethoxy, phenoxy,
substituted phenoxy, benzyloxy, phenethyloxy, t-butoxy, etc.
The term "alkylamino" denotes secondary and tertiary amines wherein the
alkyl groups may be either the same or different and may consist of straight
or branched,
saturated or unsaturated hydrocarbons.
The term "unsaturated cyclic hydrocarbon" is used to describe a non-
aromatic group with at least one double bond, such as cyclopentene,
cyclohexene, etc. and
substituted analogues thereof.
The term "heteroaryl" as used herein refers to aromatic rings in which one or
more carbon atoms of the aromatic rings) are substituted by a heteroatom such
as nitrogen,
oxygen or sulfur. Heteroaryl refers to structures which may be a single
aromatic ring,
multiple aromatic ring(s), or one or more aromatic rings coupled to one or
more non-
aromatic ring(s). In structures having multiple rings, the rings can be fused
together, linked
covalently, or linked to a common group such as a methylene or ethylene
moiety. The
common linking group may also be a carbonyl as in phenyl pyridyl ketone. As
used herein,
rings such as thiophene, pyridine, isoxazole, phthalimide, pyrazole, indole,
furan, etc. or
benzo-fused analogues of these rings are defined by the term "heteroaryl."
"Alkylheteroaryl" defines a subset of "heteroaryl" substituted with an alkyl
group, as defined herein.
"Substituted heteroaryl" refers to heteroaryl as just described wherein the
heteroaryl nucleus is substituted with one or more functional groups such as
lower alkyl,
acyl, halogen, alkylhalos (e.g. CF3), hydroxy, amino, alkoxy, alkylamino,
acylamino,
acyloxy, mercapto, etc. Thus, substituted analogues of heteroaromatic rings
such as
thiophene, pyridine, isoxazole, phthalimide, pyrazole, indole, furan, etc. or
benzo-fused
analogues of these rings are defined by the term "substituted heteroaryl."
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The term "heterocyclic" is used herein to describe a saturated or unsaturated
non-aromatic group having a single ring or multiple condensed rings from about
1 to about
12 carbon atoms and from about 1 to about 4 heteroatoms selected from
nitrogen, sulfur or
oxygen within the ring. Such heterocycles are, for example, tetrahydrofuran,
morpholine,
piperidine, pyrrolidine, etc.
The term "substituted heterocyclic" as used herein describes a subset of
"heterocyclic" wherein the heterocycle nucleus is substituted with one or more
functional
groups such as lower alkyl, acyl, halogen, alkylhalos (e.g., CF3), hydroxy,
amino, alkoxy,
alkylamino, acylamino, acyloxy, mercapto, etc.
The term "alkylheterocyclic" defines a subset of "heterocyclic" substituted
with an alkyl group, as defined herein.
The term "substituted heterocyclicalkyl" defines a subset of "heterocyclic
alkyl" wherein the heterocyclic nucleus is substituted with one or more
functional groups
such as lower alkyl, acyl, halogen, alkylhalos (e.g. CF3), hydroxy, amino,
alkoxy,
alkylamino, acylamino, acyloxy, mercapto, etc.
Introduction
This invention describes the generation of evolved dioxygenases with
enhanced performance for use in the production of chemicals of industrial
interest using any
of a variety of shuffling techniques, including, for example, gene, family and
whole genome
shuffling as described herein. In this invention, shuffling is used to enhance
properties of
dioxygenases, such as forward rate kinetics, substrate specificity and
affinity and also to
decrease susceptibility of dioxygenases to reversible inhibitors and
inactivation by solvents,
starting materials and reaction products and intermediates generated during
the catalytic
cycle.
While much of the discussion below deals explicitly with arene
dioxygenases, this is for clarity of illustration. The discussion is
representative of the
chemistries and improvements which can be made to other useful dioxygenases,
such as the
structurally and functionally similar peroxidases and chlorperoxidases, as
well as to the
structurally unrelated iron-sulfur methane dioxygenases and other enzymes
noted herein
using shuffling methodologies, e.g., shuffling of a single gene or a gene
family, as described
herein.
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In a first aspect, the present invention provides a method for obtaining a
nucleic acid that encodes an improved polypeptide possessing dioxygenase
activity. The
improved polypeptide has at least one property improved over a naturally
occurring
dioxygenase polypeptide. The method includes: (a) creating a library of
recombinant
polynucleotides encoding a recombinant dioxygenase polypeptide; and (b)
screening the
library to identify a recombinant polynucleotide that encodes an improved
recombinant
dioxygenase polypeptide that has at least one property improved over the
naturally
occurring polypeptide. Also provided are nucleic acids produced by this method
that
encode a dioxygenase polypeptide having at least one property improved over a
naturally
occurring dioxygenase polypeptide.
In a preferred embodiment, the nucleic acid libraries of the invention are
constructed by a method that includes shuffling a plurality of parental
polynucleotides to
produce one or more recombinant dioxygenase polynucleotide encoding the
improved
property. In another preferred embodiment, the polynucleotides are homologous.
A
detailed description of shuffling techniques is provided in Part A, herein
below.
In another embodiment, at least one of the parental polynucleotides is
selected from polynucleotides that encode at least one dioxygenase activity
and those that
do not encode at least one dioxygenase activity. Typically, the parental
dioxygenase
polynucleotide encodes a complete polypeptide or a polypeptide fragment
selected from an
arene dioxygenase or fragments thereof.
In a preferred embodiment, the activity catalyzed by a dioxygenase
polypeptide is a member selected from one or more reactions described in
Figures 14A-
14R. Other oxidative transformations will be apparent to those of skill in the
art.
The invention provides significant advantages over previously used methods
for optimization of dioxygenase genes. For example, nucleic acid shuffling can
result in
optimization of a desirable property even in the absence of a detailed
understanding of the
mechanism by which the particular property is mediated. In addition, entirely
new
properties can be obtained upon shuffling of DNAs, i.e., shuffled DNAs can
encode
polypeptides or RNAs with properties entirely absent in the parental DNAs
which are
shuffled.
The properties or characteristics that can be acquired or improved vary
widely, and depend on the choice of substrate. For example, for dioxygenase
genes,
properties that one can improve include, but are not limited to, increased
range of
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dioxygenase activity encoded by a particular gene, increased potency against a
dioxygenase
target, increased expression level of the dioxygenase gene, increased
tolerance of the
protein encoded by the dioxygenase gene to protease degradation (or other
natural protein or
RNA degradative processes), increased dioxygenase activity ranges for
conditions such as
heat, cold, low or high pH, reduced toxicity to the host cell, and increased
resistance of the
polypeptide and/or the organism expressing the polypeptide to organic
solvents, and
reaction feedstocks, intermediates and products.
The targets for modification vary in different applications, as does the
property sought to be acquired or improved. Examples of candidate targets for
acquisition
of a property or improvement in a property include genes that encode proteins
which have
enzymatic or other activities useful in dioxygenase reactions.
The methods typically use at least two variant forms of a starting target. The
variant forms of candidate substrates can show substantial sequence or
secondary structural
similarity with each other, but they should also differ in at least one and
preferably at least
two positions.
The initial diversity between forms can be the result of natural variation,
e.g.,
the different variant forms (homologs) are obtained from different individuals
or strains of
an organism, or constitute related sequences from the same organism (e.g.,
allelic
variations), or constitute homologs from different organisms (interspecific
variants).
Alternatively, initial diversity can be induced, e.g., the variant forms can
be generated by
error-prone transcription, such as an error-prone PCR or use of a polymerase
which lacks
proof-reading activity (see, Liao (1990) Gene 88:107-111), of the first
variant form, or, by
replication of the first form in a mutator strain (mutator host cells are
discussed in further
detail below, and are generally well known). The initial diversity between
substrates is
greatly augmented in subsequent steps of recombination for library generation.
A mutator strain can include any mutants in any organism impaired in the
functions of mismatch repair. These include mutant gene products of mutS,
mutT, mutes,
mutt, ovrD, dcm, vsr, umuC, umuD, sbcB, recJ, etc. The impairment is achieved
by
genetic mutation, allelic replacement, selective inhibition by an added
reagent such as a
small molecule or an expressed antisense RNA, or other techniques. Impairment
can be of
the genes noted, or of homologous genes in any organism.
Therefore, in carrying out the practice of the present invention, at least two
variant forms of a nucleic acid which can confer dioxygenase activity are
recombined to
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produce a library of recombinant dioxygenase genes. The library is then
screened to
identify at least one recombinant dioxygenase gene that is optimized for the
particular
property or properties of interest.
The parental polynucleotides can be shuffled in substantially any cell type,
including prokaryotes, eukaryotes, yeast, bacteria and fungi. In a preferred
embodiment, the
one or more recombinant dioxygenase nucleic acid is present in one or more
bacterial,
yeast, or fungal cells and the method involves: pooling multiple separate
dioxygenase
nucleic acids; screening the resulting pooled dioxygenase nucleic acids to
identify a distinct
or improved recombinant dioxygenase nucleic acid that exhibits distinct or
improved
dioxygenase activity compared to a non-recombinant dioxygenase activity
nucleic acid; and
cloning the distinct or improved recombinant nucleic acid.
Often, improvements are achieved after one round of recombination and
selection. However, recursive sequence recombination can be employed to
achieve still
further improvements in a desired property, or to bring about new (or
"distinct") properties.
Recursive sequence recombination entails successive cycles of recombination to
generate
molecular diversity. That is, one creates a family of nucleic acid molecules
showing some
sequence identity to each other but differing in the presence of mutations. In
any given
cycle, recombination can occur in vivo or in vitro, intracellularly or
extracellularly.
Furthermore, diversity resulting from recombination can be augmented in any
cycle by
applying prior methods of mutagenesis (e.g., error-prone PCR or cassette
mutagenesis) to
either the substrates or products for recombination.
A recombination cycle is usually followed by at least one cycle of screening
or selection for molecules having a desired property or characteristic. If a
recombination
cycle is performed in vitro, the products of recombination, i.e., recombinant
segments, are
sometimes introduced into cells before the screening step. Recombinant
segments can also
be linked to an appropriate vector or other regulatory sequences before
screening.
Alternatively, products of recombination generated in vitro are sometimes
packaged in
viruses (e.g., bacteriophage) before screening. If recombination is performed
in vivo,
recombination products can sometimes be screened in the cells in which
recombination
occurred. In other applications, recombinant segments are extracted from the
cells, and
optionally packaged as viruses, before screening.
The nature of screening or selection depends on what property or
characteristic is to be acquired or the property or characteristic for which
improvement is
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sought, and many examples are discussed below. It is not usually necessary to
understand
the molecular basis by which particular products of recombination (recombinant
segments)
have acquired new or improved properties or characteristics relative to the
starting
substrates. For example, a dioxygenase gene can have many component sequences
each
having a different intended role (e.g., coding sequence, regulatory sequences,
targeting
sequences, stability-conferring sequences, subunit sequences and sequences
affecting
integration). Each of these component sequences can be varied and recombined
simultaneously. Screening/selection can then be performed, for example, for
recombinant
segments that have increased ability to confer dioxygenase activity upon a
cell without the
need to attribute such improvement to any of the individual component
sequences of the
vector.
Depending on the particular screening protocol used for a desired property,
initial rounds) of screening are sometimes performed using bacterial cells due
to high
transfection efficiencies and ease of culture. However, for eukaryotic
dioxygenases such as
eukaryotic arene dioxygenases, bacterial expression is often not practical,
and yeast, fungal
or other eukaryotic systems are used for library expression and screening.
Similarly other
types of screening that are not amenable to screening in bacterial or simple
eukaryotic
library cells, are performed in cells selected for use in an environment close
to that of their
intended use. Final rounds of screening can be performed in the precise cell
type of
intended use.
If further improvement in a property is desired, at least one and usually a
collection of recombinant segments surviving a first round of
screening/selection are subject
to a further round of recombination. These recombinant segments can be
recombined with
each other or with exogenous segments representing the original substrates or
further
variants thereof. Again, recombination can proceed in vitro or in vivo. If the
previous
screening step identifies desired recombinant segments as components of cells,
the
components can be subjected to further recombination in vivo, or can be
subjected to further
recombination in vitro, or can be isolated before performing a round of in
vitro
recombination. Conversely, if the previous screening step identifies desired
recombinant
segments in naked form or as components of viruses, these segments can be
introduced into
cells to perform a round of in vivo recombination. The second round of
recombination,
irrespective how performed, generates further recombinant segments which
encompass
additional diversity than is present in recombinant segments resulting from
previous rounds.
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The second round of recombination is optionally followed by a further round
of screening/selection according to the principles discussed above for the
first round. The
stringency of screening/selection can be increased between rounds. Also, the
nature of the
screen and the property being screened for can vary between rounds if
improvement in more
than one property is desired or if acquiring more than one new property is
desired.
Additional rounds of recombination and screening can then be performed until
the
recombinant segments have sufficiently evolved to acquire the desired new or
improved
property or function.
In a preferred embodiment, the invention provides a recursive method for
making a nucleic acid encoding a specific dioxygenase activity. In this
method, the parental
nucleic acids are shuffled in a plurality of cells and the method optionally
further includes
one or more of: (a) recombining DNA from the plurality of cells that display
dioxygenase
activity with a library of DNA fragments, at least one of which undergoes
recombination
with a segment in a cellular DNA present in the cells to produce recombined
cells, or
recombining DNA between the plurality of cells that display dioxygenase
activity to
produce cells with modified dioxygenase activity; (b) recombining and
screening the
recombined or modified cells to produce further recombined cells that have
evolved
additionally modified dioxygenase activity; and, (c) repeating (a) or (b)
until the further
recombined cells have acquired a desired dioxygenase activity.
In another preferred embodiment, the invention provides a method for
making a nucleic acid encoding a specific dioxygenase activity. This method
includes: (a)
recombining at least one distinct or improved recombinant nucleic acid with a
further
dioxygenase activity nucleic acid, which further nucleic acid is the same or
different from
one or more of the plurality of parental nucleic acids to produce a library of
recombinant
dioxygenase nucleic acids; (b) screening the library to identify at least one
further distinct or
improved recombinant dioxygenase nucleic acid that exhibits a further
improvement or
distinct property compared to the plurality of parental nucleic acids; and,
optionally; (c)
repeating (a) and (b) until the resulting further distinct or improved
recombinant nucleic
acid shows an additionally distinct or improved dioxygenase property.
The practice of this invention involves the construction of recombinant
nucleic acids and the expression of genes in transfected host cells. Molecular
cloning
techniques to achieve these ends are known in the art. A wide variety of
cloning and in
vitro amplification methods suitable for the construction of recombinant
nucleic acids such
CA 02377669 2001-12-12
WO 01/12791 PCT/US00/22038
as expression vectors are well-known to persons of skill. General texts which
describe
molecular biological techniques useful herein, including mutagenesis, include
Berger and
Kimmel, GUIDE TO MOLECULAR CLONING TECHNIQUES, METHODS IN ENZYMOLOGY,
volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al.,
MOLECULAR
CLONING - A LABORATORY MANUAL (2nd Ed.), Vol. 1-3, Cold Spring Harbor
Laboratory,
Cold Spring Harbor, New York, 1989 ("Sambrook") and CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, F.M. Ausubel et al., eds., Current Protocols, a joint
venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented
through
1998) ("Ausubel")). Examples of techniques sufficient to direct persons of
skill through in
vitro amplification methods, including the polymerise chain reaction (PCR) the
ligase chain
reaction (LCR), Q~i-replicase amplification and other RNA polymerise mediated
techniques (e.g., NASBA) are found in Bergen Sambrook, and Ausubel, as well as
Mullis et
al., (1987) U.S. Patent No. 4,683,202; PCR PROTOCOLS A GUIDE To METHODS AND
APPLICATIONS (Innis et al. eds) Academic Press Inc., San Diego, CA (1990)
(Innis);
Arnheim & Levinson (October 1, 1990) C&EN 36-47; The Journal Of NIH Research
3:81-
94 (1991); (Kwoh et al., Proc. Natl. Acid. Sci. USA 86:1173; (1989) Guatelli
et al., Proc.
Natl. Acid. Sci. USA 87:1874 (1990); Lomell et al., J. Clin. Chem 35:1826
(1989);
Landegren et al., Science 241:1077-1080 (1988); Van Brunt, Biotechnology 8:291-
294
(1990); Wu and Wallace, Gene 4:560 (1989); Barringer et al., Gene 89:117
(1990), and
Sooknanan and Malek, Biotechnology 13:563-564 (1995). Improved methods of
cloning in
vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No.
5,426,039.
Improved methods of amplifying large nucleic acids by PCR are summarized in
Cheng et
al., Nature 369:684-685 (1994), and the references cited therein, in which PCR
amplicons
of up to 40kb are generated. One of skill will appreciate that essentially any
RNA can be
converted into a double stranded DNA suitable for restriction digestion, PCR
expansion and
sequencing using reverse transcriptase and a polymerise. See, Ausbel, Sambrook
and
Berger, all supra.
In another aspect, the present invention provides a method of increasing
dioxygenase activity in a cell. The method includes performing whole genome
shuffling of
a plurality of genomic nucleic acids in the cell and selecting for one or more
dioxygenase
activity. In this aspect of the invention, the genomic nucleic acids can be
from substantially
any source. In a preferred embodiment of this aspect of the invention, the
genomic nucleic
26
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WO 01/12791 PCT/US00/22038
acids are from a species or strain different from the cell. In a further
preferred embodiment,
the cell is of prokaryotic or eukaryotic origin.
Substantially any dioxygenase property can be selected for using the
methods of the invention. A preferred property is the activity of the
polypeptide towards a
particular class of substrates. In a preferred embodiment, the dioxygenase
property is its
ability to effect one or more reactions described in Figures 14A-14R.
In a third aspect, the invention provides a nucleic acid shuffling mixture
comprising: at least three homologous DNAs, each of which is derived from a
nucleic acid
encoding a polypeptide or polypeptide fragment which encodes dioxygenase
activity. In a
preferred embodiment of this aspect of the invention, the at least three
homologous DNAs
are present in cell culture or in vitro.
Oligonucleotides for use as probes, e.g., in in vitro amplification methods,
for use as gene probes, or as shuffling targets (e.g., synthetic genes or gene
segments) are
typically synthesized chemically according to the solid phase phosphoramidite
triester
method described by Beaucage and Caruthers, Tetrahedron Letts., 22(20):1859-
1862
(1981), e.g., using an automated synthesizer, as described in Needham-
VanDevanter et al.,
Nucleic Acids Res. 12:6159-6168 (1984). Oligonucleotides can also be custom
made and
ordered from a variety of commercial sources known to persons of skill.
A. Formats for Seguence Recombination
The methods of the invention entail performing recombination ("shuffling")
and screening or selection to "evolve" individual genes, whole plasmids or
viruses,
multigene clusters, or even whole genomes (Stemmer, BiolTechnology 13:549-553
(1995)).
Reiterative cycles of recombination and screening/selection can be performed
to further
evolve the nucleic acids of interest. Such techniques do not require the
extensive analysis
and computation required by conventional methods for polypeptide engineering.
Shuffling
allows the recombination of large numbers of mutations in a minimum number of
selection
cycles, in contrast to natural pair-wise recombination events (e.g., as occur
during sexual
replication). Thus, the sequence recombination techniques described herein
provide
particular advantages in that they provide recombination between mutations in
any or all of
these, thereby providing a very fast way of exploring the manner in which
different
combinations of mutations can affect a desired result. In some instances,
however,
structural and/or functional information is available which, although not
required for
sequence recombination, provides opportunities for modification of the
technique.
27
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WO 01/12791 PCT/US00/22038
A variety of nucleic acid shuffling protocols are available and fully
described
in the art. Descriptions of a variety of shuffling methods for generating
modified nucleic
acid sequences for use in the methods of the present invention include the
following
publications and the references cited therein: Stemmer, et al. (1999)
"Molecular breeding of
viruses for targeting and other clinical properties" Tumor Tar e~ ting 4:1-4;
Ness et al. (1999)
"DNA Shuffling of subgenomic sequences of subtilisin" Nature Biotechnolo~y
17:893-896;
Chang et al. (1999) "Evolution of a cytokine using DNA family shuffling"
Nature
Biotechnolo~y 17:793-797; Minshull and Stemmer (1999) "Protein evolution by
molecular
breeding" Current Opinion in Chemical Biolo~y 3:284-290; Christians et al.
(1999)
"Directed evolution of thymidine kinase for AZT phosphorylation using DNA
family
shuffling" Nature Biotechnolo~y 17:259-264; Crameri et al. (1998) "DNA
shuffling of a
family of genes from diverse species accelerates directed evolution" Nature
391:288-291;
Crameri et al. (1997) "Molecular evolution of an arsenate detoxification
pathway by DNA
shuffling," Nature Biotechnolo~y 15:436-438; Zhang et al. (1997) "Directed
evolution of an
effective fucosidase from a galactosidase by DNA shuffling and screening"
Proc. Natl.
Acad. Sci. USA 94:4504-4509; Patten et al. (1997) "Applications of DNA
Shuffling to
Pharmaceuticals and Vaccines" Current Opinion in Biotechnolo~y 8:724-733;
Crameri et al.
(1996) "Construction and evolution of antibody-phage libraries by DNA
shuffling" Nature
Medicine 2:100-103; Crameri et al. (1996) "Improved green fluorescent protein
by
molecular evolution using DNA shuffling" Nature Biotechnolo~y 14:315-319;
Gates et al.
(1996) "Affinity selective isolation of ligands from peptide libraries through
display on a lac
repressor 'headpiece dimer"' Journal of Molecular Biolo~y 255:373-386; Stemmer
(1996)
"Sexual PCR and Assembly PCR" In: The Encyclopedia of Molecular Biolo~y. VCH
Publishers, New York. pp.447-457; Crameri and Stemmer (1995) "Combinatorial
multiple
cassette mutagenesis creates all the permutations of mutant and wildtype
cassettes"
BioTechniques 18:194-195; Stemmer et al., (1995) "Single-step assembly of a
gene and
entire plasmid form large numbers of oligodeoxy-ribonucleotides" Gene, 164:49-
53;
Stemmer (1995) "The Evolution of Molecular Computation" Science 270: 1510;
Stemmer
(1995) "Searching Sequence Space" Bio/Technolo~y 13:549-553; Stemmer (1994)
"Rapid
evolution of a protein in vitro by DNA shuffling" Nature 370:389-391; and
Stemmer (1994)
"DNA shuffling by random fragmentation and reassembly: In vitro recombination
for
molecular evolution." Proc. Natl. Acad. Sci. USA 91:10747-10751.
28
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WO 01/12791 PCT/US00/22038
Additional details regarding DNA shuffling methods can be found in the
following U.S. patents, PCT publications, and EPO publications: USPN 5,605,793
to
Stemmer (February 25, 1997), "Methods for In Vitro Recombination;" USPN
5,811,238 to
Stemmer et al. (September 22, 1998) "Methods for Generating Polynucleotides
having
Desired Characteristics by Iterative Selection and Recombination;" USPN
5,830,721 to
Stemmer et al. (November 3, 1998), "DNA Mutagenesis by Random Fragmentation
and
Reassembly;" USPN 5,834,252 to Stemmer, et al. (November 10, 1998) "End-
Complementary Polymerase Reaction;" USPN 5,837,458 to Minshull, et al.
(November 17,
1998), "Methods and Compositions for Cellular and Metabolic Engineering;" WO
95/22625, Stemmer and Crameri, "Mutagenesis by Random Fragmentation and
Reassembly;" WO 96/33207 by Stemmer and Lipsehutz "End Complementary
Polymerase
Chain Reaction;" WO 97/20078 by Stemmer and Crameri "Methods for Generating
Polynucleotides having Desired Characteristics by Iterative Selection and
Recombination;"
WO 97/35966 by Minshull and Stemmer, "Methods and Compositions for Cellular
and
Metabolic Engineering;" WO 99/41402 by Punnonen et al. "Targeting of Genetic
Vaccine
Vectors;" WO 99/41383 by Punnonen et al. "Antigen Library Immunization;" WO
99/41369 by Punnonen et al. "Genetic Vaccine Vector Engineering;" WO 99/41368
by
Punnonen et al. "Optimization of Immunomodulatory Properties of Genetic
Vaccines;" EP
752008 by Stemmer and Crameri, "DNA Mutagenesis by Random Fragmentation and
Reassembly;" EP 0932670 by Stemmer "Evolving Cellular DNA Uptake by Recursive
Sequence Recombination;" WO 99/23107 by Stemmer et al., "Modification of Virus
Tropism and Host Range by Viral Genome Shuffling;" WO 99/21979 by Apt et al.,
"Human
Papillomavirus Vectors;" WO 98/31837 by del Cardayre et al. "Evolution of
Whole Cells
and Organisms by Recursive Sequence Recombination;" WO 98/27230 by Patten and
Stemmer, "Methods and Compositions for Polypeptide Engineering;" WO 98/13487
by
Stemmer et al., "Methods for Optimization of Gene Therapy by Recursive
Sequence
Shuffling and Selection," WO 00/00632, "Methods for Generating Highly Diverse
Libraries," WO 00/09679, "Methods for Obtaining in Vitro Recombined
Polynucleotide
Sequence Banks and Resulting Sequences," WO 98/42832 by Arnold et al.,
"Recombination of Polynucleotide Sequences Using Random or Defined Primers,"
WO
99/29902 by Amold et al., "Method for Creating Polynucleotide and Polypeptide
Sequences," WO 98/41653 by Vind, "An in Vitro Method for Construction of a DNA
29
CA 02377669 2001-12-12
WO 01/12791 PCT/US00/22038
Library," and WO 98/41622 by Borchert et al., "Method for Constructing a
Library Using
DNA Shuffling."
Certain U.S. applications provide additional details regarding shuffling
methods, including "SHUFFLING OF CODON ALTERED GENES" by Patten et al. filed
September 29, 1998, (USSN 60/102,362), January 29, 1999 (USSN 60/117,729), and
September 28, 1999, (USSN 09/407,800); "EVOLUTION OF WHOLE CELLS AND
ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION", by del Cardayre et al.
filed July 15, 1998 (USSN 09/166,188), and July 15, 1999 (USSN 09/354,922);
"OLIGONUCLEOTmE MEDIATED NUCLEIC ACID RECOMBINATION" by Crameri
et al., filed February 5, 1999 (USSN 60/118,813), June 24, 1999 (USSN
60/141,049), and
September 28, 1999 (USSN 09/408,392); "USE OF CODON-BASED
OLIGONUCLEOT>DE SYNTHESIS FOR SYNTHETIC SHUFFLING" by Welch et al.,
filed September 28, 1999 (USSN 09/408,393); "METHODS FOR MAKING
CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING
DESIRED CHARACTERISTICS" by Selifonov and Stemmer, filed February 5, 1999
(USSN 60/118854) and October 12, 1999 (USSN 09/416,375); and "SINGLE-STRANDED
NUCLEIC ACID TEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID
FRAGMENT ISOLATION" by Affholter, USSN 60/186,482 filed March 2,2000.
In brief, a variety of shuffling formats are applicable to the present
invention
and set forth, e.g., in the references above. The following exemplify some of
the different
types of formats. First, nucleic acids can be recombined in vitro by any of a
variety of
techniques discussed in the references above, including e.g., DNAse digestion
of nucleic
acids to be recombined followed by ligation and/or PCR reassembly of the
nucleic acids.
Second, nucleic acids can be recursively recombined in vivo, e.g., by allowing
recombination to occur between nucleic acids in cells. Third, whole genome
recombination
methods can be used in which whole genomes of cells or other organisms are
recombined,
optionally including spiking of the genomic recombination mixtures with
desired library
components (e.g., genes corresponding to the pathways of the present
invention). Fourth,
synthetic recombination methods can be used, in which oligonucleotides
corresponding to
targets of interest are synthesized and reassembled in PCR or ligation
reactions which
include oligonucleotides which correspond to more than one parental nucleic
acid, thereby
generating new recombined nucleic acids. Oligonucleotides can be made by
standard
nucleotide addition methods, or can be made, e.g., by tri-nucleotide synthetic
approaches.
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WO 01/12791 PCT/US00/22038
Fifth, in silico methods of recombination can be effected in which genetic
algorithms are
used in a computer to recombine sequence strings which correspond to
homologous (or
even non-homologous) nucleic acids. The resulting recombined sequence strings
are
optionally converted into nucleic acids by synthesis of nucleic acids which
correspond to
the recombined sequences, e.g., in concert with oligonucleotide synthesis/
gene reassembly
techniques. Any of the preceding general recombination formats can be
practiced in a
reiterative fashion to generate a more diverse set of recombinant nucleic
acids. Sixth,
methods of accessing natural diversity, e.g., by hybridization of diverse
nucleic acids or
nucleic acid fragments to single-stranded templates, followed by
polymerization and/or
ligation to regenerate full-length sequences, optionally followed by
degradation of the
templates and recovery of the resulting modified nucleic acids can be used.
The above references provide these and other basic recombination formats as
well as many modifications of these formats. Regardless of the shuffling
format which is
used, the nucleic acids of the invention can be recombined (with each other,
or with related
(or even unrelated) sequences) to produce a diverse set of recombinant nucleic
acids,
including, e.g., sets of homologous nucleic acids.
Following recombination, any nucleic acids which are produced can be
selected for a desired activity. In the context of the present invention, this
can include
testing for and identifying any activity that can be detected e.g., in an
automatable format,
by any of the assays in the art. A variety of related (or even unrelated)
properties can be
assayed for, using any available assay.
DNA mutagenesis and shuffling provide a robust, widely applicable, means
of generating diversity useful for the engineering of proteins, pathways,
cells and organisms
with improved characteristics. In addition to the basic formats described
above, it is
sometimes desirable to combine shuffling methodologies with other techniques
for
generating diversity. In conjunction with (or separately from) shuffling
methods, a variety
of diversity generation methods can be practiced and the results (i.e.,
diverse populations of
nucleic acids) screened for in the systems of the invention. Additional
diversity can be
introduced by methods which result in the alteration of individual nucleotides
or groups of
contiguous or non-contiguous nucleotides, i.e., mutagenesis methods. Many
mutagenesis
methods are found in the above-cited references; additional details regarding
mutagenesis
methods can be found in the references listed below.
31
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WO 01/12791 PCT/US00/22038
Mutagenesis methods include, for example, those described in
PCT/US98/05223; Publ. No. W098/42727; site-directed mutagenesis (Ling et al.
(1997)
"Approaches to DNA mutagenesis: an overview" Anal Biochem. 254(2): 157-178;
Dale et
al. (1996) "Oligonucleotide-directed random mutagenesis using the
phosphorothioate
method" Methods Mol. Biol. 57:369-374; Smith (1985) "In vitro mutagenesis"
Ann. Rev.
Genet. 19:423-462; Botstein & Shortle (1985) "Strategies and applications of
in vitro
mutagenesis" Science 229:1193-1201; Carter (1986) "Site-directed mutagenesis"
Biochem.
J. 237:1-7; and Kunkel (1987) "The efficiency of oligonucleotide directed
mutagenesis" in
Nucleic Acids & Molecular Biolo~y (Eckstein, F. and Lilley, D.M.J. eds.,
Springer Verlag,
Berlin)); mutagenesis using uracil containing templates (Kunkel (1985) "Rapid
and efficient
site-specific mutagenesis without phenotypic selection" Proc. Natl. Acad. Sci.
USA 82:488-
492; Kunkel et al. (1987) "Rapid and efficient site-specific mutagenesis
without phenotypic
selection" Methods in Enzymol. 154, 367-382; and Bass et al. (1988) "Mutant
Trp
repressors with new DNA-binding specificities" Science 242:240-245);
oligonucleotide-
directed mutagenesis (Methods in Enzymol. 100: 468-500 (1983); Methods in
EnzXmol.
154: 329-350 (1987); Zoller & Smith (1982) "Oligonucleotide-directed
mutagenesis using
M13-derived vectors: an efficient and general procedure for the production of
point
mutations in any DNA fragment" Nucleic Acids Res. 10:6487-6500; Zoller & Smith
(1983)
"Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13
vectors"
Methods in Enzymol. 100:468-500; and Zoller & Smith (1987) "Oligonucleotide-
directed
mutagenesis: a simple method using two oligonucleotide primers and a single-
stranded
DNA template" Methods in Enzymol. 154:329-350); phosphorothioate-modified DNA
mutagenesis (Taylor et al. (1985) "The use of phosphorothioate-modified DNA in
restriction enzyme reactions to prepare nicked DNA" Nucl. Acids Res. 13: 8749-
8764;
Taylor et al. (1985) "The rapid generation of oligonucleotide-directed
mutations at high
frequency using phosphorothioate-modified DNA" Nucl. Acids Res. 13: 8765-8787
(1985);
Nakamaye & Eckstein (1986) "Inhibition of restriction endonuclease Nci I
cleavage by
phosphorothioate groups and its application to oligonucleotide-directed
mutagenesis" Nucl.
Acids Res. 14: 9679-9698; Sayers et al. (1988) "Y-T Exonucleases in
phosphorothioate-
based oligonucleotide-directed mutagenesis" Nucl. Acids Res. 16:791-802; and
Sayers et al.
(1988) "Strand specific cleavage of phosphorothioate-containing DNA by
reaction with
restriction endonucleases in the presence of ethidium bromide" Nucl. Acids
Res. 16: 803-
814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) "The gapped
duplex
32
CA 02377669 2001-12-12
WO 01/12791 PCT/US00/22038
DNA approach to oligonucleotide-directed mutation construction" Nucl. Acids
Res. 12:
9441-9456; Kramer & Fritz (1987) Methods in Enz~ "Oligonucleotide-directed
construction of mutations via gapped duplex DNA" 154:350-367; Kramer et al.
(1988)
"Improved enzymatic in vitro reactions in the gapped duplex DNA approach to
oligonucleotide-directed construction of mutations" Nucl. Acids Res. 16: 7207;
and Fritz et
al. (1988) "Oligonucleotide-directed construction of mutations: a gapped
duplex DNA
procedure without enzymatic reactions in vitro" Nucl. Acids Res. 16: 6987-
6999).
Additional suitable methods include point mismatch repair (Kramer et al.
(1984) "Point Mismatch Repair" Cell 38:879-887), mutagenesis using repair-
deficient host
strains (Carter et al. (1985) "Improved oligonucleotide site-directed
mutagenesis using M13
vectors" Nucl. Acids Res. 13: 4431-4443; and Carter (1987) "Improved
oligonucleotide-
directed mutagenesis using M13 vectors" Methods in Enzymol. 154: 382-403),
deletion
mutagenesis (Eghtedarzadeh & Henikoff (1986) "Use of oligonucleotides to
generate large
deletions" Nucl. Acids Res. 14: 5115), restriction-selection and restriction-
selection and
restriction-purification (Wells et al. (1986) "Importance of hydrogen-bond
formation in
stabilizing the transition state of subtilisin" Phil. Trans. R. Soc. Lond. A
317: 415-423),
mutagenesis by total gene synthesis (Nambiar et al. (1984) "Total synthesis
and cloning of a
gene coding for the ribonuclease S protein" Science 223: 1299-1301; Sakamar
and Khorana
(1988) "Total synthesis and expression of a gene for the a-subunit of bovine
rod outer
segment guanine nucleotide-binding protein (transducin)" Nucl. Acids Res. 14:
6361-6372;
Wells et al. (1985) "Cassette mutagenesis: an efficient method for generation
of multiple
mutations at defined sites" Gene 34:315-323; and Grundstrom et al. (1985)
"Oligonucleotide-directed mutagenesis by microscale 'shot-gun' gene synthesis"
Nucl.
Acids Res. 13: 3305-3316), double-strand break repair (Mandecki (1986)
"Oligonucleotide-
directed double-strand break repair in plasmids of Escherichia coli: a method
for site-
specific mutagenesis" Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional
details on
many of the above methods can be found in Methods in Enzymolo~y Volume 154,
which
also describes useful controls for trouble-shooting problems with various
mutagenesis
methods.
In one aspect of the present invention, error-prone PCR can be used to
generate nucleic acid variants. Using this technique, PCR is performed under
conditions
where the copying fidelity of the DNA polymerase is low, such that a high rate
of point
mutations is obtained along the entire length of the PCR product. Examples of
such
33
CA 02377669 2001-12-12
WO 01/12791 PCT/US00/22038
techniques are found in the references above and, e.g., in Leung et al. (1989)
Technique
1:11-15 and Caldwell et al. (1992) PCR Methods Applic. 2:28-33. Similarly,
assembly PCR
can be used, in a process which involves the assembly of a PCR product from a
mixture of
small DNA fragments. A large number of different PCR reactions can occur in
parallel in
the same vial, with the products of one reaction priming the products of
another reaction.
Sexual PCR mutagenesis can be used in which homologous recombination occurs
between
DNA molecules of different but related DNA sequence in vitro, by random
fragmentation of
the DNA molecule based on sequence homology, followed by fixation of the
crossover by
primer extension in a PCR reaction. This process is described in the
references above, e.g.,
in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Recursive
ensemble
mutagenesis can be used in which an algorithm for protein mutagenesis is used
to produce
diverse populations of phenotypically related mutants whose members differ in
amino acid
sequence. This method uses a feedback mechanism to control successive rounds
of
combinatorial cassette mutagenesis. Examples of this approach are found in
Arkin &
Youvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815.
As noted, oligonucleotide directed mutagenesis can be used in a process
which allows for the generation of site-specific mutations in any nucleic acid
sequence of
interest. Examples of such techniques are found in the references above and,
e.g., in
Reidhaar-Olson et al. (1988) Science, 241:53-57. Similarly, cassette
mutagenesis can be
used in a process which replaces a small region of a double stranded DNA
molecule with a
synthetic oligonucleotide cassette that differs from the native sequence. The
oligonucleotide can contain, e.g., completely and/or partially randomized
native
sequence(s).
In vivo mutagenesis can be used in a process of generating random mutations
in any cloned DNA of interest which involves the propagation of the DNA, e.g.,
in a strain
of E. coli that carries mutations in one or more of the DNA repair pathways.
These
"mutator" strains have a higher random mutation rate than that of a wild-type
parent.
Propagating the DNA in one of these strains will eventually generate random
mutations
within the DNA.
Exponential ensemble mutagenesis can be used for generating combinatorial
libraries with a high percentage of unique and functional mutants, where small
groups of
residues are randomized in parallel to identify, at each altered position,
amino acids which
lead to functional proteins. Examples of such procedures are found in
Delegrave & Youvan
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WO 01/12791 PCT/US00/22038
(1993) Biotechnolo~y Research 11:1548-1552. Similarly, random and site-
directed
mutagenesis can be used. Examples of such procedures are found in Arnold
(1993) Current
Opinion in Biotechnology 4:450-455.
Kits for mutagenesis are also commercially available. For example, kits are
available from, e.g., Stratagene (e.g., QuickChange~ site-directed mutagenesis
kit; and
ChameleonTM double-stranded, site-directed mutagenesis kit), Bio/Can
Scientific, Bio-Rad
(e.g., using the Kunkel method described above), Boehringer Mannheim Corp.,
Clonetech
Laboratories, DNA Technologies, Epicentre Technologies (e.g., 5 prime 3 prime
kit);
Genpak Inc, Lemargo Inc, Life Technologies (Gibco BRL), New England Biolabs,
Pharmacia Biotech, Promega Corp., Quantum Biotechnologies, Amersham
International plc
(e.g., using the Eckstein method above), and Anglian Biotechnology Ltd (e.g.,
using the
Carter/Winter method above).
Any of the described shuffling or mutagenesis techniques can be used in
conjunction with procedures which introduce additional diversity into a
genome, e.g. a
bacterial, fungal, animal or plant genome. For example, in addition to the
methods above,
techniques have been proposed which produce nucleic acid multimers suitable
for
transformation into a variety of species (see, e.g., Schellenberger U.S.
Patent No. 5,756,316
and the references above). When such multimers consist of genes that are
divergent with
respect to one another, (e.g., derived from natural diversity or through
application of site
directed mutagenesis, error prone PCR, passage through mutagenic bacterial
strains, and the
like), are transformed into a suitable host, this provides a source of nucleic
acid diversity for
DNA diversification.
Multimers transformed into host species are suitable as substrates for in vivo
shuffling protocols. Alternatively, a multiplicity of polynucleotides sharing
regions of
partial sequence similarity can be transformed into a host species and
recombined in vivo by
the host cell. Subsequent rounds of cell division can be used to generate
libraries, members
of which, comprise a single, homogenous population of monomeric or pooled
nucleic acid.
Alternatively, the monomeric nucleic acid can be recovered by standard
techniques and
recombined in any of the described shuffling formats.
Shuffling formats employing chain termination methods have also been
proposed (see e.g., U.S. Patent No. 5,965,408 and the references above). In
this approach,
double stranded DNAs corresponding to one or more genes sharing regions of
sequence
similarity are combined and denatured, in the presence or absence of primers
specific for the
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gene. The single stranded polynucleotides are then annealed and incubated in
the presence
of a polymerase and a chain terminating reagent (e.g., ultraviolet, gamma or X-
ray
irradiation; ethidium bromide or other intercalators; DNA binding proteins,
such as single
strand binding proteins, transcription activating factors, or histones;
polycyclic aromatic
hydrocarbons; trivalent chromium or a trivalent chromium salt; or abbreviated
polymerization mediated by rapid thermocycling; and the like), resulting in
the production
of partial duplex molecules. The partial duplex molecules, e.g., containing
partially
extended chains, are then denatured and reannealed in subsequent rounds of
replication or
partial replication resulting in polynucleotides which share varying degrees
of sequence
similarity and which are chimeric with respect to the starting population of
DNA molecules.
Optionally, the products or partial pools of the products can be amplified at
one or more
stages in the process. Polynucleotides produced by a chain termination method,
such as
described above are suitable substrates for DNA shuffling according to any of
the described
formats.
Diversity can be further increased by using non-homology based shuffling
methods (which, as set forth in the above publications and applications can be
homology or
non-homology based, depending on the precise format). For example, incremental
truncation for the creation of hybrid enzymes (ITCHY) described in Ostermeier
et al. (1999)
"A combinatorial approach to hybrid enzymes independent of DNA homology"
Nature
Biotech 17:1205, can be used to generate an initial a shuffled library which
can optionally
serve as a substrate for one or more rounds of in vitro or in vivo shuffling
methods. See,
also, Ostermeier et al. (1999) "Combinatorial Protein Engineering by
Incremental
Truncation," Proc. Natl. Acad. Sci. USA, 96: 3562-67; Ostermeier et al.
(1999),
"Incremental Truncation as a Strategy in the Engineering of Novel
Biocatalysts," Biological
and Medicinal Chemistry, 7: 2139-44.
Methods for generating multispecies expression libraries have been
described (e.g., U.S. Patent Nos. 5,783,431; 5,824,485 and the references
above) and their
use to identify protein activities of interest has been proposed (U.S. Patent
5,958,672 and
the references above). Multispecies expression libraries are, in general,
libraries comprising
cDNA or genomic sequences from a plurality of species or strains, operably
linked to
appropriate regulatory sequences, in an expression cassette. The cDNA and/or
genomic
sequences are optionally randomly concatenated to further enhance diversity.
The vector
can be a shuttle vector suitable for transformation and expression in more
than one species
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of host organism, e.g., bacterial species, eukaryotic cells. In some cases,
the library is
biased by preselecting sequences which encode a protein of interest, or which
hybridize to a
nucleic acid of interest. Any such libraries can be provided as substrates for
any of the
methods herein described.
In some applications, it is desirable to preselect or prescreen libraries
(e.g.,
an amplified library, a genomic library, a cDNA library, a normalized library,
etc.) or other
substrate nucleic acids prior to shuffling, or to otherwise bias the
substrates towards nucleic
acids that encode functional products (shuffling procedures can also,
independently have
these effects). For example, in the case of antibody engineering, it is
possible to bias the
shuffling process toward antibodies with functional antigen binding sites by
taking
advantage of in vivo recombination events prior to DNA shuffling by any
described
method. For example, recombined CDRs derived from B cell cDNA libraries can be
amplified and assembled into framework regions (e.g., Jirholt et al. (1998)
"Exploiting
sequence space: shuffling in vivo formed complementarity determining regions
into a
master framework" Gene 215: 471) prior to DNA shuffling according to any of
the methods
described herein.
Libraries can be biased towards nucleic acids which encode proteins with
desirable enzyme activities. For example, after identifying a clone from a
library which
exhibits a specified activity, the clone can be mutagenized using any known
method for
introducing DNA alterations, including, but not restricted to, DNA shuffling.
A library
comprising the mutagenized homologues is then screened for a desired activity,
which can
be the same as or different from the initially specified activity. An example
of such a
procedure is proposed in U.S. Patent No. 5,939,250. Desired activities can be
identified by
any method known in the art. For example, WO 99/10539 proposes that gene
libraries can
be screened by combining extracts from the gene library with components
obtained from
metabolically rich cells and identifying combinations which exhibit the
desired activity. It
has also been proposed (e.g., WO 98/58085) that clones with desired activities
can be
identified by inserting bioactive substrates into samples of the library, and
detecting
bioactive fluorescence corresponding to the product of a desired activity
using a fluorescent
analyzer, e.g., a flow cytometry device, a CCD, a fluorometer, or a
spectrophotometer.
Libraries can also be biased towards nucleic acids which have specified
characteristics, e.g., hybridization to a selected nucleic acid probe. For
example, application
WO 99/10539 proposes that polynucleotides encoding a desired activity (e.g.,
an enzymatic
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activity, for example: a lipase, an esterase, a protease, a glycosidase, a
glycosyl transferase,
a phosphatase, a kinase, an oxygenase, a peroxidase, a hydrolase, a hydratase,
a nitrilase, a
transaminase, an amidase or an acylase) can be identified from among genomic
DNA
sequences in the following manner. Single stranded DNA molecules from a
population of
genomic DNA are hybridized to a ligand-conjugated probe. The genomic DNA can
be
derived from either a cultivated or uncultivated microorganism, or from an
environmental
sample. Alternatively, the genomic DNA can be derived from a multicellular
organism, or a
tissue derived therefrom.
Second strand synthesis can be conducted directly from the hybridization
probe used in the capture, with or without prior release from the capture
medium or by a
wide variety of other strategies known in the art. Alternatively, the isolated
single-stranded
genomic DNA population can be fragmented without further cloning and used
directly in a
shuffling format that employs a single-stranded template. Such single-stranded
template
shuffling formats are described, for example, in WO 98/27230, "Methods and
Compositions
for Polypeptide Engineering" by Patten et al.; USSN 60/186,482 filed March 2,
2000,
"Single-Stranded Nucleic Acid Template-Mediated Recombination and Nucleic Acid
Fragment Isolation" by Affholter; WO 00/00632, "Methods for Generating Highly
Diverse
Libraries" by Wagner et al.; and WO 00/09679, "Methods for Obtaining in Vitro
Recombined Polynucleotide Sequence Banks and Resulting Sequences." In one such
method the fragment population derived the genomic library(ies) is annealed
with partial,
or, often approximately full length ssDNA or RNA corresponding to the opposite
strand.
Assembly of complex chimeric genes from this population is the mediated by
nuclease-base
removal of non-hybridizing fragment ends, polymerization to fill gaps between
such
fragments and subsequent single stranded ligation. The parental strand can be
removed by
digestion (if RNA or uracil-containing), magnetic separation under denaturing
conditions (if
labeled in a manner conducive to such separation) and other available
separation/purification methods. Alternatively, the parental strand is
optionally co-purified
with the chimeric strands and removed during subsequent screening and
processing steps.
In one approach, single-stranded molecules are converted to double-stranded
DNA (dsDNA) and the dsDNA molecules are bound to a solid support by ligand-
mediated
binding. After separation of unbound DNA, the selected DNA molecules are
released from
the support and introduced into a suitable host cell to generate a library
enriched sequences
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which hybridize to the probe. A library produced in this manner provides a
desirable
substrate for further shuffling using any of the shuffling reactions described
herein.
It will further be appreciated that any of the above described techniques
suitable for enriching a library prior to shuffling can be used to screen the
products
generated by the methods of DNA shuffling.
The shuffling of a single gene and the shuffling of a family of genes provide
two of the most powerful methods available for improving and "migrating"
(gradually
changing the type of reaction, substrate or activity of a selected enzyme) the
functions of
biocatalysts. When shuffling a family of genes, homologous sequences, e.g.,
from different
species or chromosomal positions, are recombined. In single gene shuffling, a
single
sequence is mutated or otherwise altered and then recombined. These formats
share some
common principles.
The breeding procedure starts with at least two substrates that generally show
substantial sequence identity to each other (i.e., at least about 30%, 50%,
70%, 80% or 90%
sequence identity), but differ from each other at certain positions. The
difference can be
any type of mutation, for example, substitutions, insertions and deletions.
Often, different
segments differ from each other in about 5-20 positions. For recombination to
generate
increased diversity relative to the starting materials, the starting materials
must differ from
each other in at least two nucleotide positions. That is, if there are only
two substrates,
there should be at least two divergent positions. If there are three
substrates, for example,
one substrate can differ from the second at a single position, and the second
can differ from
the third at a different single position. The starting DNA segments can be
natural variants
of each other, for example, allelic or species variants. The segments can also
be from
nonallelic genes showing some degree of structural and usually functional
relatedness (e.g.,
different genes within a superfamily, such as the arene dioxygenase super
family). The
starting DNA segments can also be induced variants of each other. For example,
one DNA
segment can be produced by error-prone PCR replication of the other, or by
substitution of a
mutagenic cassette. Induced mutants can also be prepared by propagating one
(or both) of
the segments in a mutagenic strain. In these situations, strictly speaking,
the second DNA
segment is not a single segment but a large family of related segments. The
different
segments forming the starting materials are often the same length or
substantially the same
length. However, this need not be the case; for example; one segment can be a
subsequence
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of another. The segments can be present as part of larger molecules, such as
vectors, or can
be in isolated form.
The starting DNA segments are recombined by any of the sequence
recombination formats provided herein to generate a diverse library of
recombinant DNA
segments. Such a library can vary widely in size from having fewer than 10 to
more than
105, 109, 10'Z or more members. In some embodiments, the starting segments and
the
recombinant libraries generated will include full-length coding sequences and
any essential
regulatory sequences, such as a promoter and polyadenylation sequence,
required for
expression. In other embodiments, the recombinant DNA segments in the library
can be
inserted into a common vector providing sequences necessary for expression
before
performing screening/selection.
1. Use of Restriction Enzyme Sites to Recombine Mutations
In some situations it is advantageous to use restriction enzyme sites in
nucleic acids to direct the recombination of mutations in a nucleic acid
sequence of interest.
These techniques are particularly preferred in the evolution of fragments that
cannot readily
be shuffled by existing methods due to the presence of repeated DNA or other
problematic
primary sequence motifs. These situations also include recombination formats
in which it is
preferred to retain certain sequences unmutated. The use of restriction enzyme
sites is also
preferred for shuffling large fragments (typically greater than 10 kb), such
as gene clusters
that cannot be readily shuffled and "PCR-amplified" because of their size.
Although
fragments up to 50 kb have been reported to be amplified by PCR (Barnes, Proc.
Natl.
Acad. Sci. U.S.A. 91:2216-2220 (1994)), it can be problematic for fragments
over 10 kb, and
thus alternative methods for shuffling in the range of 10 - 50 kb and beyond
are preferred.
Preferably, the restriction endonucleases used are of the Class II type
(Sambrook, Ausubel
and Bergen supra) and of these, preferably those which generate nonpalindromic
sticky end
overhangs such as Alwn I, Sfi I or BstXl. These enzymes generate
nonpalindromic ends
that allow for efficient ordered reassembly with DNA ligase. Typically,
restriction enzyme
(or endonuclease) sites are identified by conventional restriction enzyme
mapping
techniques (Sambrook, Ausubel, and Bergen supra.), by analysis of sequence
information
for that gene, or by introduction of desired restriction sites into a nucleic
acid sequence by
synthesis (i.e. by incorporation of silent mutations).
The DNA substrate molecules to be digested can either be from in vivo
replicated DNA, such as a plasmid preparation, or from PCR amplified nucleic
acid
CA 02377669 2001-12-12
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fragments harboring the restriction enzyme recognition sites of interest,
preferably near the
ends of the fragment. Typically, at least two variants of a gene of interest,
each having one
or more mutations, are digested with at least one restriction enzyme
determined to cut
within the nucleic acid sequence of interest. The restriction fragments are
then joined with
DNA ligase to generate full length genes having shuffled regions. The number
of regions
shuffled will depend on the number of cuts within the nucleic acid sequence of
interest. The
shuffled molecules can be introduced into cells as described above and
screened or selected
for a desired property as described herein. Nucleic acids can then be isolated
from pools
(libraries), or clones having desired properties and subjected to the same
procedure until a
desired degree of improvement is obtained.
In some embodiments, at least one DNA substrate molecule or fragment
thereof is isolated and subjected to mutagenesis. In some embodiments, the
pool or library
of relegated restriction fragments are subjected to mutagenesis before the
digestion-legation
process is repeated. "Mutagenesis" as used herein includes such techniques
known in the
art as PCR mutagenesis, oligonucleotide-directed mutagenesis, site-directed
mutagenesis,
etc., and recursive sequence recombination by any of the techniques described
herein.
2. Reassembly PCR
A further technique for recombining mutations in a nucleic acid sequence
utilizes "reassembly PCR." This method can be used to assemble multiple
segments that
have been separately evolved into a full length nucleic acid template such as
a gene. This
technique is performed when a pool of advantageous mutants is known from
previous work
or has been identified by screening mutants that may have been created by any
mutagenesis
technique known in the art, such as PCR mutagenesis, cassette mutagenesis,
doped oligo
mutagenesis, chemical mutagenesis, or propagation of the DNA template in vivo
in mutator
strains. Boundaries defining segments of a nucleic acid sequence of interest
preferably lie
in intergenic regions, introns, or areas of a gene not likely to have
mutations of interest.
Preferably, oligonucleotide primers (oligos) are synthesized for PCR
amplification of
segments of the nucleic acid sequence of interest, such that the sequences of
the
oligonucleotides overlap the junctions of two segments. The overlap region is
typically
about 10 to 100 nucleotides in length. Each of the segments is amplified with
a set of such
primers. The PCR products are then "reassembled" according to assembly
protocols such
as those discussed herein to assemble randomly fragmented genes. In brief, in
an assembly
protocol the PCR products are first purified away from the primers, by, for
example, gel
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electrophoresis or size exclusion chromatography. Purified products are mixed
together and
subjected to about 1-10 cycles of denaturing, reannealing, and extension in
the presence of
polymerase and deoxynucleoside triphosphates (dNTP's) and appropriate buffer
salts in the
absence of additional primers ("self-priming"). Subsequent PCR with primers
flanking the
gene are used to amplify the yield of the fully reassembled and shuffled
genes.
In some embodiments, the resulting reassembled genes are subjected to
mutagenesis before the process is repeated.
In a further embodiment, the PCR primers for amplification of segments of
the nucleic acid sequence of interest are used to introduce variation into the
gene of interest
as follows. Mutations at sites of interest in a nucleic acid sequence are
identified by
screening or selection, by sequencing homologues of the nucleic acid sequence,
and so on.
Oligonucleotide PCR primers are then synthesized which encode wild type or
mutant
information at sites of interest. These primers are then used in PCR
mutagenesis to generate
libraries of full length genes encoding permutations of wild type and mutant
information at
the designated positions. This technique is typically advantageous in cases
where the
screening or selection process is expensive, cumbersome, or impractical
relative to the cost
of sequencing the genes of mutants of interest and synthesizing mutagenic
oligonucleotides.
3. Site Directed Mutagenesis (SDM) with Oligonucleotides Encoding Homologue
Mutations Followed by Shuffling
In some embodiments of the invention, sequence information from one or
more substrate sequences is added to a given "parental" sequence of interest,
with
subsequent recombination between rounds of screening or selection. Typically,
this is done
with site-directed mutagenesis performed by techniques well known in the art
(e.g., Bergen
Ausubel and Sambrook, supra.) with one substrate as template and
oligonucleotides
encoding single or multiple mutations from other substrate sequences, e.g.
homologous
genes. After screening or selection for an improved phenotype of interest, the
selected
recombinants) can be further evolved using RSR techniques described herein.
After
screening or selection, site-directed mutagenesis can be done again with
another collection
of oligonucleotides encoding homologue mutations, and the above process
repeated until
the desired properties are obtained.
When the difference between two homologues is one or more single point
mutations in a codon, degenerate oligonucleotides can be used that encode the
sequences in
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both homologues. One oligonucleotide can include many such degenerate codons
and still
allow one to exhaustively search all permutations over that block of sequence.
When the homologue sequence space is very large, it can be advantageous to
restrict the search to certain variants. Thus, for example, computer modeling
tools (Lathrop
et al., J. Mol. Biol. 255:641-665 (1996)) can be used to model each homologue
mutation
onto the target protein and discard any mutations that are predicted to
grossly disrupt
structure and function.
4. In vitro Nucleic Acid Shuffling Formats
In one embodiment for shuffling DNA sequences in vitro, the initial
substrates for recombination are a pool of related sequences, e.g., different
variant forms, as
homologs from different individuals, strains, or species of an organism, or
related sequences
from the same organism, as allelic variations. The sequences can be DNA or RNA
and can
be of various lengths depending on the size of the gene or DNA fragment to be
recombined
or reassembled. Preferably the sequences are from 50 base pairs (bp) to 50
kilobases (kb).
The pool of related substrates are converted into overlapping fragments, e.g.,
from about 5 by to 5 kb or more. Often, for example, the size of the fragments
is from
about 10 by to 1000 bp, and sometimes the size of the DNA fragments is from
about 100 by
to 500 bp. The conversion can be effected by a number of different methods,
such as
DNase I or RNase digestion, random shearing or partial restriction enzyme
digestion. For
discussions of protocols for the isolation, manipulation, enzymatic digestion,
and the like of
nucleic acids, see, for example, Sambrook et al. and Ausubel, both supra. The
concentration
of nucleic acid fragments of a particular length and sequence is often less
than 0.1 % or 1%
by weight of the total nucleic acid. The number of different specific nucleic
acid fragments
in the mixture is usually at least about 100, 500 or 1000.
The mixed population of nucleic acid fragments are converted to at least
partially single-stranded form using a variety of techniques, including, for
example, heating,
chemical denaturation, use of DNA binding proteins, and the like. Conversion
can be
effected by heating to about 80 °C to 100 °C, more preferably
from 90 °C to 96 °C, to form
single-stranded nucleic acid fragments and then reannealing. Conversion can
also be
effected by treatment with single-stranded DNA binding protein (see Wold,
Annu. Rev.
Biochem. 66:61-92 (1997)) or recA protein (see, e.g., Kiianitsa, Proc. Natl.
Acad. Sci. USA
94:7837-7840 (1997)). Single-stranded nucleic acid fragments having regions of
sequence
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identity with other single-stranded nucleic acid fragments can then be
reannealed by cooling
to 20 °C to 75 °C, and preferably from 40 °C to 65
°C. Renaturation can be accelerated by
the addition of polyethylene glycol (PEG), other volume-excluding reagents or
salt. The
salt concentration is preferably from 0 mM to 200 mM, more preferably the salt
concentration is from 10 mM to 100 mM. The salt may be KCl or NaCI. The
concentration
of PEG is preferably from 0% to 20%, more preferably from 5% to 10%. The
fragments
that reanneal can be from different substrates. The annealed nucleic acid
fragments are
incubated in the presence of a nucleic acid polymerase, such as Taq or Klenow,
and dNTP's
(i.e. dATP, dCTP, dGTP and dTTP). If regions of sequence identity are large,
Taq
polymerase can be used with an annealing temperature of between 45-65
°C. If the areas of
identity are small, Klenow polymerase can be used with an annealing
temperature of
between 20-30 °C. The polymerase can be added to the random nucleic
acid fragments
prior to annealing, simultaneously with annealing or after annealing.
The process of denaturation, renaturation and incubation in the presence of
polymerase of overlapping fragments to generate a collection of
polynucleotides containing
different permutations of fragments is sometimes referred to as shuffling of
the nucleic acid
in vitro. This cycle is repeated for a desired number of times. Preferably the
cycle is
repeated from 2 to 100 times, more preferably the sequence is repeated from 10
to 40 times.
The resulting nucleic acids are a family of double-stranded polynucleotides of
from about
50 by to about 100 kb, preferably from 500 by to 50 kb. The population
represents variants
of the starting substrates showing substantial sequence identity thereto but
also diverging at
several positions. The population has many more members than the starting
substrates. The
population of fragments resulting from shuffling is used to transform host
cells, optionally
after cloning into a vector.
In one embodiment utilizing in vitro shuffling, subsequences of
recombination substrates can be generated by amplifying the full-length
sequences under
conditions which produce a substantial fraction, typically at least 20 percent
or more, of
incompletely extended amplification products. Another embodiment uses random
primers
to prime the entire template DNA to generate less than full length
amplification products.
The amplification products, including the incompletely extended amplification
products are
denatured and subjected to at least one additional cycle of reannealing and
amplification.
This variation, in which at least one cycle of reannealing and amplification
provides a
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substantial fraction of incompletely extended products, is termed
"stuttering." In the
subsequent amplification round, the partially extended (less than full length)
products
reanneal to and prime extension on different sequence-related template
species. In another
embodiment, the conversion of substrates to fragments can be effected by
partial PCR
amplification of substrates.
In another embodiment, a mixture of fragments is spiked with one or more
oligonucleotides. The oligonucleotides can be designed to include
precharacterized
mutations of a wildtype sequence, or sites of natural variations between
individuals or
species. The oligonucleotides also include sufficient sequence or structural
homology
flanking such mutations or variations to allow annealing with the wildtype
fragments.
Annealing temperatures can be adjusted depending on the length of homology.
In a further embodiment, recombination occurs in at least one cycle by
template switching, such as when a DNA fragment derived from one template
primes on the
homologous position of a related but different template. Template switching
can be induced
by addition of recA (see, Kiianitsa (1997) supra), rad51 (see, Namsaraev, Mol.
Cell. Biol.
17:5359-5368 (1997)), rad55 (see, Clever, EMBO J. 16:2535-2544 (1997)), rad57
(see,
Sung, Genes Dev. 11:1111-1121 (1997)) or other polymerises (e.g., viral
polymerises,
reverse transcriptase) to the amplification mixture. Template switching can
also be
increased by increasing the DNA template concentration.
Another embodiment utilizes at least one cycle of amplification, which can
be conducted using a collection of overlapping single-stranded DNA fragments
of related
sequence, and different lengths. Fragments can be prepared using a single
stranded DNA
phage, such as M13 (see, Wang, Biochemistry 36:9486-9492 (1997)). Each
fragment can
hybridize to and prime polynucleotide chain extension of a second fragment
from the
collection, thus forming sequence-recombined polynucleotides. In a further
variation,
ssDNA fragments of variable length can be generated from a single primer by
Pfu, Taq,
Vent, Deep Vent, UlTma DNA polymerise or other DNA polymerises on a first DNA
template (see, Cline, Nucleic Acids Res. 24:3546-3551 (1996)). The single
stranded DNA
fragments are used as primers for a second, Kunkel-type template, consisting
of a uracil-
containing circular ssDNA. This results in multiple substitutions of the first
template into
the second. See, Levichkin, Mol. Biology 29:572-577 (1995); Jung, Gene 121:17-
24 (1992).
In some embodiments of the invention, shuffled nucleic acids obtained by
use of the recursive recombination methods of the invention, are put into a
cell and/or
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organism for screening. Shuffled dioxygenase genes can be introduced into, for
example,
bacterial cells (including cyanobacteria), yeast cells, fungal cells,
vertebrate cells,
invertebrate cells or plant cells for initial screening. Bacterial species,
such as E. coli,
Pseudomonas sp, Bacillus. subtilis, Burkholderia cepacia, Alcaligenes,
Acinetobacter,
Rhodococcus Arthrobacter, Sphingomonas are preferred examples of suitable
bacterial cells
into which one can insert and express shuffled dioxygenase genes which provide
for
convenient shuttling to other cell types (a variety of vectors for shuttling
material between
these bacterial cells and eukaryotic cells are available; see, Sambrook,
Ausubel and Bergen
all supra). The shuffled genes can be introduced into bacterial, fungal or
yeast cells either
by integration into the chromosomal DNA or as plasmids.
Although bacterial and yeast systems are most preferred in the present
invention, in one embodiment, shuffled genes can also be introduced into plant
cells for
production purposes (it will be appreciated that transgenic plants are,
increasingly, an
important source of industrial enzymes). Thus, a transgene of interest can be
modified using
the recursive sequence recombination methods of the invention in vitro and
reinserted into
the cell for in vivolin situ selection for the new or improved dioxygenase
property, in
bacteria, eukaryotic cells, or whole eukaryotic organisms.
5. In vivo Nucleic Acid Shuffling Formats
In some embodiments of the invention, DNA substrate molecules are
introduced into cells, wherein the cellular machinery directs their
recombination. For
example, a library of mutants is constructed and screened or selected for
mutants with
improved phenotypes by any of the techniques described herein. The DNA
substrate
molecules encoding the best candidates are recovered by any of the techniques
described
herein, then fragmented and used to transfect a plant host and screened or
selected for
improved function. If further improvement is desired, the DNA substrate
molecules are
recovered from the host cell, such as by PCR, and the process is repeated
until a desired
level of improvement is obtained. In some embodiments, the fragments are
denatured and
reannealed prior to transfection, coated with recombination stimulating
proteins such as
recA, or co-transfected with a selectable marker such as Neon to allow the
positive selection
for cells receiving recombined versions of the gene of interest. Methods for
in vivo
shuffling are described in, for example, PCT application WO 98/13487 and WO
97/20078.
The efficiency of in vivo shuffling can be enhanced by increasing the copy
number of a gene of interest in the host cells. For example, the majority of
bacterial cells in
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stationary phase cultures grown in rich media contain two, four or eight
genomes. In
minimal medium the cells contain one or two genomes. The number of genomes per
bacterial cell thus depends on the growth rate of the cell as it enters
stationary phase. This is
because rapidly growing cells contain multiple replication forks, resulting in
several
S genomes in the cells after termination. The number of genomes is strain
dependent,
although all strains tested have more than one chromosome in stationary phase.
The number
of genomes in stationary phase cells decreases with time. This appears to be
due to
fragmentation and degradation of entire chromosomes, similar to apoptosis in
mammalian
cells. This fragmentation of genomes in cells containing multiple genome
copies results in
massive recombination and mutagenesis. The presence of multiple genome copies
in such
cells results in a higher frequency of homologous recombination in these
cells, both
between copies of a gene in different genomes within the cell, and between a
genome within
the cell and a transfected fragment. The increased frequency of recombination
allows one
to evolve a gene more quickly to acquire optimized characteristics.
In nature, the existence of multiple genomic copies in a cell type would
usually not be advantageous due to the greater nutritional requirements needed
to maintain
this copy number. However, artificial conditions can be devised to select for
high copy
number. Modified cells having recombinant genomes are grown in rich media (in
which
conditions, multicopy number should not be a disadvantage) and exposed to a
mutagen,
such as ultraviolet or gamma irradiation or a chemical mutagen, e.g.,
mitomycin, nitrous
acid, photoactivated psoralens, alone or in combination, which induces DNA
breaks
amenable to repair by recombination. These conditions select for cells having
multicopy
number due to the greater efficiency with which mutations can be excised.
Modified cells
surviving exposure to mutagen are enriched for cells with multiple genome
copies. If
desired, selected cells can be individually analyzed for genome copy number
(e.g., by
quantitative hybridization with appropriate controls). For example, individual
cells can be
sorted using a cell sorter for those cells containing more DNA, e.g., using
DNA specific
fluorescent compounds or sorting for increased size using light dispersion.
Some or all of
the collection of cells surviving selection are tested for the presence of a
gene that is
optimized for the desired property.
In one embodiment, phage libraries are made and recombined in mutator
strains such as cells with mutant or impaired gene products of mutS, mutT,
mutes, mutt,
ovrD, dcm, vsr, umuC, umuD, sbcB, recJ, etc. The impairment is achieved by
genetic
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mutation, allelic replacement, selective inhibition by an added reagent such
as a small
compound or an expressed antisense RNA, or other techniques. High multiplicity
of
infection (MOI) libraries are used to infect the cells to increase
recombination frequency.
Additional strategies for making phage libraries and or for recombining
DNA from donor and recipient cells are set forth in U.S. Pat. No. 5,521,077.
Additional
recombination strategies for recombining plasmids in yeast are set forth in WO
97 07205.
6. Whole Genome Shuffling
In one embodiment, the selection methods herein are utilized in a "whole
genome shuffling" format. An extensive guide to the many forms of whole genome
shuffling is found in the pioneering application to the inventors and their co-
workers
entitled "Evolution of Whole Cells and Organisms by Recursive Sequence
Recombination,"
Attorney Docket No. 018097-020720US filed July 15, 1998 by del Cardayre et al.
(USSN
09/161,188).
In brief, whole genome shuffling makes no presuppositions at all regarding
what nucleic acids may confer a desired property. Instead, entire genomes
(e.g., from a
genomic library, or isolated from an organism) are shuffled in cells and
selection protocols
applied to the cells.
The fermentation of microorganisms for the production of natural products is
the oldest and most sophisticated application of biocatalysis.
The methods herein allow dioxygenase biocatalysts to be improved at a
faster pace than conventional methods. Whole genome shuffling can at least
double the rate
of strain improvement for microorganisms used in fermentation as compared to
traditional
methods. This provides for a relative decrease in the cost of fermentation
processes. New
products can enter the market sooner, producers can increase profits as well
as market share,
and consumers gain access to more products of higher quality and at lower
prices. Further,
increased efficiency of production processes translates to less waste
production and more
frugal use of resources. Whole genome shuffling provides a means of
accumulating
multiple useful mutation per cycle and thus eliminate the inherent limitation
of current
strain improvement programs (SIPs).
Nucleic acid shuffling provides recursive mutagenesis, recombination, and
selection of DNA sequences. A key difference between nucleic acid shuffling-
mediated
recombination and natural sexual recombination is that nucleic acid shuffling
effects both
the pairwise (two parents) and the poolwise (multiple parents) recombination
of parent
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molecules. Natural recombination is more conservative and is limited to
pairwise
recombination. In nature, pairwise recombination provides stability within a
population by
preventing large leaps in sequences or genomic structure that can result from
poolwise
recombination. However, for the purposes of directed evolution, poolwise
recombination is
appealing since the beneficial mutations of multiple parents can be combined
during a
single cross to produce a superior offspring. Poolwise recombination is
analogous to the
crossbreeding of inbred strains in classic strain improvement, except that the
crosses occur
between many strains at once. In essence, poolwise recombination is a sequence
of events
that effects the recombination of a population of nucleic acid sequences that
results in the
generation of new nucleic acids that contains genetic information from more
than two of the
original nucleic acids.
There are a few general methods for effecting efficient recombination in
prokaryotes. Bacteria have no known sexual cycle per se, but there are natural
mechanisms
by which the genomes of these organisms undergo recombination. These
mechanisms
include natural competence, phage-mediated transduction, and cell-cell
conjugation.
Bacteria that are naturally competent are capable of efficiently taking up
naked DNA from
the environment. If homologous, this DNA undergoes recombination with the
genome of
the cell, resulting in genetic exchange. Bacillus subtilis, the primary
production organism
of the enzyme industry, is known for the efficiency with which it carnes out
this process.
In generalized transduction, a bacteriophage mediates genetic exchange. A
transducing phage will often package headfulls of the host genome. These phage
can infect
a new host and deliver a fragment of the former host genome which is
frequently integrated
via homologous recombination. Cells can also transfer DNA between themselves
by
conjugation. Cells containing the appropriate mating factors transfer episomes
as well as
entire chromosomes to an appropriate acceptor cell where it can recombine with
the
acceptor genome. Conjugation resembles sexual recombination for microbes and
can be
intraspecific, interspecific, and intergeneric. For example, an efficient
means of
transforming Streptomyces sp., a genera responsible for producing many
commercial
antibiotics, is by the conjugal transfer of plasmids from Echerichia coli.
For many industrial microorganisms, knowledge of competence, transducing
phage, or fertility factors is lacking. Protoplast fusion has been developed
as a versatile and
general alternative to these natural methods of recombination. Protoplasts are
prepared by
removing the cell wall by treating cells with lytic enzymes in the presence of
osmotic
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stabilizers. In the presence of a fusogenic agent, such as polyethylene glycol
(PEG),
protoplasts are induced to fuse and form transient hybrids or "fusants."
During this hybrid
state, genetic recombination occurs at high frequency allowing the genomes to
reassort.
The final step is the successful segregation and regeneration of viable cells
from the fused
protoplasts. Protoplast fusion can be intraspecific, interspecific, and
intergeneric and has
been applied to both prokaryotes and eukaryotes. In addition, it is possible
to fuse more
than two cells, thus providing a mechanism for effecting poolwise
recombination. While no
fertility factors, transducing phages or competency development is needed for
protoplast
fusion, a method for the formation, fusing, and regeneration of protoplasts is
typically
optimized for each organism.
Modifications can be made to the method and materials as hereinbefore
described without departing from the spirit or scope of the invention as
claimed, and the
invention can be put to a number of different uses, including:
The use of an integrated system to test dioxygenase in shuffled DNAs,
including in an iterative process.
7. Shuffling families of arene dioxygenases
For identifying homologous genes used to shuffle a family of genes,
representative alignments of monooxygenase enzymes can be found in the
literature or
generated from sequences retrieved from GeneBank or an associated public
database
(a). Tar eg t sequences for nucleic acid shuffling of ADOs and variations
The following discussion focuses on ADO for clarity of illustration. Those
of skill in the art will recognize that this discussion is illustrative of DOs
in general, and is
not limited to ADOs.
Because all known ADOs are multicomponent enzymes having from 2 to 4
functionally different subunits, any of the sequence components comprising a
particular
ADO or a family of homologous ADOs can be shuffled. For the purposes of
evolving
ADOs with changes in specificity, regioselectivity and mode of action (e.g.
dioxygenase or
monooxygenase activity) for oxidizing a particular compound of interest, it is
often
preferred to shuffle nucleic acids encoding the iron-sulfur protein of
terminal oxygenase
(where terminal oxygenase is made of two polypeptides, the large subunit is
the preferred
target sequence for shuffling).
For the purpose of improving overall turnover rates and efficiency of
electron transfer between all components of dioxygenases, shuffling of the
polynucleotide
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sequences encoding other functional polypeptides (reductase, ferredoxin, small
terminal
oxidase component) is a preferred embodiment. These sequences can be shuffled
individually, in sub-sets or as a part of a gene cluster which encodes all of
the ADO
polypeptides. This allows for changes in both the coding polynucleotide
sequences, and
also for generating combinations of functional chimeric ADOs with various
functions
assembled from two or more parental sequences (family gene cluster shuffling).
Many natural dioxygenases are in effect fusion proteins where the functions
of reductase and ferredoxin are combined in one polypeptide. Moreover, the
terminal
oxygenase component is presented by one polypeptide instead of two. For one
skilled in the
art, it is apparent that polynucleotides encoding natural and artificial
fusions of ADO
components can be also used for shuffling. For example, in a recursive
shuffling process, in
principle, all desired characteristics of an ADO can be altered and improved
in the desired
directions regardless of which particular polynucleotide subsequence is
selected from a
complete ADO coding sequence.
To illustrate shuffling a family of genes to improve arene dioxygenase
enzymes, one or more of the more than 50 members of this superfamily is
selected, aligned
with similar homologous sequences, shuffled against these homologous sequences
and
screened.
The screening is done most easily in a bacterial system. DNA from clones
with improved activity can be shuffled together in subsequent rounds of
shuffling and
screened for further improvement.
8. Codon Modification Shuffling
Procedures for codon modification shuffling are described in detail in
SHUFFLING OF CODON ALTERED GENES, Phillip A. Patten and Willem P.C.
Stemmer, filed September 29, 1998, USSN 60/102362 and in SHUFFLING OF CODON
ALTERED GENES, Phillip A. Patten and Willem P.C. Stemmer, filed January 29,
1999,
USSN 60/117729. In brief, by synthesizing nucleic acids in which the codons
encoding
polypeptides are altered, it is possible to access a completely different
mutational cloud
upon subsequent mutation of the nucleic acid. This increases the sequence
diversity of the
starting nucleic acids for shuffling protocols, which alters the rate and
results of forced
evolution procedures. Codon modification procedures can be used to modify any
nucleic
acid described herein, e.g., prior to performing nucleic acid shuffling, or
codon modification
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approaches can be used in conjunction with oligonucleotide shuffling
procedures as
described supra.
In these methods, a first nucleic acid sequence encoding a first polypeptide
sequence is selected. A plurality of codon altered nucleic acid sequences,
each of which
encode the first polypeptide, or a modified or related polypeptide, is then
selected (e.g., a
library of codon altered nucleic acids can be selected in a biological assay
which recognizes
library components or activities), and the plurality of codon-altered nucleic
acid sequences
is recombined to produce a target codon altered nucleic acid encoding a second
protein.
The target codon altered nucleic acid is then screened for a detectable
functional or
structural property, optionally including comparison to the properties of the
first
polypeptide and/or related polypeptides. The goal of such screening is to
identify a
polypeptide that has a structural or functional property equivalent or
superior to the first
polypeptide or related polypeptide. A nucleic acid encoding such a polypeptide
can be used
in essentially any procedure desired, including introducing the target codon
altered nucleic
acid into a cell, vector, virus, attenuated virus (e.g., as a component of a
vaccine or
immunogenic composition), transgenic organism, or the like.
9. Oligonucleotide and in silico shuffling formats
In addition to the formats for shuffling noted above, at.least two additional
related formats are useful in the practice of the present invention. The
first, referred to as
"in silico" shuffling utilizes computer algorithms to perform "virtual"
shuffling using
genetic operators in a computer. As applied to the present invention, gene
sequence strings
are recombined in a computer system and desirable products are made, e.g., by
reassembly
PCR of synthetic oligonucleotides. In silico shuffling is described in detail
in Selifonov and
Stemmer in "METHODS FOR MAKING CHARACTER STRINGS,
POLYNUCLEOT>DES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS"
filed February 5, 1999, USSN 60/118854. In brief, genetic operators
(algorithms which
represent given genetic events such as point mutations, recombination of two
strands of
homologous nucleic acids, etc.) are used to model recombinational or
mutational events
which can occur in one or more nucleic acid, e.g., by aligning nucleic acid
sequence strings
(using standard alignment software, or by manual inspection and alignment) and
predicting
recombinational outcomes. The predicted recombinational outcomes are used to
produce
corresponding molecules, e.g., by oligonucleotide synthesis and reassembly
PCR.
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The second useful format is referred to as "oligonucleotide mediated
shuffling" in which oligonucleotides corresponding to a family of related
homologous
nucleic acids (e.g., as applied to the present invention, interspecific or
allelic variants of a
dioxygenase nucleic acid) which are recombined to produce selectable nucleic
acids. This
format is described in detail in Crameri et al. "OLIGONUCLEOTIDE MEDIATED
NUCLEIC ACID RECOMBINATION" filed February 5, 1999, USSN 60/118,813 and
Crameri et al. "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID
RECOMBINATION" filed June 24, 1999, USSN 60/141,049. The technique can be used
to
recombine homologous or even non-homologous nucleic acid sequences.
One advantage of the oligonucleotide-mediated recombination is the ability
to recombine homologous nucleic acids with low sequence similarity, or even
non-
homologous nucleic acids. In these low-homology oligonucleotide shuffling
methods, one
or more set of fragmented nucleic acids are recombined, e.g., with a with a
set of crossover
family diversity oligonucleotides. Each of these crossover oligonucleotides
have a plurality
of sequence diversity domains corresponding to a plurality of sequence
diversity domains
from homologous or non-homologous nucleic acids with low sequence similarity.
The
fragmented oligonucleotides, which are derived by comparison to one or more
homologous
or non-homologous nucleic acids, can hybridize to one or more region of the
crossover
oligos, facilitating recombination.
When recombining homologous nucleic acids, sets of overlapping family
gene oligonucleotides (which are derived by comparison of homologous nucleic
acids and
synthesis of oligonucleotide fragments) are hybridized and elongated (e.g., by
reassembly
PCR), providing a population of recombined nucleic acids, which can be
selected for a
desired trait or property. Typically, the set of overlapping family genes
include a plurality
of oligonucleotide member types which have consensus region subsequences
derived from a
plurality of homologous target nucleic acids.
Typically, family gene shuffling oligonucleotides are provided by aligning
homologous nucleic acid sequences to select conserved regions of sequence
identity and
regions of sequence diversity. A plurality of family gene shuffling
oligonucleotides are
synthesized (serially or in parallel) which correspond to at least one region
of sequence
diversity.
Sets of fragments, or subsets of fragments, used in oligonucleotide shuffling
approaches can be provided by cleaving one or more homologous nucleic acids
(e.g., with a
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DNase), or, more commonly, by synthesizing a set of oligonucleotides
corresponding to a
plurality of regions of at least one nucleic acid (typically oligonucleotides
corresponding to
a full-length nucleic acid are provided as members of a set of nucleic acid
fragments). In
the shuffling procedures herein, these cleavage fragments (e.g., fragments of
dioxygenases)
can be used in conjunction with family gene shuffling oligonucleotides, e.g.,
in one or more
recombination reaction to produce recombinant dioxygenase nucleic acids.
10. Chimeric shuffling templates
In addition to the naturally occurnng, mutated and synthetic oligonucleotides
discussed above, polynucleotides encoding chimeric polypeptides can be used as
substrates
for shuffling in any of the above-described shuffling formats. Preferred
chimeras have a
shuffled active site or a shuffled active site region. Art-recognized methods
for preparing
chimeras are applicable to the methods described herein (see, for example,
Shimoji et al.,
Biochemistry 37: 8848-8852 (1998)).
B. Reactions of Improved Dioxy~enases
In another aspect, the invention provides a method for obtaining a
polynucleotide encoding an improved dioxygenase polypeptide acting on an
organic
substrate. Presently preferred substrates include a target group selected from
classes of
substrates shown in Figures 14A-14R. The improved polypeptide exhibits one or
more
improved properties, compared to a naturally occurring polypeptide acting on
the
substrate(s). The method involves: (a) creating a library of recombinant
polynucleotides
encoding a dioxygenase polypeptide acting on the substrate; and (b) screening
the library to
identify a recombinant polynucleotide encoding an improved polypeptide that
exhibits one
or more improved properties compared to a naturally occurring dioxygenase
polypeptide.
In a preferred embodiment, the library of recombinant polynucleotides is
created by recombining at least a first form and a second form of a nucleic
acid. At least
one of these forms encodes the naturally occurring polypeptide or a fragment
thereof.
Preferably, the first form and the second form differ from each other in two
or more
nucleotides. In a further preferred embodiment, the first and second forms of
the nucleic
acid are homologous.
In addition to the methods described above for producing the encoding
polynucleotides, the present invention also provides the polypeptides encoded
by these
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polynucleotides and methods of using these peptides for synthesizing valuable
organic
compounds. Some of these polypeptides and methods of using them are set forth
below.
It is noted that the basic chemistry described below with reference to
oxygenases in general and dioxygenases in particular is known. In addition to
Ortiz de
Montellano, supra, general guides to dioxygenase product chemistry include S.
Brown and
T. Hudlicky, in ORGANIC SYNTI-~sls. THEORY AND APPLICATIONS (T.Hudlicky,
Ed.,),
volume 2, p. 113-176, JAI Press, Greenwich Connecticut (1993). Moreover, the
various
chemistries involved are found in Stryer BIOCHEMISTRY, third edition (or later
editions)
Freeman and Co. New York, NY (1988); Pine et al. ORGANIC CHEMISTRY FOURTH-
EDTTION
(1980) McGraw-Hill, Inc. (USA) (or later editions); March, ADVANCED ORGANIC
CHEMISTRY REACTIONS, MECHANISMS and Structure 4th ed J. Wiley and Sons (New
York,
NY, 1992) (or later editions); Greene, et al., PROTECTIVE GROUPS IN ORGANIC
CHEMISTRY,
2nd Ed., John Wiley & Sons, New York, NY, 1991 (or later editions); Lide (ed)
THE CRC
HANDBOOK OF CHEMISTRY AND PHYSICS 75TH EDITION (1995) (or later editions); and
in the
references cited in the foregoing. Furthermore, an extensive guide to many
chemical and
industrial processes applicable to the present invention is found in the KIRK-
OTHMER
ENCYCLOPEDIA OF CHEMICAL TECHNOLOGY (third edition and fourth edition, through
year
1998), Martin Grayson, Executive Editor, Wiley-Interscience, John Wiley and
Sons, NY,
and in the references cited therein ("Kirk-Othmer")
The following chemistries illustrate those generally accessible through the
arene dioxygenase superfamily.
Shuffling approaches, such as shuffling a family of genes, apply to
enhancing performance of dioxygenase polypeptides useful in each of the
following classes
of industrial chemical transformation. Other dioxygenase enzyme classes are
also useful in
practicing the present invention. Moreover, other polypeptides accessible
through the
present invention, and method of using these polypeptides will be apparent to
those of skill
in the art.
1. Oxidation of ~c-bonds to diols
In a preferred embodiment, the present invention provides improved
polypeptides that can mediate the oxidation of ~-bonds to vicinal diols. Also
provided are
host organisms expressing such improved polypeptides and methods of using
these
polypeptides and organisms in synthetic processes.
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Among the enzymes known to oxidize ~-bonds to the corresponding vicinal
diols are the bacterial arene dioxygenases (ADOs). In the presence of oxygen,
and of a
reducing compound such as NAD(P)H, these enzymes catalyze the reductive
dioxygenation
of compounds as diverse as aromatic rings and non-aromatic multiple bonds.
Arene dioxygenases, such as toluene 2,3-dioxygenase, isopropylbenzene 2,3-
dioxygenase, benzene-1,2-dioxygenase, biphenyl-2,3-dioxygenase, naphthalene-
1,2-
dioxygenase, and many homologous and/or functionally similar enzymes,
constitute
members of a class of enzymes useful in the manufacture of vicinal diols in a
highly
regioselective fashion. Moreover, the action of this class of enzymes on
aromatic substrates
does not involve formation of reactive arene epoxides or phenols. While
potentially
interesting from an academic standpoint, these enzymes have not been generally
utilized
due to several shortcomings. For example, these enzymes do not exhibit
sufficient turnover
numbers nor are they known to provide satisfactory regioselectivity for
dihydroxylation of
~-bonds in a substrates having more than one ~-bond, or with more than one
type of ~-bond
(e.g., styrene).
Arene dioxygenases of various specificity, regioselectivity and
enantiospecificity are capable of forming vicinal diols from a large array of
substituted
aromatic compounds and non-aromatic alkenes. For example, toluene dioxygenase
has
recently been implicated in dihydroxylation of several non-aromatic alkenes
with
concomitant formation of the glycol compounds of known and unknown
stereochemistry.
(Lange and Wackett, JBacteriol.,179(12):3858-3865 (1997)). Similarly, purified
naphthalene dioxygenase has been shown to catalyze dihydroxylation of styrene
to (R)1-
phenyl-1,2-ethanediol with enantiomeric excess of about 79% (Lee et al., Appl.
Environ.
Microbiol., 62(9):3101-3106 (1996); Lee et al., J. Bacteriol., 178(11):3353-
3356 (1996)).
The non-phenolic nature of ring cis-dihydroxylation products arising from
action of arene dioxygenases offers a significant advantage for manufacturing
diols and
other compounds by avoiding the accumulation toxic and reactive epoxide
intermediates
which may significantly impair the performance of the biocatalyst.
a. Oxidation of exocyclic and acyclic ~t-bonds
The present invention also provides improved polypeptides capable of
oxidizing exocyclic and acyclic ~t-bonds for producing oxygen-containing
species. For
clarity of illustration, the following discussion focuses on the oxidation of
olefins. This
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focus is intended to be illustrative and not limiting of the scope of the
invention. Many
other appropriate substrates for oxidation using the methods of the invention
will be
apparent to those of skill in the art.
An exemplary oxidation of an olefin to the corresponding vicinal diol uses a
dioxygenase to obtain the glycol directly from the olefin. This is best
accomplished by
recruiting a dioxygenase, such as an arene dioxygenase.
Arene dioxygenases are multi-component enzymes for which substrate
specificity is primarily determined by the non-heme iron-sulfur cluster
containing terminal
oxidase protein(s), and, in the cases where terminal oxidase is comprised of
two proteins, by
the large subunit of the terminal oxidase. Other proteins such as ferredoxins
and ferredoxin
reductases provide transfer of electrons from reducing equivalents such as
NAD(P)H to the
terminal oxidase. Accordingly, where it is required to obtain a significant
change in the
substrate specificity, as compared to specificities of known arene
dioxygenases existing in
nature, nucleic acids that encode the terminal oxidase components) are the
preferred
substrate for the recombination and selection methods of the invention. Upon
producing a
library of recombinant polynucleotides as described herein, one can then
select to identify
those polynucleotides that encode an enzyme that has the desired change in
substrate
specificity. Such changes in the substrate specificity can include, for
example, a gain of
turnover of a novel substrate, a change in regioselectivity of oxidation, and
a change in
chirality of product formed. If the goal is primarily to obtain enzymes that
have an
increased catalytic turnover with an already acceptable substrate, shuffling
of the nucleic
acids encoding all of the components of arene dioxygenase is preferred.
Many arene dioxygenase genes that can be used as substrates for the
recombination and selection methods of the invention are described in the art.
Suitable
arene dioxygenase-encoding polynucleotides can be obtained from many organisms
using
cloning methods known to one skilled in the art. The following list provides
examples of
polynucleotides that encode arene dioxygenases and are suitable for use in the
methods of
the invention. The loci are identified by GenBank ID and encode complete or
partial
protein components of the arene dioxygenases. Suitable loci include:
[PSETODC1C] toluene-1,2-dioxygenase;
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[AF006691], [PJU53507], [PSECUMA], (REU24277] isopropylbenzene-
2,3-[E04215], [PSEBDO] dioxygenase; benzene-1,2-dioxygenase;
[U78099] tetrachlorobenzene dioxygenase;
(AEBPHA1F], [CTU47637], [D78322], [D88020], [D88021], [PSEBPHA],
[PSEBPHABC], [PSEBPHABCC], [PSU95054], [RERBPHA1],
[RGBPHA], [RSU27591] biphenyl-2,3-dioxygenase; [PSU15298]
chlorobenzene dioxygenase;
(AB004059], [AF010471], [AF036940], [AF053735], [AF053736],
pAF079317], [AF004283], [AF004284], [PSENAPDOXA],
[PSENAPDOXB], [PSENDOABC], [PSEORF1], [PSU49496]
naphthalene-1,2-dioxygenase; [AF009224], [PSEBEDC12A] benzoate-
1,2-dioxygenase; [PWWXYL] toluate dioxygenase; [ASCBAABC],
[U18133] 3-chlorobenzoate-3,4-dioxygenase; [PCCBDABC] 2-
chlorobenzoate-1,2-dioxygenase; [BSU62430] 2,4-dinitrotoluene
dioxygenase; [PSU49504] 2-nitrotoluene dioxygenase; [PPU24215] p-
cumate-2,3-dioxygenase; [U18133] 3-chlorobenzoate 3,4-(4,5)-
dioxygenase; [PSEPHT] phthalate-4,5-dioxygenase; [AB008831],
[ACCANI], [D85415] aniline 1,2-dioxygenase; [D90884]
phenylpropionic acid 2,3-dioxygenase; [PPPOBAB] phenoxybenzoate
dioxygenase; [AF060489], [AB001723], [D89064]carbazole
dioxygenase.
Also of utility are organisms whose genomes contain genes encoding other
dioxygenases, including tetralin-5,6-dioxygenase, Sikkema et al., Appl.
Eviron. Microbiol.
59:567-573, (1993); p-cumate-2,3-dioxygenase DeFrank et al., J. Bacteriol.
129:1356-1364
(1977); fluorenone 1,1a-dioxygenase, Selifonov et al, Biochem. Biophys. Res.
Comm.
193:67-76(1993); dibenzofuran-4,4a dioxygenase, Trenz et al, J. Bacteriol.
176:789-795
(1994); phthalate-3,4-dioxygenase, Eaton et al., J. Bacteriol. 151:48-58
(1982); and 2-
chlorobenzoate-1,2-dioxygenase (Selifonov et al, Biochem Biophys. Res. Comm.
213(3):759-767 (1995).
Solely for the purpose of illustrating this invention with respect to
oxidation
of non-aromatic ~t-bonds, nucleic acids encoding arene dioxygenases, or any
fragments
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thereof, are preferably selected from the following non-limiting set of genes
and organisms:
naphthalene 1,2-dioxygenase, 2,4-dinitro-toluene-4,5-dioxygenase, 2-
nitrotoluene 2,3-
dioxygenase, toluene-2,3-dioxygenase, isopropylbenzene-2,3-dioxygenase,
benzene-1,2-
dioxygenase and biphenyl-2,3-dioxygenase, chlorobenzene and tetrachlorobenzene
dioxygenases.
Additional suitable homologous arene dioxygenase genes can be found in
many microorganisms which one skilled in the art can isolate from various
sources
including, for example, soil, sediment, air, and aqueous samples by enrichment
culture
techniques in mineral media using aromatic compounds such as alkyl and halogen-
substituted benzenes, biphenyls, indans, naphthalenes and tetralins as carbon
sources.
b. Oxidation of aromatic ~-bonds
The present invention also provides improved polypeptides capable of
oxidizing aromatic ~-bonds for producing oxygen-containing aromatic species.
Although
substantially any oxidized aromatic or heteroaromatic species can be obtained
using the
polypeptides of the invention, in a presently preferred embodiment, the
species include
hydroxylated aromatic carboxylic acids and hydroxy alkyl arenes.
Hydroxylated aromatic compounds, such as hydroxylated aromatic
carboxylic acids, and alkyl hydroxyarenes (e.g., di- and tri-methyl phenols)
are an important
group of industrial chemicals. For example, the methylphenols find use in the
industrial
synthesis of vitamin E, and, also in the synthesis of various polymers and
resins, where they
can be used individually or as part of more complex compositions that include
other
phenolic and non-phenolic compounds. Typically, dimethylphenols and
trimethylphenols
are generated by successive methylation of phenol and cresols. However,
current synthetic
chemical methods based on methylation of phenol do not offer sufficiently high
selectivity
for preparing isomeric dimethyl- and trimethyl-phenols individually.
Therefore, complex
mixtures of methylated products having a varying degree of methylation and
various
substitution patterns are generally obtained. The presence of these mixtures
necessitates the
use of expensive separation procedures to obtain pure compounds.
Hydroxylated aromatic carboxylic acids (HCA) and their esters and lactones
are useful components of various polymers and co-polymers, such as polyesters.
Their
utility stems largely from their bifunctional reactive nature (e.g., hydroxyl
and carboxyl
groups) and the hydrophobic aromatic ring, which often imparts desirable
physical and
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chemical properties to the polymers (p-hydroxybenzoate, m-hydroxybenzoate).
HCAs are
also used in, for example, anti-microbial additives to pharmaceuticals (esters
of para-
hydroxybenzoic acid, parabens) and to fragrances (esters of salicylic acid,
coumarins and
3,4-dihydrocoumarin).
While many chemical synthetic methods for HCAs and their derivatives are
known in the art, typically, these compounds are manufactured from a non-
oxidized
aromatic precursor by multistep processes, requiring both the harsh conditions
and the
extensive product purification from by-products, arising from non-selective
reactions.
The present invention solves many of these problems by providing a
dioxygenase polypeptide that oxidizes one or more aromatic ~t-bonds to the
corresponding
diol. The diols can, if desired, be subsequently dehydrated to restore the
aromatic system
and yield a hydroxylated aromatic ring.
C. Accessory Polyneutides
In conjunction with the oxidative pathways utilizing polypeptides having
dioxygenase activity, as discussed above, the present invention provides
accessory non-
dioxygenase polypeptides. As used herein, "accessory polypeptides" refers to
those
polypeptide that do not carry out the initial dioxidation step in the methods
of the invention.
Exemplary accessory polypeptide include, ligases, transferases,
dehydrogenases, and the
like. Although both shuffled and non-shuffled accessory polypeptides can be
used,
preferred accessory polypeptides are those that have been shuffled.
The non-dioxygenase polypeptides can be used at any step of a pathway
using a dioxygenase of the invention. In a preferred embodiment, the accessory
polypeptides are used to further transform an oxidation product. Although it
will generally
be preferred to utilize oxidized substrates that are produced by a dioxygenase
of the
invention, those of skill will appreciate that these routes can be practiced
with analogous
substrates that are, for example chemically synthesized, commercially
available, etc.
Moreover, the present invention provides methods using both the improved
accessory peptides and unimproved accessory peptides to further elaborate the
dioxygenase
mediated reaction product. The method involves contacting the product of the
dioxygenase
mediated reaction with one or more of the accessory polypeptides. In a
preferred
embodiment, the product is contacted with an organism that expresses the
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polypeptide(s). When the accessory polypeptides are improved polypeptides,
they will
generally be produced by the methods described herein.
The improved dioxygenase and the accessory polypeptide(s) can be
expressed by the same host cell, or they can be expressed by different host
cells. In a
preferred embodiment, the accessory polypeptide and the improved dioxygenase
are
expressed by the same host cell.
By utilizing accessory polypeptides, the present invention makes possible the
synthesis of a great variety of industrially valuable compounds via the
methods disclosed
herein.
1. Dehydrogenases
In a preferred embodiment, an alcohol or diol is converted to an aldehyde or
carboxylic acid by the action of a dehydrogenase. The substrate for the
dehydrogenase is
preferably the product of an improved oxygenase of the invention.
Polynucleotides encoding many known dehydrogenases can be used as
substrates for nucleic acid shuffling. Exemplary dehydrogenases useful in
practicing the
present invention include, but are not limited to:
[ECOALDB, ECAE000436, ECAE000239, D90780, D90781, ECOFUCO,
ECOFUCO] dehydrogenase of Escherichia coli; [AF029734 and
AF029733] dehydrogenase of Xanthobacter autotrophicus; [AREXOYGEN]
dehydrogenase of Agrobacterium radiobacter; [AB003475] dehydrogenase
of Deinococcus radiodurans; [AF034434, VIBTAGALDA] dehydrogenase
of Vibrio cholerae; [D32049] dehydrogenase of Synechococcus sp.;
[AE001154] dehydrogenase of Borrelia burgdorferi (BB0528); [ABY17825]
dehydrogenase of Agaricus bisporus; [ASNALDAA] dehydrogenase of
Aspergillus niger; [EMEALDA, EMEALCA] dehydrogenase of Aspergillus
nidulans; [AF019635, PPU15151] dehydrogenase of Pseudomonas putida
TOL plasmid, xylW, xyl C; [AF031161] dehydrogenase of Pseudomonas sp.
VLB120, (stdD); [PFSTYABCD] dehydrogenase of P. fluorescens, styD;
[PPU24215] dehydrogenase of P. putida, Flp-cymene alcohol and aldehyde
dehydrogenases.
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2. Conversion of hydroxyls and/or acids to esters
In another preferred embodiment, a method is provided for converting
carboxylic acid and hydroxyl groups to adducts such as esters and ethers.
Useful
polypeptides include, for example, ligases and transferases (see, Fig.l3). For
the purposes
of the discussion below, these polypeptides are referred to as "adduct-
forming"
polypeptides.
The adduct-forming polypeptides are useful for enhancing and controlling
the production of biotransformation products. These polypeptides, which
convert a diol, for
example, to a monoacyl or monoglycosyl derivative can enhance control over the
regioselectivity of subsequent reactions (e.g., chemical dehydration). For
example, the
regioselectivity of chemical dehydration in certain cases can be controlled by
converting the
compounds to their diacyl derivatives by means of chemical reaction, and then
selectively
removing one of the acyl groups using an polypeptide of the invention.
Alternatively, one
can control the regioselectivity of the dehydration by using an esterase or a
trans-acylase
polypeptide to convert the compounds to monoacyl derivatives, preferably in
the presence
of an excess of another carboxylic acid ester. In addition, the isolation of
certain products is
simplified by their conversion to more hydrophobic species. For example, the
acylation of a
diol to the corresponding carboxylic ester provides a more efficient recovery
of such diols,
in the form of an ester, by organic solvent extraction of the adduct.
Preferred organic
solvents are those that can be used in an immiscible biphasic organic-aqueous
biotransformation with whole cells, whether in a batch or in a continuous
mode.
An adduct-forming polypeptide is optionally expressed by the same host cell
that expresses the dioxygenase, dehydrogenase, racemase, etc., or by a
different host cell.
Moreover, an adduct-forming polypeptide can be a naturally occurnng
polypeptide, or it can
be improved by the method of the invention.
When the adduct-forming polypeptide is an improved polypeptide, in
presently preferred embodiments, the polypeptide demonstrates increased
efficiency in the
formation of the monoacyl- or monoglycosyl- derivatives of a desired compound
(e.g., a
glycol, carboxylic acid, etc.). Other improved adduct-forming polypeptides
include
transferases and ligases that selectively modify only one of the hydroxyl
groups of a diol,
thus providing a means for controlling the regioselectivity of dehydration of
such
derivatives to either of two possible isomeric a-hydroxycarboxylic acid
compounds.
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a. Acyltransferases
Other enzymes useful in practicing the present invention are the
acyltransferases. These polypeptides are optionally evolved to enhance certain
catalytic
properties of the encoded polypeptides such as, specificity for a particular
hydroxyl and/or
acid, enantiomeric and/or diastereomeric selectivity.
More specifically, these polypeptides catalyze acyl transfer reactions as
shown in Fig. 13. Acyltransferases are ubiquitous in nature, and many
organisms (e.g.,
microbes, plants, mammals, etc.) can be used as sources of genes encoding
these
polypeptides. No matter their origin, the acyltransferase genes are preferably
selected from
those encoding functional polypeptides that catalyze active (CoA) ester
transfer reactions in
the biocatalytic processes described herein. Preferred acyltransferase genes
are selected
from those encoding functional polypeptides catalyzing reactions of small non-
biopolymeric
molecules.
Examples of various acyltransferases useful in the present invention include
polypeptides that catalyze the methylation of a-hydroxycarboxylic acids. A
list of
exemplary polynucleotides that can be recruited for this purpose are listed
below by the
corresponding GenBank identification:
[AF043464] acetyl-CoA: benzylalcohol acetyltransferase of Clarkia breweri,
and benzoyl-CoA benzyl alcohol acetyltransferase present in the same
organism, (Dudareva et al, Plant Physiol. 116(2):599-604 (1998));
[DCANTHRAN, DCHCBT1, DCHCBT1A, DCHCBT1B, DCHCBT2,
DCHCBT3] hydroxycinnamoyl/benzoyl-CoA:anthranilate N-acyltransferase
of Dianthus caryophyllus; [E08840] homoserine o-acetyltransferase of
Acremonium chrysogenum; [E12754] anthocyanin 5-aromatic
acyltransferase, of Gentiana triflora; [HUMBCAT] branched chain
acyltransferase (human, J03208, J04723); [MG396;D02 °orf 152(lacA);
MJ1064(lacA) MJ1678, MTH1067]; galactoside 6-O acetyl transferase EC
2.3.1.18, lac A of E.coli ; B0342(lacA); or of other organisms;
[B3607(cysE), HI0606(cysE), HP1210(cysE), SLR1348(cysE)] serine O-
acetyltransferase EC 2.3.1.30; [YGR177C, YOR377W] alcohol O-
acetyltransferase, EC 2.3.1.84, of Saccharomyces cerevisiae; [e.g.,
Q00267,D90786,Z92774,I78931 AF030398, AF008204, AF042740]
arylamine N-acetyltransferase, EC 2.3.1.118; [YAR035(YAT1),
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YM8054.01(CAT2)] carnitine O-acetyltransferase, EC 2.3.1.7, or
mammalian origin of from yeast; [CHAT] choline O-acetyltransferase, EC
2.3.1.6, of mammalian origin; acetyl CoA:deacetylvindoline 4-O-
acetyltransferase (EC 2.3.1.107) St-Pierre et al, Plant J. 14(6): 703-713
(1998); and [ECOPLSC] 1-acyl-sn-glycerol-3-phosphate acyltransferase
(plsC) of Escherichia coli.
b. Acyl CoA ligases
In another embodiment an accessory polypeptide having acyl CoA ligase
activity is provided.
Specificity of acyl-CoA ligases towards a particular exogenous substrate or a
group of substrates is preferably optimized by screening or selecting for the
acylation of a
substrate by shuffled and co-expressed acyl-CoA ligases and acyltransferases.
Utilizing
these polypeptides in tandem allows the combined effect of both polypeptides
to be
exploited.
To illustrate the single gene shuffling or shuffling of a family of genes
approach to improve acyl-CoA ligases or acyltransferases, one or more of the
members of
the corresponding superfamilies of these polypeptides are selected, aligned
with similar
homologous sequences, and shuffled against these homologous sequences.
An exemplary list of useful acyl-CoA ligase genes for inclusion into an
organism of the invention is provided below:
[AF029714, ECPAA, AJ000330, PSSTYCATA] phenylacetate-CoA ligase,
EC 6.2.1.30; [Y11070, Y11071] phenylpropionate-CoA ligase;
[B2260(menE), SLR0492(menE), SAU51132(menE)] O-succinylbenzoate-
CoA ligase, EC 6.2.1.26; [RPU75363, RBLBADA, AA532705, AA664442,
AA497001, AF042490, ARGFCBABC] (chloro)benzoate-CoA ligase, EC
6.2.1.25; [SBU23787, VPRNACOAL, POTST4C11, RIC4CL2R, OS4CL,
AF041051, AF041052, GM4CL14, GM4CL16, LEP4CCOALA,
LEP4CCOALB, PC4CL1A, PC4CL1AA, PC4CL2A, PC4CL2AA,
TOB4CCAL, TOBTCL2, TOBTCL6, ECO110K, AF008183, AF008184,
AF041049, AF041050, ATU18675, NTU5084, NTU50846, PTU12013,
PTU39404, PTU39405, ATF13C5, ORU61383, AF064095, AA660600,
AA660679, STMPABA] 4-coumarate-CoA ligase EC 6.2.1.12; [RPU02033]
4-hydroxybenzoate-CoA ligase; [PSPPLAS] 2-aminobenzoate-CoA ligase.
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In some embodiments of the invention, a carboxylic acid is fed exogenously
to an organism that expresses the ligase or transferase. Preferably, the
carboxylic acid is
selected from those compounds that cannot be altered by the polypeptide used
to produce
the substrate acted upon by the adduct forming polypeptide. Such carboxylic
acids include,
for example, both substituted and non-substituted benzoic acid, phenylacetic
acid,
naphthoic, phenylpropionic acid, phenoxyacetic acid, cycloalkanoic acid,
carboxylic acids
derived from terpenes, pivalic acid, substituted acrylic acids, and the like.
To facilitate the utilization of exogenously supplied carboxylic acids, and
for
enhancing the variety of compounds suitable for use in this process, the
invention also
provides microorganisms in which one or more mutations are introduced.
Preferred
mutations are those that effectively block metabolic modifications of such
acids beyond
their conversion to a suitable active ester (e.g., as a derivative of coenzyme
A). Such
mutations in the host organism are optionally introduced by classical
mutagenesis methods,
by site-directed mutagenesis, by whole genome shuffling, and other methods
known to
those of skill in the art. One can also introduce mutations that minimize host
endogenous
esterase activity.
In a presently preferred embodiment, the acyl transferase-encoding nucleic
acids used as substrates for creating recombinant libraries encode
polypeptides that transfer
an acetyl group from an endogenous pool of acetyl-CoA in the cells of the
host. The
endogenous pools of acetyl-CoA can also be enhanced by nucleic acid shuffling
of an
acetyl-CoA ligase and by supplying an exogenous acetate in the medium.
While using acetyl-CoA transferases or other acyltransferase or
glycosyltransferase does not necessarily require expression of a corresponding
acetyl-CoA
or other ligase, in a presently preferred embodiment, the organisms produce a
sufficient
amount of an acyl-CoA ligase so as to activate the carboxylic acids to CoA
thioesters,
which in turn serve as substrates for acyl-CoA transferases that utilize the
oxidation
products as substrates. The specificity of an acyl-CoA ligase towards a
desired exogenous
carboxylic acid can be optimized using the recombination and
screening/selection methods
of the invention. Preferably, the screening or selecting is performed using co-
expressed
acyl-CoA ligases and acyltransferases, thus permitting one to screen on the
basis of the
combined effect of both polypeptides in the pathway for provision of
monoacylated
derivatives of the oxidation products.
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Nucleic acids that encode acyl-CoA ligases and other acyltransferases useful
as substrates for the recombination and selection/screening methods of the
invention
include, for example, one or more members of the superfamilies of these
polypeptides. In a
presently preferred embodiment, the nucleic acids are selected, aligned with
similar
homologous sequences, and shuffled against these homologous sequences.
c. Glycosxltransferases
Similarly, one or more glycosyltransferases can be expressed by the host
cells of the invention. Alternatively, one or more glycosyltransferases can be
selected from
the glycosyltransferase superfamily, aligned with similar homologous
sequences, and
shuffled against these homologous sequences. Glycosyl transfer reactions are
ubiquitous in
nature, and one of skill in the art can isolate such genes from a variety of
organisms, using
one or more of several art-recognized methods. The following are illustrative
examples of
glycosyltransferase-encoding nucleic acids that can be used as substrates for
creation of the
recombinant libraries. The libraries are then screened to identify those
polypeptides that
exhibit an improvement in the glycosylation of compounds such as alcohols,
diols and a-
hydroxycarboxylic acids:
[EC 2.4.1.123] inositol 1-a-galactosyltransferase; [NTU32643, NTU32644]
phenol (3-glucosyltransferase, EC 2.4.1.35; flavone 7-O-beta-
glucosyltransferase, EC 2.4.1.81; [AB002818, ZMMCCBZ1, AF000372,
AF028237, AF078079, D85186, ZMMC2BZ1, VVUFGT]; flavonol 3-O-
glucosyltransferase, EC 2.4.1.91; o-dihydroxycoumarin 7-O-
glucosyltransferase, EC 2.4.1.104; vitexin beta-glucosyltransferase, EC
2.4.1.105; coniferyl-alcohol glucosyltransferase, EC 2.4.1.111; monoterpenol
beta-glucosyltransferase, EC 2.4.1.127; arylamine glucosyltransferase, EC
2.4.1.71; sn-glycerol-3-phosphate 1-galactosyltransferase, EC 2.4.1.96;
[RNUDPGTR, AA912188, AA932333] glucuronosyltransferase, EC
2.4.1.17; the human UGT and isoenzymes (~35 genes); salicyl-alcohol
glucosyltransferase, EC 2.4.1.172; 4-hydroxybenzoate 4-O-beta-D-
glucosyltransferase, EC 2.4.1.194; zeatin O-beta-D-glucosyltransferase, EC
2.4.1.203; [VFAUDPGFTA] D-fructose-2-glucosyltransferase; and
[MBU41999] ecdysteroid UDP-glucosyltransferase (egt).
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In presently preferred embodiments, the glycosyltransferases are selected
from those which transfer hexose residues from UDP-hexose derivatives.
Preferred hexoses
include, for example, D-glucose, D-galactose and D-N-acetylglucosamine.
d. Methyltransferases
In a still further preferred embodiment, the host cells of the present
invention
express a polypeptide capable of converting a carboxylic acid to a carboxylic
acid methyl
ester. Presently preferred polypeptides include methyltransferases.
For the purpose of this invention, genes encoding S-adenosylmethionine-
dependent methyltransferases are preferred. In a preferred embodiment, these
polypeptides
are evolved to enhance selected properties of the encoded polypeptides such
as, specificity
for a particular substrate and enantiomeric and/or diastereomeric selectivity
and/or solvent
resistance.
More specifically, these polypeptides can be evolved to catalyze the O-
methylation of carboxyl groups of a caroxylic acid substrate thus forming the
corresponding
methyl esters. Methyltransferases are ubiquitous in nature, and many organisms
(e.g.,
microbes, plants, mammals, etc.) can be used as sources of genes encoding
these
polypeptides. No matter their origin, the methyltransferase genes are
preferably selected
from those which encode functional polypeptides that catalyze the methylation
of small
non-biopolymeric molecules. Preferably, the methyltransferases are those which
act on the
carboxyl groups of organic acids.
Examples of various methyltransferases that can be expressed by host cells
of the invention and which are useful for nucleic acid shuffling-based
directed evolution of
polypeptides catalyzing the methylation of carboxylic acids are listed below
by the
corresponding GenBank identification:
[SCCCAGC3] methyltransferase of Streptomyces clavuligerus
methyltransferase CmcJ; [SEERYGENE] methyltransferase of S.erythraea
methyltransferases; [SEU77454] methyltransferase of Saccharopolyspora
erythraea; erythromycin O-methyltransferase (eryG); [SGY08763]
methyltransferase of S.griseus; [SKZ86111] methyltransferase of S.lividans;
[STMDNRDKP] methyltransferase of Streptomyces peucetius;
carminomycin o-methyltransferase (dnrK); [MDAJ39670] methyltransferase
of Streptomyces ambofaciens; [SEY14332] methyltransferase of
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Saccharopolyspora erythraea; [SPU10405] methyltransferase of
Streptomyces purpurascens ATCC 25489; [STMDAUA] methyltransferase
of Streptomyces sp.; aklanonic acid methyltransferase (dauC), and
carminomycin 4-O-methyltransferase (dauK); [SC2A11 and SC3F7]
methyltransferase of Streptomyces coelicolor; [SHGCPIR] methyltransferase
of S.hygroscopicus; [STMCARMETH] methyltransferase of Streptomyces
peucetius carminomycin 4-O-methyltransferase; [STMODPOMT]
methyltransferase of Streptomyces alboniger O-demethylpuromycin-O-
methyltransferase (dmpM); [STMTCREP]; methyltransferase of
Streptomyces glaucescens; [SLLMRBG] methyltransferase of S. lincolnensis
lmrB methyltransferase; [SSU65940] 31-O-demethyl-FK506
methyltransferase (fkbM) of Streptomyces sp.; [STMDAUABCE] aklanonic
acid methyltransferase (dauC) of Streptomyces sp.; [STMMDMBC] O-
methyltransferase (mdmC) of Streptomyces mycarofaciens; [STMTYLF]
macrocyn-O-methyltransferase (tylF) of S.fradiae; [E08176] Gene of
mycinamicin III-O-methyltransferase; [AF040571] methyltransferase of
Amycolatopsis mediterranei; [ECU56082] S-adenosylmethionine:2-
demethylmenaquinone methyltransferase (menG) of Escherichia coli;
[RHANODABC] methyltransferase (nods) of Azorhizobium caulinodans;
[YSCSTE14] isoprenylcysteine carboxyl methyltransferase (STE14) of
Saccharomyces cerevisiae; [YSCMTSW] farnesyl cysteinecarboxyl-
methyltransferase (STE14) of Saccharomyces cerevisiae; [YSCDHHBMET]
3,4-dihydroxy-5-hexaprenylbenzoate methyltransferase (COQ3) of
S.cerevisiae; [AF004112 and AF004113] phospholipid methyltransferases
(chol+), (cho2+) of Schizosaccharomyces pombe; [ASNOMT,
ASNOMT1A, ASNOMT1B, ASNOMT1C and AF036808-AF036830] O-
methyltransferases of Aspergillus; [MSU20736] S-adenosyl-L-methionine;
trans-caffeoyl-CoA3-O-methyltransferase of Medicago sativa; [ALFIOM]
isoliquiritigenin 2'-O-methyltransferase of Medicago sativa; [MSU20736] S-
adenosyl-L-methionine; trans-caffeoyl-CoA3-O-methyltransferase
(CCOMT) of Medicago sativa; [MSAF000975] 7-O-methyltransferase (7-
IOMT(6)) of Medicago sativa; [MSAF000976] 7-O-methyltransferase (7-
IOMT(9)) of Medicago sativa; [MSU97125] of isoflavone-O-
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methytransferase Medicago sativa; [NTCCOAOMT] caffeoyl-CoA O-
methyltransferase of Nicotiniana tabacum; [NTZ82982] caffeoyl-CoA O-
methyltransferase 5 of N.tabacum; [NTDIMET] o-diphenol-O-
methyltransferase of N.tabacum; [PCCCOAMTR, PUMCCOAMT] trans-
caffeoyl-CoA 3-O-methyltransferase of Petroselinum crispum; [PTOMT1] s
caffeic acid/5-hydroxyferulic acid O-methyltransferase (PTOMT1) of
Populus tremuloide; [PBTAJ4894-PBTAJ4896] caffeoyl-CoA 3-O-
methyltransferases of Populus balsamifera subsp. trichocarpa; [ZEU19911]
S-adenosyl-L-methionine: caffeic acid 3-O-methyltransferase of Zinnia
elegans; [SLASADEN] S-adenosyl-L-methioninearans-caffeoyl-CoA 3-O-
methyltransferase of Stellaria longipes; [VVCCOAOMT] caffeoyl-CoA O-
methyltransferase of V vinifera; [D88742] O-methyltransferase of
Glycyrrhiza echinata; [AF046122] caffeoyl-CoA 3-O-methyltransferase
(CCOMT) of Eucalyptus globulus; [ATCOQ3]
dihydroxypolyprenylbenzoate: methyltransferase of Arabidopsis thaliana
[CSJSALMS90] S-adenosyl-L-methionine:scoulerine 9-O-methyltransferase
of Coptis japonica; [HVU54767] caffeic acid O-methyltransferase
(HvCOMT) of Hordeum vulgare; [MCU63634] inositol methyltransferase
(Imtl) of Mesembryanthemum crystallinum; [PSU69554] 6a-
hydroxymaackiain methyltransferase (hmm6) of Pisum sativum;
[CAU83789] O-diphenol-O-methyltransferase of Capsicum annuum;
[U16794] 3' flavonoid O-methyltransferase (fomtl) of Chrysosplenium
americanum; [CBU86760] SAM:(Iso)eugenol O-methyltransferase(IEMT1)
of Clarkia breweri; salicylic acid carboxyl SAM-O-methyltransferase
(Dudareva et al, Plant Physiol. 116(2):599-604 (1998)); [HSHIOMT9]
hydroxyindole-O-methyltransferase (HIOMT) of Homo Sapiens;
[HSCOMT2] gene catechol O-methyltransferase of Homo Sapiens;
[HCTMPNMTA] phenylethanolamine N-methyltransferase gene of Homo
Sapiens; [HUMCOMTA] catechol-O-methyltransferase of Homo Sapiens;
[HUMCOMTC] catechol-O-methyltransferase of Homo Sapiens;
[HUMPNMT] phenylethanolamine N-methyltransferase of Homo Sapiens;
[AF064084] prenylcysteine carboxyl methyltransferase (PCCMT) of Homo
Sapiens; [HUMCMT] carboxyl methyltransferase of Homo Sapiens;
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[FfUMI~IMA] histamine N-methyltransferase of Homo sapiens;
[RATCATAA, RATCATAB] catechol-O-methyltransferase of R.norvegicus;
[RATDHNPBMT] dihydroxypolyprenylbenzoate methyltransferase of
Rattus norvegicus; [BOVPNMTB] of Bovine phenylethanolamine N-
methyltransferase; [MPEMT7] phosphatidylethanolamine-N-
methyltransferase of Mus musculus 2; [MMU86108] nicotinamide N-
methyltransferase (NNMT) of Mus musculus; [MUSCMT] carboxyl
methyltransferasease protein of Mouse; [GDHOMT] hydroxyindole-O-
methyltransferase of G.domesticus; [DRU37434] L-isoaspartate (D-
aspartate) O-methyltransferase (PCMT)of Danio rerio; [DMU37432] protein
D-aspartyl, L-isoaspartylmethyltransferase of Drosophila melanogaster; and
[HAU25845 and HAU25846] farnesoic acid o-methyl-transferases of
Homarus americanus.
3. Enantiomeric interconversion.
In a still further preferred embodiment, the present invention provides a
nucleic acid encoding a polypeptide capable of converting a particular
enantiomer of a
chiral compound such as an alcohol, diol or a-hydroxycarboxylic acid or a
precursor or
analogue thereof to its antipode.
Presently preferred polypeptides include racemases, such as the mandelate
racemase of Pseudomonas putida (PSEMDLABC). These polypeptides can expressed
by
hosts of the invention in their natural form or, alternatively, they can be
evolved to enhance
certain catalytic properties of the encoded polypeptides such as, specificity
for a particular
substrate and enantiomeric and/or diastereomeric selectivity.
The nucleic acids encoding the mandelate racemase of Pseudomonas putida,
which catalyzes the interconversion of mandelate R and S enantiomers, is a
typical
preferred example of genes selected for use in this invention. The nucleic
acids encoding
this gene, and any homologs of thereof, are subjected to nucleic acid
shuffling to evolve
polypeptides having improved or optimal performance and specificity towards
particular
substrates such as a-hydroxycarboxylic acids. In a preferred embodiment, the
polypeptide
has a performance and/or specificity that is enhanced over the wild type.
Preferred
polypeptides act on a-hydroxycarboxylic acid substrates, such as those
displayed in Fig. 11.
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4. Solvent resistance polypeptides
The invention also provides organisms expressing one or more of the
improved polypeptides of the invention and that are also resistant to
solvents, organic
substrates and reaction products (e.g., epoxides, glycols, a-hydroxyaldehydes,
a-
hydroxycarboxylic acids and a-hydroxycarboxylic acid derivatives (e.g.,
esters)) according
to the methods of the invention.
The solvent resistance of organisms and polypeptide used in the biocatalytic
conversion of organic compounds is important for enhancing the productivity of
such
processes. Increased solvent resistance of the organisms can enhance
longevity, viability
and catalytic activity of the microbial cells, and can simplify the
administration of the
feedstock compounds to the reactor and the recovery or separation of desired
products by
means of, for example, continuous or semi-continuous liquid-liquid extraction.
In another aspect, the invention provides microbial cells that are useful in
the
synthetic methods described herein, which express proteins conferring
resistance to solvents
(in particular, organic solvents) upon the microbial cells. This allows the
use of whole
microbial cells in a organic-aqueous mixture (e.g., a biphasic mixture). In
presently
preferred embodiments, the invention provides microbial strains including at
least two of
the polypeptide systems described herein. For example, a microorganism of the
invention
can contain both a dioxygenase gene and a transferase gene. In other
embodiments, the
microorganism can contain both an arene dioxygenase gene and a solvent
resistance gene.
The microbial cells thus provide a significant improvement in productivity of
the synthesis
processes, selectivity of product formation, operational simplicity, ease of
product recovery
and minimizing any by-product streams.
Several microorganisms are known to possess high resistance to hydrophobic
compounds such as benzene and lower alkylbenzenes. Recently, genes encoding a
solvent
efflux pump (srpABC) have been identified in Pseudomonas putida strains
(Kieboom et al.,
J. Biol. Chem. 273:85-91 (1998)). Similarly, various genes that encode
polypeptides that
confer organic solvent resistance can be found in bacterial strains such as
Pseudomonas
putida GM73 (Kim et al., J. Bacteriol. 180: 3692-3696 (1998)), Pseudomonas
putida DOT-
T1E (Ramos et al., J. Bacteriol. 180: 3323-3329 (1998)), Pseudomonas Idaho
(Pinkart and
White, J. Bacteriol. 179: 4219-4226 (1997)). These and other genes, such as
those that
encode many proton-dependent multidrug efflux systems, e.g., MexA-MexB-OprM,
MexC
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MexD-OprJ, and MexE-MexF-OprN of Pseudomonas aeruginosa (Li et al., J.
Bacteriol.
180: 2987-2991 (1998)), or the tolC, acrAB, marA, soxS, and robA loci of
Escherichia coli
(Aono et al., J. Bacteriol. 180: 938-944 (1998); White et al., J. Bacteriol.
179: 6122-6126
(1997)), and in many other microorganisms, can be used to confer solvent
resistance upon a
host microbial strain used in the oxidative biocatalytic conversion of olefins
by action of
dioxygenases.
In presently preferred embodiments, the ability of a polypeptide to confer
solvent resistance is enhanced by subjecting nucleic acids encoding solvent
resistance
polypeptides, or the genomes of the microorganisms themselves, to the
recombination and
selection/screening methods described herein. The nucleic acids listed above,
as well as
similar genes, provide a source of substrates for incorporation into organisms
of the
invention and/or use in nucleic acid shuffling and other methods of
constructing libraries of
recombinant polynucleotides. The libraries can then be screened to identify
those nucleic
acids that encode polypeptides confernng improved solvent tolerance on a host.
For
example, one can select for improved tolerance to compounds such as olefins,
AHAs,
aldehydes, esters and hydrophobic solvents, including alkanes, cycloalkanes,
alcohols and
halocarbon derivatives, for example, which are used for performing
biotransformation (e.g.,
two-phase oxidation) of olefins to glycols, AHAs and to their corresponding
acyl- and
glycosyl- derivatives, etc. Similarly, shuffling of nucleic acids that encode
these
polypeptides can be used to confer and to improve resistance of the microbial
cell to high
concentrations of biotransformation substrates, intermediates and endproducts,
thus
improving biocatalyst performance and productivity.
In addition to each of the methods set forth above, the present invention
provides polypeptides produced according to these disclosed methods. Moreover,
the
invention provides organisms that express the polypeptides produced by the
method of the
invention. The organisms of the invention can express one or more of the
improved
polypeptides. Also provided by the present invention are methods of
synthesizing a desired
compound. This method involves contacting an appropriate substrate with a
polypeptide of
the invention. In a preferred embodiment, the substrate is contacted with an
organism of the
invention that expresses a polypeptide of the invention.
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D. Methods of Using Improved Polypeptides to Prepare Organic Compounds
In addition to the methods discussed above, the present invention provides a
range of methods for preparing useful organic compounds by the oxidation and
further
elaboration of appropriate precursors. Among the methods provided by the
present
invention are, for example, the oxidation of alkylarene compounds to the
corresponding
unsaturated diols and the subsequent dehydration of these diols to hydroxy
alkylarenes.
Additionally, there is provided an analogous method for preparing hydroxylated
aromatic
carboxylic acids. Moreover, the invention provides methods for preparing
exocyclic and/or
acyclic diols from molecules having alkene bonds. These diols can be readily
converted to
a-hydroxycarboxylic acids.
The reaction types and sequences set forth below are illustrative of the scope
of the invention. The dioxygenases of the invention are capable of oxidizing
any organic
substrate comprising an oxidizable moiety. Additional reaction sequences
utilizing the
polypeptides of the invention will be apparent to those of skill in the art.
1. Preparation of vicinal diol,r
The formation of vicinal diols by oxidizing a ~-bond using a dioxygenase of
the invention provides ready access to a wide array of compounds that are
useful as both
final products and as intermediates in mufti-step reaction pathways. The
dioxygenases of
the invention are capable of converting to vicinal diols an array of
structurally distinct
compounds comprising one or more ~-bonds.
Although the method can be practiced with essentially any ~-bond, in
essentially any compound, in a preferred embodiment, the method involves.
preparing a
vicinal diol group by contacting a substrate comprising a carbon-carbon double
bond with
an improved dioxygenase polypeptide, or an organism expressing an improved
dioxygenase
polypeptide.
In another preferred embodiment, the substrate comprising the carbon-carbon
~-bond is selected from styrene, substituted styrene, divinylbenzene,
substituted
divinylbenzene, isoprene, butadiene, diallyl ether, allyl phenyl ether,
substituted allyl phenyl
ether, allyl alkyl ether, allyl aralkyl ether, vinylcyclohexene,
vinylnorbornene, and acrolein.
In yet another preferred embodiment, the vicinal diol produced by the action
of the improved dioxygenase polypeptide has the structure:
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OH
R
HO R1
R6 ~ 5 . . n
R
wherein R' and RS are each independently selected from alkyl, substituted
alkyl, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic,
substituted heterocyclic,
NR2R3, -ORZ, -CN, C(R4)NRzR3 and C(R4)ORZ groups, or R' and RS are joined to
S form a ring system selected from saturated hydrocarbon rings, unsaturated
hydrocarbon
rings, optionally substituted, saturated or unsaturated heterocyclic rings; RZ
and R3 are
members independently selected from H, alkyl, substituted alkyl, aryl,
substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic
groups; R4 is
selected from =O and =S; R~ and R' are independently selected from H and
alkyl; and n is
an integer from 0 to 10, inclusive.
In certain preferred vicinal diols, R' is selected from phenyl, substituted
phenyl, pyridyl, substituted pyridyl, NRZR3, -ORZ, --CN, C(R4)NRZR3 and
C(R4)ORZ
groups, RZ and R3 are members independently selected from H, alkyl,
substituted alkyl, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and
substituted heterocyclic
groups; and R4 is selected from =O and =S.
In another preferred embodiment, the diol includes a six-member ring having
at least one endocyclic double bond and at least one substituent selected from
methyl,
carboxyl and combinations thereof. Preferred diols having this structure are
displayed in
Fig.l and are the compounds having the structures III, IV, V, VI, VII, VIII,
and Fig. 4 and
are compounds having the structures XXIII, XXIV, XXV, XXVI, XXVII, XXVIII,
XXIX,
XXX.
2. Preparation of hydroxy alkylarenes
In another preferred embodiment, the invention provides methods for
preparing hydroxy arenes and hydroxy arenes that are further functionalized
with, for
example, alkyl and substituted alkyl groups. The method involves contacting a
substrate
comprising an aryl group with an improved dioxygenase of the invention to from
a diol.
The diol intermediate is subsequently dehydrated, thereby producing an
aromatic ring
functionalized with a hydroxy radical. Methods for carrying out the
dehydration are known
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in the art. Both enzymatic and chemical/physical means are appropriate.
Preferred
chemical/physical techniques include acid, base, heat and combinations
thereof.
In another preferred embodiment, the substrate includes a member selected
from arylalkyl groups, substituted arylalkyl groups, heteroarylalkyl groups,
and substituted
heteroarylalkyl groups.
In a still further preferred embodiment, the substrate has the structure
~R)n
wherein, each of the n R groups is a member selected from the group consisting
of alkyl
groups, substituted alkyl groups, alkynyl groups, aralkyl groups, alkoxy
groups, aryloxy,
alkylthio groups, cycloalkyl groups, alkenyl groups, halogens, CF3, CN, N02,
trimethylsilyl,
trimethylgermanyl, trimethylstannyl, and alkylamines; and n is an integer from
0 to 5,
inclusive. Typically R contains about 1 to about 15 carbons, preferably R is a
lower alkyl
group, more preferably methyl; and n is an integer from 1 to 5, inclusive,
more preferably, n
is an integer from 1 to 4, inclusive.
The enzymes, bioengineered pathways, and microorganisms of the invention
are useful for the synthesis of a wide variety of compounds, including many
that are of
commercial importance for purposes such as vitamin production. The methods and
enzymes of the invention provide a means by which commercially valuable
compounds can
be formed using relatively inexpensive compounds as precursors. An
illustrative example
of the use of the enzymes and methods of the invention is a new selective
process for
making isomeric trimethylphenols which can further be converted to
trimethylhydroquinone
(a key vitamin E intermediate). In the case of 2,4,5-trimethylphenol, the new
route uses
similar conditions as are typically applied to conversion of 2,4,6-
trimethylphenol (Fig. 2).
The use of arene dioxygenases that are optimized using the methods of the
invention to synthesize precursors of vitamin E, for example, provide
significant advantages
over previously available methods for obtaining vitamin E. Natural tocopherols
are isolated
from common oils such as soybean, corn, canola, cottonseed, safflower, and the
like, by
means of repeated vacuum distillation and alkali treatment of the oils,
followed by multi-
step processes for removal of impurities such as sterols and fatty acids.
Microbial
production of tocopherols has also been reported, from e.g., Aspergillus,
Lactobacter,
Euglene and Mycobacterium, but fermentation titers are low, rendering these
processes
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commercially insignificant. Chemical synthesis of vitamin E from
petrochemicals, for
example, typically results in an equal mixture of eight different isomers,
with the mixture
containing 12.5 percent of each isomer. The recombination and selection
methods of the
invention can be used to obtain arene dioxygenases that produce predominantly
the desired
RRR-a-tocopherol by virtue of the enhanced enantiospecificity and
regiospecificity of the
improved recombinant enzymes.
Key intermediates in the synthesis of D-a-tocopherol (vitamin E), for
example, are 2,3,5-trimethylphenol (Compound IX in Fig. 2), 2,4,5-
trimethylphenol
(Compound X), 2,3,6-trimethylphenol (Compound XI) and 2,4,6-trimethylphenol
(Compound XII). From these trimethylphenol compounds, one can use catalytic
oxidation
to obtain 2,3,5-trimethylhydroquinone as shown in Fig. 2. One method that is
suitable for
the catalytic oxidation is described in, for example, US Patent No. 4,250,335.
The 2,3,5-
trimethylhydroquinone, in turn can be used to prepare vitamin E by any of
several known
methods. For example, a natural or synthetic phytol side chain can be attached
to the 2,3,5-
trimethylhydroquinone using Friedel-Craft acidic catalytic conditions (see,
e.g., US Patent
No. 5,468,883).
The 2,3,5-, 2,4,5, 2,4,6- and 2,3,6-trimethylphenols can be synthesized using
the arene dioxygenases and microbial cells of the invention as shown in Fig.
1. For
example, 1,2,4-trimethylbenzene (Compound I) is oxidized to any of Compounds
III, IV, V,
VI, or VII using an arene dioxygenase. These arene cis-dihydrodiols can then
be subjected
to chemical dehydration to obtain the corresponding trimethylphenols as shown
in Fig. 1.
Alternatively, 1,3,5-trimethylbenzene (Compound II) can be oxidized using an
arene
dioxygenase to obtain Compound VIII, which in turn can be dehydrated to obtain
2,4,6-
trimethylphenol (Compound XII). In a presently preferred embodiment, an arene
dioxygenase exhibits enhanced regiospecificity for the addition of the
hydroxyl residues to
the appropriate carbon atoms and/or enhanced enantiospecificity to obtain the
desired
chirality.
The invention also provides methods in which acyltransferases or
glycosyltransferases are used to facilitate the production of a desired isomer
of a
dialkylphenol or a trialkylphenol from the cis-dihydrodiol intermediates that
are formed
upon arene dioxygenase-mediated biocatalysis. An example of this reaction is
shown in
Fig. 3, in which an acyltransferase or a glycosyltransferase is employed to
acylate or
glycosylate one of the cis-hydroxyl groups on a cis-dihydrodiol (Compound IV).
Upon
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chemical dehydration of the resulting compound (Compound XVI), the acylated or
glycosylated group is preferentially lost, leaving a hydroxyl at the non-
acylated position. By
using an acyltransferase or glycosyltransferase that has been subjected to
recombination and
screening for those that are regiospecific for a particular hydroxyl group,
using the methods
of the invention, one can achieve specific production of the desired isomer of
trimethylphenol (Compound IX).
Alternatively, one can employ chemical acylation in conjunction with an
esterase to convert a cis-dihydrodiol di- or trialkylbenzene intermediate into
a desired
isomer of trialkyl or dialkylphenol. In this embodiment of the invention, the
cis-dihydrodiol
product of an arene dioxygenase reaction is subjected to chemical acylation
with an
anhydride, resulting in acylation of both hydroxyl groups (e.g., Compound
XVIII in Fig. 3).
An esterase is then employed to release one of the acyl groups, thus producing
the
monohydroxyl derivative (e.g., Compound XVI), which can then be converted to a
desired
dialkyl- or trialkylphenol by chemical dehydration (e.g., Compound IX). In
presently
preferred embodiments, the esterase is a recombinant esterase that has been
enhanced, using
the methods of the invention, for improved properties such as regiospecificity
and
enantiospecificity, and the like.
Tocopherol derivatives that lack a methyl group, and thus use
dimethylphenols as precursors, find use as antioxidants, among other uses.
Such
compounds can be synthesized using the methods, enzymes, and microorganisms of
the
invention. Fig. 4 shows the various dimethylphenol compounds that one can
produce by
arene dioxygenase-catalyzed oxidation of xylenes, preferably in conjunction
with whole cell
biocatalysis, followed by chemical dehydration. For example, the invention
provides
methods in which o-xylene (Compound XXI) is oxidized by an arene dioxygenase
to form
one or more of the cis-dihydrodiols shown as Compounds XXV, XXVI, and XXVII.
Chemical dehydration of Compounds XXV and XXVII can then be used to obtain 2,3
dimethylphenol (Compound XXXII) and 3,4-dimethylphenol (Compound XXXIV),
respectively, while dehydration of Compound XXVI results in both of these
compounds
being produced. Accordingly, where a particular isomer is desired, in a
presently preferred
embodiment, the arene dioxygenase that is employed in the reaction is one that
is optimized
for the desired regiospecificity and/or enantiospecificity.
In additional embodiments, the invention provides methods in which an
arene dioxygenase is used to catalyze the oxidation of m-xylene (Compound
XXII) to one
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or more of the arene cis-dihydrodiols Compound XXVIII, Compound XXIX, and
Compound XXX. The arene cis-dihydrodiols can then, in turn, be subjected to
chemical
dehydration to obtain one or more dihydrophenols. For example, by dehydrating
Compounds XXIX and XXX, respectively, 2,4-dimethylphenol (Compound XXXV) and
2,6-dimethylphenol (Compound XXXVI) are obtained. Dehydration of Compound
XXVIII
results in a mixture of the 2,4- and 2,6-dimethylphenols. Again, a particular
isomer can be
obtained by using an arene dioxygenase that has been optimized for the desired
regiospecificity and/or enantiospecificity using the recombination and
selection/screening
methods of the invention.
p-Xylene (Compound XX) can also serve as a substrate for the arene
dioxygenase-mediated oxidation. The resulting arene cis-dihydrodiols
(Compounds XXIII
and XXIV) can be chemically dehydrated to obtain 2,5-dimethylphenol. In these
methods
in particular, it is preferred that the arene dioxygenase is expressed by a
cell that is of a
species other than that from which the arene dioxygenase gene was obtained, or
that the
arene dioxygenase is expressed from a recombinant arene dioxygenase-encoding
polynucleotide that has been optimized for improved properties using the
recombination and
selection/screening methods of the invention.
3. Preparation of hydroxylated aromatic carboxylic acids
Hydroxylated aromatic carboxylic acids have many diverse uses, including
as antimicrobial additives, UV protectants (e.g. esters of p-hydroxybenzoic
acid, parabens),
pharmaceutical compositions (e.g., esters of salicylic acid, coumarins and 3,4-
dihydroxcoumarin).
Thus, in another preferred embodiment, the present invention provides a
method for preparing hydroxylated aromatic carboxylic acids. The method
involves
contacting a substrate comprising an aryl carboxylic acid with a dioxygenase
polypeptide of
the invention. The polypeptide is preferably expressed by an organism of the
invention.
a. Carboxylic acid substrates
The carboxylic acids used as substrates in the present invention can be
obtained from commercial sources, or they can be prepared by methods known in
the art. In
a preferred embodiment, the carboxylic acids are prepared by contacting a
substrate
comprising an aryl alkyl group with an oxygenase polypeptide to produce the
corresponding
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aryl alkyl alcohol. The alcohol is subsequently acted upon by a dehydrogenase
polypeptide
to produce the desired carboxylic acid. Alternatively, the alcohol can be
converted to
COOH by chemical means.
For clarity of illustration, the discussion herein focuses on the oxidation of
arylmethyl groups to carboxylic acids. This focus is intended to be
illustrative and not
limiting.
(i). Monooxygenation
The first step in the biotransformation processes for conversion of methylaryl
compounds, such as toluene and isomeric xylenes involves the selective
oxidation of at least
one methyl group present in the aromatic substrate to the corresponding
carboxylic acid
(e.g., benzoic, toluic acids). In an exemplary embodiment, the substrate is
toluene, p- or, m-
or o-xylene or 1,2,4-trimethylbenzene, or a mixture thereof, and preferably,
only one of the
methyl groups is oxidized.
Following the oxygenation step, the resulting alcohol is dehydrogenated,
generally by the action of a dehydrogenase polypeptide to produce the desired
carboxylic
acid.
The invention provides for polypeptides that selectively oxidize only one
alkyl group of an arene bearing two or more alkyl substituents. This
embodiment is
illustrated in Fig. 6, with the monooxidation of various xylenes. For example,
p-xylene (2)
is selectively converted to a monocarboxylic acid (22). Alternatively, the
invention
provides polypeptides that are capable of oxidizing more than one alkyl
substituent of a
species substituted with two or more alkyl groups. For example, certain
polypeptides of the
invention are capable of oxidizing both of the methyl substituents of a
xylene, such as o-
xylene (4) to the corresponding benzenedimethanol (4a).
In a preferred embodiment, the monoxygenation/dehydrogenation pathway
produces a carboxylic acid having the structure:
~R~n
COOH
wherein each of the n R groups is independently selected from H, alkyl and
substituted alkyl
groups; and n is an integer from 1 to 5, inclusive, more preferably R is
methyl, and more
preferably still, n is an integer from 1 to 3, inclusive.
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In a still further preferred embodiment, the carboxylic acid group is selected
from:
C02H C02H C02H C02H
H3C / ~ / ~ / ~ and
\ ~ HsC \ ~ \ HaC \
CH3 CH3
Many enzymes for effecting these reactions are well known in the art, and
are suitable for use in the construction of useful polypeptides and host
strains. To achieve
the initial oxidation of the methyl groups, certain enzymes are presently
preferred, including
non-heme multicomponent monooxygenases of toluene and xylenes, and p-cymene,
as well
as certain arene dioxygenases which act on these substrate in a monooxygenase
mode. The
latter are exemplified by naphthalene dioxygenase, 2-nitrotoluene 2,3-
dioxygenase and 2,4-
dinitrotoluene 4,5-dioxygenase. These dioxygenases do not oxidize the aromatic
ring of
methylbenzenes, but are capable of oxidizing methyl groups of a variety of
aromatic
compounds in a monooxygenase mode (Selifonov, et al., Appl. Environ.
Microbiol, 62(2):
507-514 (1996); Lee et al., Appl. Environ. Microbiol. ;62(9):3101-3106 (1996);
Parales, et
al, J.Bacteriol., 180(5):1194-1199 (1998); Suen et al, J.Bacteriol.,
178(16):4926-4934
( 1996).
The following list provides examples of polynucleotides that encode a
monooxygenase and are suitable for use in the methods of the invention. The
loci are
identified by GenBank >D and encode complete or partial protein components of
the arene
dioxygenases. Suitable loci include:
[PSEXYLMA], [AF019635], [D63341], [E02361] xylene/toluene
monooxygenase of Pseudomonas putida TOL plasmid (xyl M, xylA);
[PPU24215] p-cymene monooxygenase of P. putida; [AF043544]
nitrotoluene monooxygenase of Pseudomonas sp. TW3, NtnMA (ntnM,
ntnA).
Additional monooxygenase polynucleotides useful in practicing the present
invention are disclosed in USSN 09/373,928 entitled DNA SHUFFLING OF
MONOXYGENASE GENES FOR PRODUCTION OF INDUSTRIAL CHEMICALS, to
Joseph A. Affholter, Sergey A. Selifonov and S. Christopher Davis, Attorney
Docket No.
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018097-025810, filed on August 12, 1999, and incorporated herein by reference
in its
entirety.
In another preferred embodiment, the monooxygenase used is actually a
dioxygenase that exhibits monooxygenase activity. As with the other
polypeptide activities
discussed herein, the ability of a dioxygenase to act as a monooxygenase is a
property that
can be optimized by shuffling the nucleic acids encoding these dioxygenases.
The following list provides examples of polynucleotides that encode
dioxygenases acting as monooxygenases and which are suitable for use in the
methods of
the invention. The loci are identified by GenBank ID and encode complete or
partial
protein components of the arene dioxygenases. Suitable loci include:
[AB004059], [AF010471], [AF036940], [AF053735], [AF053736],
[AF079317], [AF004283], [AF004284], [PSENAPDOXA],
[PSENAPDOXB], [PSENDOABC], [PSEORF1], [PSU49496] naphthalene-
1,2-dioxygenase; [BSU62430] 2,4-dinitrotoluene dioxygenase; [PSU49504]
2-nitrotoluene dioxygenase.
A polypeptide that catalyzes monooxygenation can be a naturally occurnng
polypeptide, or it can have one or more properties that are improved relative
to an
analogous naturally occurring polypeptide. In a preferred embodiment, the
polypeptides are
expressed by one or more host organisms. Moreover, the polypeptide that
catalyzes the
monooxygenation can be co-expressed by the same host expressing a polypeptide
used for
further structural elaboration of the oxidation substrate or product (e.g., a
dioxygenase
polypeptide that oxidizes the ~-bond). Alternatively, the mono- and di-
oxygenase
polypeptides can be expressed in different hosts.
(ii). Oxidation of adkylarenes having alkyl groups with > C2
While much of the discussion above highlighting pathway and organism
construction for oxidation of methylbenzenes is directly applicable to the set
of processes
dealing with alkyl benzenes bearing other alkyl group (Fig. 7), the essential
distinguishing
feature of the processes discussed in this section is in the provision of
alternative enzymes
for oxidation of these alkyl chains.
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Thus, in a preferred embodiment, at least one alkyl group of the alkylarene
has at least two carbon atoms. Preferred species produced in the
monoxygenation step have
the structure:
(R)m
(CH2)r,-C02H
wherein each of the m R groups is selected from H, alkyl, substituted alkyl,
aryl, substituted
aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic; m is an
integer from 0 to 5, inclusive; and n is an integer from 1 to 10, inclusive.
Preferred aryl
groups are those substituted on the aryl group with at least one methyl
moiety.
In another preferred embodiment, the compound has the structure:
(CH2)~ C02H
wherein n is an integer from 1 to 6, inclusive.
Certain exemplary embodiments are provided in Fig.7 which illustrates the
oxidation to a carboxylic acid of the terminal methyl groups of alkylbenzene
compounds 5-
9. The oxidation is accomplished by recruiting one or more genes encoding
oxygenase
activity. Generally, this is best accomplished by expressing a suitable
cytochrome P450
type enzyme system. The enzymes of this class are ubiquitous in nature, and
they can be
found in a variety of organisms. For example, n-propylbenzene is known to
undergo a-
oxidation in strains of Pseudomonas desmolytica S449B 1 and Pseudomonas
convexa
S107B1(Jigami et al., Appl Environ Microbiol. 38(5):783-788 (1979)) which can
utilize this
hydrocarbon in either of two alternative oxidation pathways.
Similarly, well known in the art, alkane monooxygenases of bacterial origin,
or cytochromes P450 for camphor oxidation, whether wild-type or mutant, can be
recruited
for the purpose of introducing the oxygen at the terminal methyl group of
alkylarenes (Lee
et al.,Biochem. Biophys. Res. Commun. 218(1):17-21 (1996); van Beilen et al.,
Mol.
Microbiol. 6(21):3121-3136 (1992); Kok et al., J. Biol. Chem. 264(10):5435-
5441 (1989);
Kok et al., J. Biol. Chem. 264(10):5442-5451 (1989); Loida et al., Protein
Eng. 6(2):207-
212 (1993).
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(iii) Oxygenation of arenes with exocyclic ~c-bonds
In another preferred embodiment, the starting material for the carboxylic acid
is an arene bearing an exocyclic ~t-bond. This class of compounds is
exemplified by
styrene. Other analogous species are set forth in Fig. 11.
The conversion of the exocyclic ~-bond is best accomplished by recruiting a
cluster of bacterial styrene oxidation genes well known in the art (Marconi et
al., Appl.
Environ. Microbiol. 62(1):121-127 (1996); Beltrametti et al., Appl. Environ.
Microbiol.
63(6):2232-2239 (1997); O'Connor et al., Appl. Environ. Microbiol. 63(11):4287-
4291
(1997); Velasco et al., J. Bacteriol. 180(5):1063-1071 (1998); Itoh, et al.,
Biosci.
Biotechnol. Biochem. 60(11):1826-1830 (1996). Alternatively, the styrene
epoxidation step
can be accomplished by using monooxygenases of methyl substituted aromatic
compounds,
such as toluene or xylenes (Wubbolts, et al., Enzyme Micro.b Technol.
16(7):608-615
( 1994).
(iv). Dehydrogenation
To produce the desired carboxylic acid, the alcohol from (i-iii), above, is
preferably treated with a dehydrogenase polypeptide. The dehydrogenase enzymes
can be
endogenous to a host that expresses one or more of the oxygenase polypeptides,
or it can
exhibit properties that are improved relative to an endogenously expressed
dehydrogenase.
The polypeptide that catalyzes the dehydrogenation can be a naturally
occurring polypeptide, or it can have one or more properties that are improved
relative to an
analogous naturally occurring polypeptide. In a preferred embodiment, the
polypeptides are
expressed by one or more host organisms. Moreover, the polypeptide that
catalyzes the
dehydrogenation can be co-expressed by the same host expressing one or more of
the
dioxygenase polypeptide. Alternatively, the dehydrogenase and oxygenase
polypeptides
can be expressed in different hosts.
In yet another preferred embodiment, the invention provides a method for
altering or controlling the regiospecificity of the dehydrogenation reaction
of a vicinal diol.
This method "blocks" one of the vicinal diol hydroxyl groups by forming an
ester, for
example. The method involves contacting the vicinal diol with a polypeptide,
preferably
expressed by a host organism, having an activity selected from ligase,
transferase and
combinations thereof, thereby forming a a-hydroxycarboxylic acid adduct. As
with the
other polypeptides discussed above, this polypeptide can be expressed by the
same host cell
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that expresses other polypeptides of the reaction cascade. Moreover, this
polypeptide can
be a naturally occurring polypeptide, or it can be improved using the method
of the
invention.
The combined steps of oxygenation are illustrated in Fig. 5, with the
preparation of compound 10 from compound 1, compound 22 from compound 2,
compound
23 from 3 and compound 24 from compound 4.
b. Dioxy~enation of aromatic ~-bonds
In the synthesis of hydroxyaryl carboxylic acids using the methods of the
invention, once the carboxylic acid moiety is in place, the molecule is
submitted to a
dioxygenation cycle. The dioxygenation of the aromatic ring is preferably
accomplished by
recruiting one or more arene dioxygenase genes, preferably of bacterial
origin. Exemplary
dioxygenase genes are disclosed herein. The method of the invention can be
practiced using
essentially any type of aromatic ring system. Exemplary aromatic systems
include,
benzenoid and fused benzenoid ring systems (e.g., benzene, napthalene, pyrene,
benzopyran, benzofuran, etc.) and heteroaryl systems (pyridine pyrrole, furan,
etc.). In a
preferred embodiment, the substrate includes a benzenoid hydrocarbon.
Similar to the embodiments discussed above, in this embodiment, the
polypeptide that catalyzes the dioxygenation can be coexpressed with one or
more
polypeptides used in this synthetic pathway. For example, the monooxygenase,
dehydrogenase and dioxygenase polypeptides can all be coexpressed in a single
host. Other
functional combinations of coexpression will be apparent to those of skill in
the art.
In a preferred embodiment, benzoate-1,2-dioxygenase or toluate-1,2-
dioxygenase are used to catalyze the formation of compound 14, p-cumate 2,3-
dioxygenase to catalyze formation of compounds 13, 25, 26, and phthalate 4,5-
dioxygenase or phthalate 3,4-dioxygenase to catalyze the formation of compound
12 (see, Fig. 5).
c. Exemplary embodiments
For the purpose of further illustration, methods of the invention for
preparing
coumarin derivatives and o-cinnamic acids are described below. These examples
are
intended to illustrate, not to limit, the scope of the present invention.
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(i). Synthesis of 3,4-dihydrocoumarin
The present invention provides both a chemoenzymatic route to 3,4-
dihydrocoumarin (43) from n-propylbenzene (steps as shown in Fig. 8), and
means for the
subsequent conversion of this compound to other lactone derivatives, such as
coumarin (58)
and 4-oxygenated derivatives of coumarin. While compound 43 can be converted
to 58 by
chemical methods known in the art (e.g. by reaction with sulfur, or by
catalytic
dehydrogenation over Pd or Pt catalyst), the purpose of this invention is also
in the
provision of alternative biocatalytic means for effecting such reaction.
In a preferred embodiment, one or more arene dioxygenase, such as
naphthalene dioxygenase, toluene 2,3-dioxygenase or other structurally and
functionally
related dioxygenases, are shuffled, as described herein, to produce an
improved polypeptide.
In the method of the invention, the improved polypeptide is used to catalyze
benzylic
monooxygenation reactions with a variety of benzocycloalkanes, and benzylic
desaturation
reactions of compounds exemplified by 1,2-dihydronapthalene, indan, and
ethylbenzene.
For the purpose of converting 3,4-dihydrocoumarin (43) to coumarin, these
catalytic activities of the arene dioxygenase enzymes can be used to make
either coumarin
itself (desaturation), or 4-hydroxy-3,4-dihydrocoumarin (59). Regardless of
enantiomeric
composition, the latter compound can be chemically dehydrated (under acidic or
heat
conditions) to coumarin.
Alternatively, compound 59 can be microbially converted to acyl or glycosyl
derivative 60 with the provision of corresponding transferase genes. When a
dehydrogenase
activity specific toward compound 59 is displayed by the host microbial
strain, the variation
of this route provides access to 4-hydroxycoumarin (70, Rg=H) (spontaneous
tautomer of
dehydrogenase product, 4-keto-3,4-dihydrocoumarin), or with provision of a
suitable
transferase gene to its 4-O-glycosyl, 4-O-Methyl or 4-O-acyl derivative 70.
Availability of
these routes is beneficial for making a range of other products originating in
n-
propylbenzene oxidative biotransformation pathway discussed above.
(ii). Conversion of naphthalene to coumarin and o-hydroxycinnamic acid
The invention also provides methods of making coumarin and o-hydroxy-
cinnamic acids from naphthalene, using a whole-cell microbial biocatalyst, and
methods for
constructing microbial strains effecting such conversions in their entirety,
or in part. In the
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latter case, the last steps for making coumarin are accomplished by chemical
methods.
Exemplary reaction sequences used in these processes are shown in Fig. 9.
To construct an improved polypeptide, the invention preferably uses a subset
of genes encoding catabolism of naphthalene by bacteria. In a preferred
embodiment, at
least four polypeptides (and the corresponding genes) are used to catalyze a
series of
reactions. These polypeptides include, naphthalene 1,2-dioxygenase (compound
10 to 61),
NahA (a multicomponent enzyme); cis-1,2-dihydro-1,2-dihydroxynaphthalene
dehydrogenase (compound 61 to 62), NahB; 1,2-dihydroxynaphthalene 1,1a
dioxygenase
(compound 62 to 63) and NahC.
Compound 63 is known to be labile, readily undergoing a series of
tautomerization and intramolecular ring closure reactions. The cis-trans
equilibrium
between compounds 63 and 64 is preferably effected by 2-hydroxybenzalpyruvate
isomerase (NahD) which can be used to impose a degree of control on the
isomerization of
the double bond. Preferably, NahE, 2-hydroxybenzalpyruvate hydratase/aldolase
is
preferably not used in this pathway, and preferred host strains are those
essentially lacking
activity of this enzyme.
In a preferred embodiment, the step of this process which allows for the
preparation of either coumarin 58, or cis/trans o-hydroxycinnamic acids (66,
67) is the
provision of an alpha-ketoacid decarboxylase enzyme with specific activity
towards either
compound 63 or 64 or both.
Several alpha-ketoacid decarboxylase enzymes are known, and in the
preferred embodiment, the benzoylformate decarboxylase of Pseudomonas putida,
or an
enzyme structurally or functionally similar to it, is used (Gen Bank
PSEMDLABC,
benzoylformate decarboxylase (mdlC)).
Ring closure of compound 66 to 58 can be effected by enzymatic or chemical
means (e.g. extraction under acidic conditions). Similarly, cis-trans
isomerization of 67 to
66 can be effected by enzymatic or chemical means (e.g. by Pt or Pd- catalyzed
hydrogenation/dehydrogenation under acidic conditions).
Biocatalytic variations of these processes, which can be used to produce
coumarin from o-hydroxycinnamic acids, preferably involve the use of acyl-CoA
ligase and
transferase enzymes (pathway for conversion of 66 to 68 to 58), or conversion
of 67 to
glycoside 69 with subsequent isomerization later in an enzymatic process akin
to that of
coumarin biosynthesis in plants.
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4. Preparation of a-hydroxycarboxylic acids
a-hydroxycarboxylic acids (AHAs) are an important group of industrial
chemicals. One of the simplest representatives of this class of compounds is
lactic acid
which find many uses, including synthesis of polyester polymers (polylactic
acid). Other
representative AHAs, such as mandelic acid can also be used as a constituent
of polymers
or co-polymers with lactic acid. Enantiomerically pure AHAs are also used as
resolving
reagents for separating racemates of chiral molecules.
AHAs are typically generated chemically by hydrolysis of a cyanohydrin,
generally prepared from an aldehyde. Aldehydes, however, are relatively
expensive starting
materials, and the cyanohydrin pathway does not readily provide for direct
preparation of
AHAs in high enantiomeric excess. One pathway for the synthesis of AHAs in
high
enantiomeric excess is through the use of one or more enzymatic reactions
starting from an
inexpensive and readily available starting material such as an alkene.
Arene dioxygenases (ADOs) are known to oxidize alkenes to the
corresponding vicinal diols. ADOs, such as toluene 2,3-dioxygenase,
isopropylbenzene 2,3-
dioxygenase, benzene-1,2-dioxygenase, biphenyl-2,3-dioxygenase naphthalene-1,2-
dioxygenase, and many homologous and/or functionally similar enzymes can be
used to
manufacture AHAs in a highly regioselective fashion. An example of this
dioxygenation
reaction is provided in Fig. 12, with the conversion of alkene (I) to the
vicinal diol (II).
The present invention provides general methods for the biocatalytic
manufacture of AHAs and their esters, and also for the construction and
optimization of the
biocatalytic properties of enzymes and host strains that effect oxidative cis-
dihydroxylation
or epoxidation reactions of a variety of alkenes. Moreover, the invention
provides for the
subsequent enzymatic conversion of the dioxygenation products to AHAs and
ester
derivatives of AHAs.
Thus, in another preferred embodiment, the invention provides a method for
converting an olefin into an a-hydroxyacid. The method involves: (a)
contacting the olefin
with an improved dioxygenase polypeptide to form a vicinal diol; and (b)
contacting the
vicinal diol with a dehydrogenase polypeptide to form the a-hydroxyacid.
The polypeptide that catalyzes the dehydrogenation can be a naturally
occurring polypeptide, or it can have one or more properties that are improved
relative to an
analogous naturally occurring polypeptide. In a preferred embodiment, the
polypeptides are
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expressed by one or more host organisms. Moreover, the polypeptide that
catalyzes the
dehydrogenation can be co-expressed by the same host expressing the
dioxygenase
polypeptide that oxidizes the ~t-bond. Alternatively, the dehydrogenase and
dioxygenase
polypeptides can be expressed in different hosts. An example of the
dehydrogenation is
provided in Fig. 12, with the conversion of diol (II) to aldehyde (III) and
its conversion to
a-hydroxycarboxylic acid (IV). As shown in Figs. 14A-14R, this same method can
be
carried out on a substrate comprising an aromatic moiety. The exemplary two-
step
dehydrogenation can equally well be carried out using a one step process.
Although the method of the invention can be used to produce AHAs having
substantially any structure, in another preferred embodiment, the a-
hydroxycarboxylic acid
has the structure:
OH
R'
HOOC n
wherein R1 is selected from aryl, substituted aryl, heteroaryl, substituted
heteroaryl,
heterocyclic, substituted heterocyclic, NRZR3, ~R2, ~N, C(R4)NRZR3 and
C(R4)OR2
groups; RZ and R3 are members independently selected from H, alkyl,
substituted alkyl, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and
substituted heterocyclic
groups; R4 is selected from =O and =S, and n is an integer between 0 and 10,
inclusive.
In a further preferred embodiment, R' is selected from phenyl, substituted
phenyl, pyridyl, substituted pyridyl, NRZR3, -OR2, -CN, C(R4)NRZR3 and
C(R4)ORZ
groups, R2 and R3 are members independently selected from H, alkyl,
substituted alkyl, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and
substituted heterocyclic
groups; and R4 is selected from =O and =S.
a. a-Hydroxycarboxylic acid adducts
AHAs are bifunctional molecules with two chemically and enzymatically
distinguishable functional groups, carboxyl and hydroxyl. In the biocatalytic
modifications
of AHAs described in this invention, either of these groups can be derivatized
by bond
formation. While these reactions do not change the oxidation state of the AHA
molecule,
recruitment of the enzymes effecting modification of AHAs provides the
opportunity to
generate biotransformation endproducts with substantially different physical
and chemical
properties than that of a free AHA. Generally desirable properties include an
increase of
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hydrophobicity, a decrease of aqueous solubility and, for an ester formed
through a
carboxylic group of an AHA, a decrease in acidity of the process end-products.
In a preferred embodiment, the adduct-forming polypeptide produces an a-
hydroxycarboxylic acid adduct selected from esters and ethers. The method
involves
contacting an a-hydroxycarboxylic acid with a polypeptide having an activity
selected from
ligase, transferase and combinations thereof, thereby forming a a-hydroxyacid
adduct. The
adduct forming polypeptides useful in this embodiment can be naturally
occurring
polypeptides or, alternatively, they can be polypeptides improved using the
methods of the
invention, as discussed generally, above.
Exemplary adduct forming reactions are provided in Fig. 13. This Figure
shows the use of a methyltransferase to convert carboxylic acid (X) to the
corresponding
methyl ester (XI), acyltransferase I to convert compound X to ester XIII, and
acyl-CoA
ligase to convert X to intermediate XIV. This intermediate can then be
transformed into a
simple alkyl ester (XIX) or to structures having greater complexity of
structure in the
alcohol-derived component (e.g., XV). Species such as XV can be further
elaborated using
other polypeptides including, for example, acyltransferase III to produce
compound XVII,
thioesterase II to produce compound XVIII and thioesterase I to produce
compound XVI.
In a further preferred embodiment, the a-hydroxycarboxylic acid adduct has
the structure:
O R'
R1
R600C
n
wherein, R' is selected from aryl, substituted aryl, heteroaryl, substituted
heteroaryl,
heterocyclic, substituted heterocyclic, NRZR3(R4)m, -OR2, ~N, C(RS)NRZR3 and
C(RS)OR2 groups, RZ, R3 and R4 are members independently selected from the
group
consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,
substituted
heteroaryl, heterocyclic and substituted heterocyclic groups; RS is selected
from =O and
=S; R6 is selected from H, alkyl and substituted alkyl groups; R' is C(O)Rg,
wherein R8 is
selected from H alkyl and substituted alkyl groups and R' and R8 are not both
H; m is 0 or
1, such that when m is 1, an ammonium salt is provided; and n is an integer
between 0 and
l0,inclusive.
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In yet another preferred embodiment, R' is selected from phenyl, substituted
phenyl, pyridyl, substituted pyridyl NR2R3, ~RZ, -CN, C(RS)NRZR3 and C(RS)OR2
groups; RZ and R3 are members independently selected from the group consisting
of H, C1-
C6 alkyl and allyl; and RS is =O.
In yet another preferred embodiment of this invention, the described
reactions and pathways are utilized for biocatalytic whole-cell conversion of
styrene to
mandelic acid and its ester derivatives. The pathway for styrene conversion,
all of its
intermediates and reactions are shown in Figs. 14A- 14R.
The esterified adducts provide an increase in the overall efficiency of the
biotransformation process as they simplify end-product recovery. The esters
are easily
isolated by organic solvent extraction and partitioning. Moreover, the adducts
obviate the
need for pH adjustment in the aqueous fermentation media to prevent the
accumulation of
the high levels of acidic biotransformation products.
There are several biochemically distinct means by which AHAs can be
biocatalytically esterfied in a substantially aqueous environment. In one
preferred
embodiment of this invention, expression of genes encoding an S-
adenosylmethionine
(SAM)-dependent O-methyltransferase is used to effect conversion of AHAs to
their methyl
esters (e.g., Fig. 13, conversion of compound X to compound XI). SAM-dependent
methyltransferases of differing substrate specificity are common in nature,
and suitable
enzymes and corresponding genes can be found and used directly for the purpose
of this
invention. Alternatively, these species can be further evolved and optimized
for specific
activity with the AHAs using one or more nucleic acid shuffling methods
described herein.
The invention also provides means for HTP screening for the presence, and
quantitative
determination, of the AHA-specific O-methyltransferase catalytic activities in
microorganisms, cells, tissues or extracts of tissues of higher eukaryotic
organisms. These
methods can be used either to identify sources of corresponding genes or to
evolve the
desired specificity of known methyltransferases towards the AHAs by nucleic
acid shuffling
as described herein.
In another embodiment acyltransferase enzymes which specifically esterify
the sec-hydroxyl of AHAs by means of active carboxyl transfer from either acyl-
coenzyme
A or acylated acyl carrier protein (ACP) are incorporated into the reaction
pathway. This
pathway is depicted in Fig 13, as shown by the coupling of compounds X and XII
to yield
compound XIII. A preferred embodiment of this pathway, involves recruiting and
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expressing genes) encoding acyl-CoA-dependent acyltransferases, including
those which
utilize as substrates acetyl-CoA and CoA derivatives of fatty acids, as well
as lactoyl-CoA,
CoA-thioesters with other AHAs, and CoA derivatives of aromatic, arylalkanoic,
branched
chain alkanoic carboxylic acids, and alpha-aminoacids. Where carboxylic acids
(either in
the form of a free acid, salt or ester), intended for esterification of AHAs,
are supplied
exogenously, or are co-produced by another co-functioning biotransformation or
fermentative pathway in the same host organism, or a different host organism,
the invention
provides a means for facilitating ester formation by recruiting and co-
expressing those acyl-
CoA ligases or ACPs which effect in-vivo activation of these acids forming
suitable
substrates for the acyl transferase enzymes that act on the AHAs.
The invention also provides for another type of biochemical transformation
of AHAs to AHA carboxylic esters wherein free AHAs are first converted to
their active
ester form by means of the enzymatic formation of a derivative with CoA or ACP
(Fig. 13,
compound XIV). Several alternative acyltransferase enzymes (and genes encoding
them)
can be recruited for effecting subsequent transformations of compound XIV to
esters of
different compositions. These preferably include AHA-CoA transferases acting
(a) on
alcohols (XX) to produce esters, or (b) on molecule of compound XIV or
compound XV to
produce acyclic homo- and hetero- oligomers (n=2-5) of AHAs. By recruiting an
additional
thioesterase enzymes, the activated forms of these oligomeric esters can be
converted to free
carboxylic oligomers (e.g., XVIII) or to the cyclic substituted glycolides
(XVI).
In another preferred embodiment, the formation of an a-hydroxycarboxylic
acid ester is catalyzed by an acyl CoA-ligase that is evolved by nucleic acid
shuffling. In a
preferred embodiment, shuffling of nucleic acids encoding acyl-CoA ligase
activities results
in an increase in the synthesis of esters. In another preferred embodiment,
the esters are
selected from structures XIII-XVIII (Fig. 13). The synthesis of these and
other esters will
generally rely on the provision of a corresponding a-hydroxycarboxylic acid
precursor. In a
preferred embodiment, the a-hydroxycarboxylic acid precursor is present in an
amount
sufficient to establish intracellular pools of CoA-activated carboxylic
derivatives of a-
hydroxycarboxylic acids.
In still another preferred embodiment, the transferase polypeptide is selected
from glycosyltransferase and methyltransferase, more preferably
methyltransferase and
more preferably still a S-adenosylmethionine dependent O-methyltransferase.
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5. Enzymes effecting chiral switch at the level of AHAs.
Another object of this invention is the effective control of the enantiomeric
composition of the compounds prepared by the methods of the invention. For
clarity of
illustration, the discussion below focuses on AHA esters made by the
biotransformation
process from alkenes. This focus is intended to be illustrative and not
limiting of the scope
of this embodiment of the invention.
Means of enantiomeric control, when integrated as part of the multistep
biocatalytic pathway, constitutes an important advantage as it allows
selective production of
either enantiomer of the AHA. The enantiomerically pure AHAs can be used as
resolving
reagents, chiral synthons, or monomers for polyesters or co-polyesters with
lactic acid.
In a preferred embodiment, the AHA is mandelic acid, or an analogue
thereof, and the chiral switch is effected by recruiting mandelate a racemase
gene.
Mandelate reacemase catalyzes the interconversion of the R and S
enantiomers of mandelic acid and its derivatives. An exemplary mandelate
racemase is that
of PseudomonaS putida (the sequence of the gene can be found in the GenBank
database
under the locus [PSEMDLABC]). Preferred mandelate racemases are those of the
P. putida
strain ATCC 12633, however, mandelate racemases from any other organism can be
used.
Although, in a preferred embodiment, the chiral switch is made at the level
of the AHA, this switch can be made with any of the precursors or adducts of
the AHA as
well. Thus, in yet another preferred embodiment, the AHA is modified by at
least one of
the ester-forming enzymes discussed herein. Preferred ester forming enzymes
are those
which specifically, or preferentially, act on one enantiomer of the AHA, thus
allowing
enantiospecific resolution of the racemate in-vivo. The activity of the above
racemases
provides an enantiomeric equilibrium at the expense of the non-esterified
enantiomer. The
combined action of the racemase and the AHA esterifying enzymes provides a
chiral switch
which allows preparation of one desired enantiomer, whether R or S, from AHAs
of any
enantiomeric composition.
E. Antioxidant and Impurity Modification and Detoxification
In another embodiment, the invention provides methods of degrading or
modifying organic materials which lead to their detoxification. Exemplary
compounds
include stabilizing agents, antioxidizing agents, environmental pollutants and
the like. This
method is applicable to substantially any compound that can be detoxified by,
for example,
oxidation, either with or without additional structural elaboration. For
clarity of illustration,
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the discussion below focuses on the detoxification of agents commonly found in
organic
solvents and in ~t-bonded compounds of use in the present invention.
Many commercially available compounds (e.g., alkylbenzenes, alkenes, etc.)
are stabilized with small amounts of antioxidants such as 4-tert-butylcatechol
or
alkylphenols (e.g. BHT) to prevent polymerization during storage and
transportation. While
the amount of these compounds is usually relatively small (10-15 ppm), they
can inhibit
biocatalyst performance as they accumulate in aqueous fermentation medium
during
prolonged incubations required to obtain satisfactory endproduct
concentrations.
Several types of enzymes for modifying the phenolic stabilizing compounds
can be used to alleviate any negative effects of these compounds on the whole
cell
biocatalyst performance. Their genes can be introduced in the same host
organism used to
produce endproducts or intermediates of relevance to this invention.
Alternatively, they can
be incorporated into a separate host organism. This obviates any need for
additional steps
in the process to remove these stabilizers. Optimization of one or several of
these enzymes
for the efficient removal of these stabilizing compounds is a target for
nucleic acid
shuffling.
Exemplary enzymes for modifying phenolic and diphenolic stabilizers
include, but not limited to, acyltransferase, methyltransferase,
glycosyltransferase, lactase
and peroxidase. In addition to these enzymes, catecholic stabilizers also can
be modified to
innocuous products by catechol dioxygenases effecting meta- or ortho-ring
cleavage. Many
of these enzymes show a significant breadth of activity towards compounds
related to
phenolic stabilizers. Thus, nucleic acid shuffling can be applied to optimize
enzyme
parameters such as:
a) increased turnover with particular phenolic stabilizer,
b) increased functional expression, by obviating the requirements for certain
post-transitional modifications of those enzymes which require such
modifications (e.g.
glycosylation of peroxidases and lactases); and
c) alleviation of inhibition of these enzymes by high concentration of co-
occurnng feedstock compounds and intermediates and endproducts of the
biocatalytic
process.
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F. Analytical Methodology
A number of analytical techniques are useful in practicing the present
invention. These analytical techniques are used to measure the extent of
conversion of a
particular substrate to product. These techniques are also used to analyze the
regioselectivity and/or the enantiomeric selectivity of a particular reaction
catalyzed by a
polypeptide of the invention. Moreover, these techniques are employed to
assess the effect
of nucleic acid shuffling experiments on the efficiency and selectivity of the
polypeptides
produced following the shuffling. The discussion below focuses on those
aspects and
embodiments of the invention in which an olefin precursor is oxidized by a
dioxygenase.
The analytical techniques discussed in the this context are generally of broad
applicability to
other aspects and embodiments of the invention. This is particularly true of
the
spectroscopic and chromatographic methods discussed below. Thus, in the
interest of
brevity, the following discussion focuses on analyzing the products of the
oxidation of an
olefin, but the utility of the methods discussed is not limited to this
embodiment.
The generation and screening of high quality shuffled libraries provides for
nucleic acid shuffling (or "directed evolution"). The availability of
appropriate high-
throughput analytical chemistry to screen the libraries permits integrated
high-throughput
shuffling and screening of the libraries to achieve a desired dioxygenase
activity.
Moreover, this discussion is also generally applicable to those dioxygenases
that act as
monooxygenases.
Selecting for Dioxygenase activity
Dioxygenase activity can be monitored by HPLC, chiral HPLC, gas
chromatography, NMR spectrometry, and mass spectrometry, as well as a variety
of other
analytical methods available to one of skill. Incorporation of 1g0 from
radiolabeled
molecular oxygen can be monitored directly by mass shift by MS methods and by
an
appropriate radioisotope detector with HPLC and GC devices. For example,
oxidation of 1-
hexadecene to 1,2-hexadecanediol can be monitored by 180 incorporation either
in intact
whole cells or lysate. This has been used, for example by Bruyn et al. with
Candida
lipolytica.
In addition, epoxide formation can be indirectly measured by various
reactive colorimetric reactions. When H202 is used as the oxidant,
disappearance of
peroxide over time can be monitored directly either potentiometrically or
colorimetrically
using a number of commercially available peroxide reactive dyes.
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In a high-throughput modality, a preferred method is high-throughput MS, or
MS operating in a coordination ion spray and/or electrospray-based mode. In
addition,
selection protocols in which the organism uses a given alkene or aromatic
system as a sole
carbon source can be used. In some systems this will be most readily
accomplished by
using the dioxygenase to generate a metabolizable diol.
2. Automation for Strain Improvement
One key to strain improvement is having an assay that can be dependably
used to identify a few mutants out of thousands that have potentially subtle
increases in
product yield. The limiting factor in many assay formats is the uniformity of
library cell (or
viral) growth. This variation is the source of baseline variability in
subsequent assays.
Inoculum size and culture environment (temperature/humidity) are sources of
cell growth
variation. Automation of all aspects of establishing initial cultures and
state-of-the-art
temperature and humidity controlled incubators are useful in reducing
variability.
In one aspect, library members, e.g., cells, viral plaques, spores or the
like,
are separated on solid media to produce individual colonies (or plaques).
Using an
automated colony picker (e.g., the Q-bot, Genetix, U.K.), colonies are
identified, picked,
and 10,000 different mutants inoculated into 96 well microtitre dishes
containing two 3 mm
glass balls/well. The Q-bot does not pick an entire colony but rather inserts
a pin through
the center of the colony and exits with a small sampling of cells, (or
mycelia) and spores (or
viruses in plaque applications). The time the pin is in the colony, the number
of dips to
inoculate the culture medium, and the time the pin is in that medium each
effect inoculum
size, and each can be controlled and optimized. The uniform process of the Q-
bot decreases
human handling error and increases the rate of establishing cultures (roughly
10,000/4
hours). These cultures are then shaken in a temperature and humidity
controlled incubator.
Glass or, preferably, stainless steel balls in the microtiter plates act to
promote uniform
aeration of cells and the dispersal of mycelial fragments similar to the
blades of a fermenter.
a. Prescreen
The ability to detect a subtle increase in the performance of a shuffled
library
member over that of a parent strain relies on the sensitivity of the assay.
The chance of
finding the organisms having an improvement is increased by the number of
individual
mutants that can be screened by the assay. To increase the chances of
identifying a pool of
sufficient size, a prescreen that increases the number of mutants processed by
10-fold can be
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used. The goal of the primary screen will be to quickly identify mutants
having equal or
better product titres than the parent strains) and to move only these mutants
forward to
liquid cell culture for subsequent analysis.
For the purpose of preparing a shuffled ADO library screening and sorting
out non-functional variants, several general activity detection methods for
ADOs can be
used, including cases for direct screening and colony picking on agar medium
plates.
Certain presently preferred examples of general methods are:
(a) the formation of indigo from indole, and similarly, from substituted
indoles and indole-carboxylic acids (to produce indigo or substituted indigo).
The
development of blue or blue-grey hued growing colonies signifies the
expression of
catalytically functional ADOs. Most of the ADOs exhibit some activity with
either indole
(e.g. toluene dioxygenase, biphenyl dioxygenase, naphthalene dioxygenase and
homologous
enzymes), or with indole carboxylates (e.g. toluate-1,2-dioxygenase, p-cymate
2,3-
dioxygenase);
(b) the detection of catechol formation, which can be enhanced in the
presence of an aromatic amine (e.g. p-toluidine) and iron salts. Many
catechols oxidize
readily under oxygen, or in the presence of other oxidants, forming various
colored products
in the media surrounding the colonies expressing catalytically active ADOs.
This assay method is applicable for cases where a cis-diol is unstable
(angular dihydroxylation products at aromatic ~-bonds substituted with
heteroatoms such
as N, O, S, and halogens) and rearomatized spontaneously with concomitant
elimination of
a leaving substituent. The leaving substituent (e.g. halide, nitrite or
ammonia) can also be
detected by a variety of qualitative assays known in the art, or by detecting
changes in pH of
surrounding medium (pH indicator).
The assay can also be used in cases of stable dihydrodiol formation, where a
suitable gene of arene cis-diol dehydrogenase is co-expressed (in many cases
such genes are
indeed available and well known in the art). In this case, the accumulated
catechol can be
detected by the presence of colored oxidation products, whether enhanced by p-
toluidine/Fe
or not; and
(c) color formation due to enzyme activities encoded by accessory genes for
subsequent metabolism of arene cis-dihydrodiols. In this case, a yellow (or
orange) color is
developed by colonies in the medium which signifies the expression of
catalytically active
ADO.
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A combination of the methods can be used. The advantage of the
indole/indigo assay is that the color does not diffuse into areas of medium
surrounding the
colonies. The advantage of other assays is that they often can provide
information about
both the general activity and also about the regioselectivity of the reaction
catalyzed by
ADO.
Positive clones selected by either of the above-described exemplary methods
can be examined by subsequent tier assay.
3. Screening for improved dioxygenase activity.
In each of the aspects and embodiments discussed herein, the concept of
screening the library of recombinant polypeptides to enable the selection of
improved
members of the library is set forth. Although it will be apparent to those of
skill in the art
that many screening methodologies can be used in conjunction with the present
invention,
the invention provides a screening process comprising:
(a) introducing the library of recombinant polynucleotides into a
population of test microorganisms such that the recombinant polynucleotides
are expressed;
(b) placing the organisms in a medium comprising at least one
substrate; and
(c) and identifying those organisms exhibiting an improved property
compared to microorganisms without the recombinant polynucleotide.
a. Oxidation of olefins
Depending on the specific outcome desired from a particular course of
shuffling of nucleic acids encoding oxygenases for biocatalytic oxidation of
olefins, the
invention provides several methods for detecting and measuring catalytic
properties
encoded by the recombinant polynucleotides. These are exemplified by the
following
methods.
Optimizing individual reactions and whole pathways for producing oxidized
compounds, their derivatives, analogues and precursor compounds described in
this
invention can be monitored by virtually any analytic technique known in the
art. In
preferred embodiments, the production of the desired compound is monitored
using one or
more techniques selected from thin layer chromatography (TLC), high
performance liquid
chromatography (HPLC), chiral HPLC, mass-spectrometry, mass spectrometry
coupled
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with a chromatographic separation modality, NMR spectroscopy, radioactivity
detection
from a radioactively labeled compounds (e.g., olefins, diols, carboxylic
acids, aldehydes,
AHAs, etc.), scintillation proximity assays, and by UV-spectroscopy. In a high
throughput
modality, the preferred methods are selected from one or any combination of
these methods.
(i). Methods for measuring glycol formation from ~ bonded species
The methods of the invention are used to improve polypeptides that catalyze
the initial oxidation of ~-bonded species. Methods using dioxygenase-based
pathways are
encompassed herein. The oxidation product from the conversion of a substrate
comprising
a ~t-bond (e.g., arenes, alkylarenes, alkenes, etc.) can be detected by
numerous methods well
known to those of skill in the art. Certain preferred methods are set forth
herein.
In a preferred embodiment, the vicinal diol derived from oxidation of an
olefin is quantitated using a radioactively labeled substrate. Although any
radioactive
isotope commonly used in the art can be incorporated into a substrate,
preferred isotopic
labels include, for example, 14C and/or 3H. Differences in the volatility of
the olefin
substrate and the corresponding diol can be exploited to quantitate the
radioactively labeled
product. This method can easily be applied to aqueous samples of culture
fluids obtained
by incubating individual clones of cells expressing libraries of a recombinant
polynucleotide
obtained using the methods of the invention.
In an exemplary embodiment, cells expressing libraries of recombinant
polynucleotides encoding a dioxygenase can be grown in a multiwell dish with a
radioactive
substrate administered directly to the aqueous medium. After incubation of the
cells with
the radioactive olefin substrate, any residual uncoverted substrate is removed
by
evaporation, with or without application of vacuum. After removing the
unconverted
substrate, the culture fluid (or aliquots thereof) is mixed with a suitable
scintillation
cocktail, and the radioactivity in the samples is quantitatively measured. In
a preferred
embodiment, selection of the most active clones is based on the amount of
radioactivity
incorporated into the compounds produced by the organisms expressing the
clone.
Alternatively, radioactively labeled substrate can be administered as a vapor
phase to colonies growing on a surface of a membrane filter overlaying agar-
solidified
medium. After incubation, the membrane is removed from the agar surface, and
any
residual hydrocarbon is evaporated from the membrane. The membrane is
autoradiographed, or a scintillation dye is sprayed over the membrane for
radioactivity
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detection. A modification of this assay that is particularly suitable for 14C
label detection in
and/or around colonies capable of oxidizing ~-bonds to the corresponding
glycols involves
using a porous membrane that has scintillation dye incorporated in the
membrane
composition by covalent or adsorption means. This assay is termed
"scintillation proximity
assay on membrane" or "SPA."
In another embodiment of this invention, a variation of SPA is used to
selectively quantify the glycol derived from the substrate. This variation
involves adding
beads for scintillation proximity assay to the samples of culture fluids or
extracts obtained
by incubation of cells with radiolabeled substrate as described above.
Alternatively, the
sample can be applied to a membrane. The beads or membrane are functionalized
with
groups that interact with a glycol.
In a preferred embodiment of this assay, the beads or membranes contain a
suitable scintillating dye and their surfaces are modified by chemical groups
that interact
readily with diols. Such materials can be prepared by known chemical methods
from
commercially available SPA materials and they can be used to trap free diols
directly in the
aqueous medium or culture broths obtained by incubation of the microbial cells
with the
radiolabeled substrates.
In another preferred embodiment, the surface of the beads used in this assay
is functionalized with a sufficient amount of a compound that interacts with a
glycol, such
as compounds containing aryl or alkylboronate (boronic acid). Such beads can
be obtained
by chemical modification of commercially available SPA beads by reactions
known to one
skilled in the art. In a preferred embodiment, the reactions used to modify
the beads are
analogous to those used for the preparation of arylboronate-modified resins
for solid-phase
extraction or chromatography. After incubation, the beads are washed with a
sufficient
amount of water or other suitable solvent and subjected to quantitative
determination of
radioactivity.
One can also determine amounts of glycol produced by oxidation of an ~-
bond by taking advantage of the reactive nature of the substrate. Samples of
culture fluids,
or extracts in an appropriate solvent, can be treated with known excess
amounts of dilute
solutions of, for example, a halogen (C12, Br2, I2), permanganate salts. The
residual excess
amount of those reagents, left after reaction with any substrate present, can
be measured by
chemical methods known in the art for determination of these compounds (see,
for example,
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VOGEL'S PRACTICAL ORGANIC CHEMISTRY 5'h Ed., Furniss et al., Eds., Longman
Scientific
and Technical, Essex, 1989).
Mass spectrometry can also be used to determine the amount of a vicinal
glycol formed due to species encoded by the libraries of shuffled oxygenase
genes. Mass
spectrometric methods allow ion peaks to be detected. The ion peaks derived
from the
vicinal glycol can be readily distinguished from peaks derived from olefin
substrates. In a
preferred embodiment, coordination ion spray or electrospray mass spectrometry
is utilized.
In another preferred embodiment, a compound that interacts with a
component of the mixture, preferably the glycol, is utilized to enhance the
sensitivity and
selectivity of the method. In a presently preferred embodiment, the sample
analyzed
contains excess arylboronic or alkylboronic acid. Preferred boronic acids are
those
containing at least one nitrogen atom and include, but are not limited to,
dansylaminophenylboronic acid, aminophenylboronic acid, pyridylboronic acid.
The ions detected in the mass spectrum derive from cyclic boronate ester
derivatives of the glycols with a boronic acid. The samples are preferably
analyzed in non-
acidic and non-basic organic solvent or aqueous phase, substantially free of
alcohols and
other glycols. Other appropriate analytical conditions will be apparent to
those of skill in
the art.
Another preferred method for quantitating the glycols uses periodic acid or
its salts, preferably the sodium salts, to cleave the vicinal glycols to the
corresponding
aldehydes. In a preferred embodiment, vicinal diols other than the analyte
(e.g.,
carbohydrates) are excluded from the aqueous or organic solvent samples. This
is easily
attained by using non-carbohydrate carbon sources to grow the microbial cells,
and/or by
removal of the cells from the media by centrifugation or filtration prior to
contacting of the
sample with periodate reagent. The periodate reagent can be used in solution,
or preferably,
immobilized on a solid phase (e.g. anion exchange resin). After reacting the
glycol with an
excess of periodate ion, the amount of free aldehyde groups can be measured by
a variety of
assays know in the art. In a preferred method, the aldehydes are quantitated
by a method
based on the formation of a colored hydrazone derivative. Alternatively, when
using
radioactively labeled olefins for biotransformation, the free aldehydes
obtained by this
method can be trapped by aldehyde reactive groups (e.g., free amines) on the
surface of an
appropriately modified SPA beads or membranes.
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(ii). Methods for detecting alternative regioselectivity of oxidation of
species
with multiple ~c-bonds
In one embodiment, the substrate includes more than one ~-bond (e.g.,
styrene, butadiene, etc.). In a preferred embodiment, one of the ~-bonds
undergoes reaction
more readily than the other. In this embodiment, it is generally preferred to
determine
which of the ~-bonds underwent reaction. The preferred method for making this
determination is'H or'3C NMR, although other methods can be used. Other
methods
include, for example, chromatography (e.g., TLC, GC, HPLC, etc.), UV/vis
spectroscopy
and IR spectroscopy. In an embodiment wherein the reaction is operating in a
high
throughput mode, the method of choice is flow-through'H or'3C NMR
spectroscopy.
When'3C NMR is used, the substrates are preferably labeled with'3C. ~-
bonded species can be synthesized by methods know in the art. from a'3C
enriched material
to incorporate one, or any combination of several, labeled carbon atoms) into
the structure
of these compounds (by synthetic methods known in the art as exemplified by
Selifonov et
al, in Appl Environ Microbiol. 64(4):1447-53 (1998)). The enrichment levels
for the
labeled positions are preferably at least 5% of'3C, more preferably 50% and
more
preferably still 95% for any given labeled position. Incorporation of a'3C
label provides a
number of advantages, such as increasing the NMR signal and decreasing time
required for
spectral acquisition. Moreover, labeled compounds allow for a quantitative or
semi-
quantitative interpretation of the composition of a mixture of isomeric
oxidation products.
Preferably, incubations with'3C labeled olefins are conducted in multi-well
plates, and
aliquots of culture fluids or their extracts are sampled with an autosampler
communicating
with the NMR probe. In another preferred embodiment, the reaction components
are not
chromatographed or otherwise purified prior to obtaining a NMR spectrum.
Determining the absolute configuration and the enantiomeric composition of
the glycols formed from ~-bonded species, preferably employs a variation of
the method
described above for determining regioselectivity of dihydroxylation of the
olefinic
substrates by a dioxygenase using'H or'3C NMR. In a preferred embodiment, the
substrates are labeled with'3C and'3C NMR, is employed. This method preferably
involves the use of a chiral and essentially enantiomerically pure
derivatizing reagent such
as a substituted arylboronic acid which forms a cyclic boronate derivatives
with vicinal
glycols, as know in the art (Burgess and Porte, Angew. Chem Intl Ed. Engl.
33(41):1182-4
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(1994)). In a preferred embodiment, both the substrates and one or more carbon
atoms of
the boronic acid is labeled with'3C. Although a broad range of boronic acids
are of use in
the present invention, a currently preferred boronic acid is shown below:
CH30 ~3CH30H
B~OH
The absolute configuration of any chiral center of the compounds produced
by the methods of the invention can be either R or S. In presently preferred
embodiments,
the enantiomeric excess of the product is preferably 98% or more. NMR signals
of different
enantiomers of the reaction products can be distinguished in diastereomeric
products using
substantially enantiomerically pure boronate compounds as discussed above.
Moreover, the
relative intensity of the NMR signals arising from corresponding atoms of the
diastereomeric products can be used for estimating the enantiomeric
composition of the
products) present in the sample.
(iii). Methods for detecting alternative regioselectivity of oxidation of
alkylarenes
Useful methods for determining the regioselectivity of the oxidation of
alkylarene compounds are substantially similar to those described in section
(ii), supra.
4. AHA formation from glycols
Among methods for specifically measuring the free AHAs produced in the
biocatalytic process, those which are particularly preferred are methods using
a variation of
the scintillation proximity assay described above. These methods preferably
use an excess
of beads or membranes bearing one or more positively charged functional groups
(e.g
quaternary or tertiary or primary amines). In preferred embodiments, these
beads or
membranes act as an anion exchange medium and they selectively trap free AHAs,
thereby
removing them from aqueous culture broths. In another preferred embodiment,
this method
employs a radioactively labeled starting material, or subsequent intermediate,
(e.g., glycol,
epoxide, etc.). The radioactively labeled compound interacts with the beads or
membrane.
Prior to measuring the radioactivity associated with the beads or the
membrane, non-
specifically adsorbed label is preferably removed by evaporating excess
radioactive
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compound and/or washing with an aqueous solution which does not cause elution
of the
AHAs from the anion-exchange beads or membrane.
Preferred methods for determining the chirality and absolute configuration of
AHAs formed in the described biotransformation process are substantially
similar to those
methods employed in making these determinations with respect to the glycols,
as discussed
above.
5. Methods for determination of HCAs
In HTP mode, a preferred analytical method is flow-through'H or'3C NMR
spectroscopy. In the'3C NMR mode, the aromatic substrate for oxidation by a
dioxygenase
is preferably labeled by the'3C isotope. Alkylaryl compounds or the
corresponding
arylalkanoic acids are synthesized by methods known in the art from a'3C
enriched material
to incorporate one, or any combination of several, labeled carbon atoms) into
the structure
of these compounds. The enrichment levels for any labeled position are
preferably at least
5% of'3C, and more preferably at least 95%. Incorporation of'3C label
increases
sensitivity of the NMR measurement, decreases time required for acquisition of
spectrum
per sample, and allows for quantitative or semi-quantitative interpretation of
compositions
of mixtures of isomeric oxidation products. Preferably, incubations with'3C
labeled
precursors are conducted in mufti-well plates, and aliquots of culture fluids
or their extracts
are sampled with autosampler connected to the solvent line passing through NMR
probe
without any column separation.
For determining absolute configuration and enantiomeric composition of the
HCAs, a variation of the methods described above for determining reaction
regioselectivity
by'H or'3C NMR is used. In conjunction with the preferred use of'3C labeled
substrates,
'3C NMR is preferably employed.
The absolute configuration of any chiral center may be either R or S. In a
preferred embodiment, the enantiomeric excess is 98% or more. NMR signals of
different
enantiomers of HCAs can be distinguished in diastereomeric products using
known
methods, such as NMR in conjunction with lanthanide shift reagents.
In another preferred embodiment, a variation of the SPA method is used. In
this version, a solid support, such as beads or a membrane containing a
suitable scintillation
dye is used. The solid support is modified with positively charged groups such
that it acts
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like an anion-exchange material. These materials can be prepared from
commercially
available SPA materials and they can be used to trap free acids directly in
the aqueous
medium or culture broths obtained by incubation of the host cells with a
radiolabeled
alkylarene.
6. Methods for determination of esters
In the interest of brevity, the following discussion focuses on the
determination of esters of AHAs. One of skill will appreciate that the same,
or similar,
methods can be used to determine esters of other compounds formed using the
methods of
the invention.
Both spectroscopic and non-spectroscopic methods can be used to quantitate
the extent of ester synthesis and to characterize the esters. The preferred
non-spectroscopic
method for assaying AHA methyl ester formation catalyzed by methyl
transferases is based
on use of a radioactively labeled precursors to AHA methyl esters. 14C or 3H
methyl labeled
SAM (or its in-vivo precursor, methionine) can be used as a probe. In another
preferred
embodiment, the labeled substrate is the free a-hydroxycarboxylic acid itself.
Using the methods of the invention, methyltransferases that are selective for
a particular AHA enantiomer can be selected and further improved by iterative
cycles of
shuffling and this assay. The selectivity of the methyltransferases of the
invention towards
a particular enantiomeric configuration of an AHA is preferably measured using
samples of
the a-hydroxycarboxylic acids that are substantially enantiomerically pure.
Host cells
employed in this biocatalytic cycle will preferably lack AHA racemase activity
(e.g.
mandelate racemase). In another preferred embodiment, both AHA enantiomers
have a
different radioactive label, e.g. one enantiomer is labeled with 14C, and
another with 3H (at
one or more H positions which do not readily exchange with water). Measurement
of the
radioactivity incorporated into the product is performed using a radioactivity
detector that
allows for the selective measurement of at least two different isotopes. This
variation
allows the evaluation of the enantioselectivity of a methyltransferases in a
single sample.
The radioactivity associated with methyl esters of AHAs is preferably
measured in samples which are obtained by selective extraction or partitioning
of the methyl
esters from neutral or moderately basic (pH about 6-10) aqueous culture
samples. These
samples can contain varying amounts of free, labeled AHA, of AHA salts and
other non-
labeled organic compounds. The samples are preferably obtained by incubating
individual
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clones expressing methyltransferase libraries with the labeled AHAs. The
incubation
medium is subsequently extracted by adding a defined amount of a preferably
water-
immiscible organic solvent, or by contacting the broth with an extraction
medium (e.g.
XAD-1180, or similar beads, or membrane).
In those embodiments employing an extraction medium, following its
removal from contact with the broth, the extraction media is preferably washed
to remove
adventitiously bound compounds. Preferred wash solutions are aqueous solutions
that do
not elute the AHA methyl esters from the extraction medium, but which remove
other
molecules adsorbed onto the medium. The radioactivity of the extracted
material is then
measured by methods well known in the, art. In embodiments using beads or a
membrane
an appropriate scintillating dye is preferably used for detecting the
radioactivity.
Substantially similar methods can also be employed for detecting other
neutral esters of AHAs, such as those exemplified by glycolides (e.g., XVI,
Fig. 13) and
esters of type XX. Thus the same approach is useful for assaying and
characterizing the
ester forming activity of polypeptides represented by libraries of acyl-
transferases, or by a
combination of AHA-CoA: alcohol acyltransferases and AHA-CoA ligases.
Variations on
this method can include the use of a radioactively labeled alcohol (e.g., XIX)
or any of its
in-vivo metabolic precursor.
In another preferred embodiment, the method for detecting polypeptide
activity leading to the formation of neutral AHA esters employs UV or
fluorescence
spectroscopy. This method is applicable to those embodiments in which the
transferase
activity yields products exhibiting distinct UV and/or fluorescent
characteristics.
Exemplary compounds include, for example, substituted or non-substituted
esters of
aromatic carboxylic acids (e.g., mandelic acid). In preferred embodiments of
this method, a
solvent or solid-phase extraction under neutral or moderately basic conditions
(pH about 6-
12) is performed on the cell culture medium. Compounds thus isolated are
detected by
measurement of their UV absorption or fluorescence. These spectral parameters
are
evaluated to determine relative amounts and identities of the products formed
by the
transferase reactions.
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a. Screenin fg o~proved transferase activity
The screening of the transferase libraries, obtained by nucleic acid shuffling
or other methods as described above, is done most easily in bacterial or yeast
systems by
one or more of the screening methods described below.
(i). Methods for detecting increased activity of transferase reactions
The methods for detection of increased formation of monoacyl- and
monoglycosyl-derivatives of, for example, glycols and a-hydroxycarboxylic
acids include
methods in which physical differences between the substrates, the cis-diols
and the
derivatives arising from the transferase-catalyzed reactions are measured.
Preferred
methods include HPLC and mass-spectrometry. In a high throughput modality, a
method of
choice is mass-spectrometry, preferably, coordination ion and/or electrospray
mass-
spectrometry.
For acyl transferases, another presently preferred method uses a labeled acyl-
donor precursor, e.g. labeled carboxylic acid or its derivative, administered
to the cells that
express libraries of shuffled genes encoding acyl ligases and/or acyl
transferases, e.g., acyl-
CoA ligases and acyl-CoA transferases. The amount of label in the hydrophobic
reaction
products is measured after extraction of the labeled derivatives into a
suitable organic
solvent, or after solid-phase extraction of these compounds by addition of a
sufficient
amount of hydrophobic porous resin beads (e.g., XAD 1180, XAD-2, -4, -8). In
the case of
a radiolabeled compound, scintillating dye can be present in the organic
solvent, added to
the samples, or chemically incorporated in the bead polymer. The latter
constitutes a
modification of scintillation proximity assay method.
(ii) Methods for detecting regioselectivity of transferase reactions.
The methods for detecting regioselectivity of the transferase reactions
include HPLC, and in an HTP modality, flow-through NMR spectroscopy. When NMR
spectroscopy is used for determining relative amounts of different regiomeric
monoacyl or
monoglycosyl derivatives of oxidized substrates, the latter are preferably
obtained by action
of the arene dioxygenases on isotopically (13C and/or 2H) labeled substrate.
Another
variation of the NMR technique includes use of isotopically labeled precursors
of acyl- or
glycosyl- donor intermediates.
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7. Selecting for enhanced organic solvent resistance.
Selection for recombinant polynucleotides that provide improved organic
solvent resistance can be accomplished by introducing a library of recombinant
polynucleotides into a population of microorganism cells and subjecting the
population to a
medium that contains various concentrations of the organic hydrophobic
compounds of
interest. The medium can contain, for example, carbon, nitrogen and minerals,
and
preferably does not otherwise limit growth and viability of the cells in the
absence of the
solvent, thus ensuring that solvent resistance is essentially the only
limiting factor affecting
growth of the cells expressing variants of the genes encoding solvent
resistance traits.
In other embodiments, one can employ a screening strategy to identify those
recombinant polynucleotides that encode polypeptides that confer improved
solvent
resistance. For example, one can screen based on the in vivo expression of a
reporter gene,
such as those encoding fluorescent proteins (exemplified by the green
fluorescent protein,
GFP). Preferably, for the purpose of detecting the best solvent resistant
genes under
essentially stationary growth phase conditions, those reporter genes are used
which display
their function in a fashion dependent on availability of intracellular
reducing pools, such as
NADH and NADPH, and essentially unimpaired ribosomal biosynthesis of proteins.
Such genes and can be exemplified by several bacterial luciferase gene
clusters (lux) which contain not only luciferase components, but also all
polypeptides
required for in-vivo regeneration of the aldehyde substrate for luciferase.
A variety of methods can be used to detect and to pick or to enrich for the
clones with the most efficient solvent resistant traits as judged by display
of the properties
associated with the in-vivo reporter genes. These methods include, for
example,
fluorescence activating cell sorting of liquid cell suspensions (e.g., cells
that express GFP)
and CCD camera imaging of individual colonies grown on a solidified) medium
(e.g., for
cells that express lux).
If additional improvement in solvent resistance is desired, one can carry out
a
series of cycles of iterative nucleic acid shuffling and selection by growing
the cells in the
presence of the organic solvent. Concentrations of the solvents used for
selective growth
conditions are incrementally increased after each round of recursive mode
nucleic acid
shuffling in order to provide more stringent selective pressure for those
organisms
expressing solvent resistance genes.
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For use in a high throughput screening protocol, the increase in the solvent
resistance to a particular compound of interest and relevance to the
biocatalytic synthesis of
interest can also be directly measured by administering a radioactively
labeled compound
and determining relative distribution of radioactivity between cell biomass
and extracellular
medium components, similar to the method described by Ramos et al., J.
Bacteriol.
180:3323-3329 (1998).
G. Bioreactors
In another aspect, the invention provides a bioreactor system for carrying out
biotransformations using the improved polypeptides of the invention. The
bioreactor
includes: (a) an improved dioxygenase polypeptide of the invention; (b) a
redox partner
source; (c) oxygen; and (d) a substrate for oxidation.
In a preferred embodiment, the dioxygenase polypeptide is an arene
dioxygenase polypeptide.
In another preferred embodiment, the bioreactor further includes another
useful polypeptide, such as a transferase, ligase, dehydrogenase and the like.
The additional
useful polypeptide(s) can be co-expressed by a host cell also expressing the
improved
dioxygenase or expressed by a host cell that does not express the improved
dioxygenase.
Moreover, each of the polypeptides incorporated into the reactor can be
provided as a
constituent of a whole cell preparation, a polypeptide extract or as a
substantially pure
polypeptide. The cells and/or polypeptides are optionally in suspension, in
solution, or
immobilized on an insoluble matrix, bead or other particle. Additional
considerations are
discussed below. This discussion is intended as illustrative and not limiting.
Other
bioreactor formats, conditions, etc. will be apparent to those of skill in the
art.
General growth conditions for culturing the particular organisms are obtained
from depositories and from texts known in the art such as BERGEY~S MANUAL of
SYSTEMATIC BACTERIOLOGY, Vol.l, N. R. Krieg, ed., Williams and Wilkins,
Baltimore/London (1984).
For clarity of illustration, the discussion below focuses on the preferred
conditions for the oxidation of an organic substrate using the polypeptides of
the invention.
It is understood that this focus is for the purpose of illustration and that
similar conditions
are applicable to pathways of the invention other than oxidation.
The nutrient medium for the growth of any oxidizing microorganism should
contain sources of assimilable carbon and nitrogen, as well as mineral salts.
Suitable sources
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of assimilable carbon and nitrogen include, but are not limited to, complex
mixtures, such as
those constituted by biological products of diverse origin, for example soy
bean flour,
cotton seed flour, lentil flour, pea flour, soluble and insoluble vegetable
proteins, corn steep
liquor, yeast extract, peptones and meat extracts. Additional sources of
nitrogen are
ammonium salts and nitrates, such as ammonium chloride, ammonium sulfate,
sodium
nitrate and potassium nitrate. Generally, the nutrient medium should include,
but is not
limited to, the following ions: Mg2+, Na+, K+, Ca2+, NH4+, Cl-, 5042-, P042-
and N03- and
also ions of the trace elements such as Cu, Fe, Mn, Mo, Zn, Co and Ni. The
preferred
source of these ions are mineral salts.
If these salts and trace elements are not present in sufficient amounts in the
complex constituents of the nutrient medium or in the water used it is
appropriate to
supplement the nutrient medium accordingly.
The microorganisms employed in the process of the invention can be in the
form of fermentation broths, whole washed cells, concentrated cell
suspensions, polypeptide
extracts, and immobilized polypeptides and/or cells. Preferably concentrated
cell
suspensions, polypeptide extracts, and whole washed cells are used with the
process of the
invention (S. A. White and G. W. Claus, J. Bacteriology, 150:934-943 (1982)).
Methods of immobilizing polypeptides and cells are well known in the art
and include such techniques as microencapsulation, attachment to alginate
beads, cross-
linked polyurethane, starch particles, polyacrylamide gels and the use of
coacervates, which
are aggregates of colloidal droplets. In a presently preferred embodiment, the
polypeptide
and/or cell is immobilized onto a glass particles having a porous outer
surface, such as that
described in Dubin , et al., U.S. Patent No. 5,922,531, issued July 13, 1999.
Concentrated washed cell suspensions may be prepared as follows: the
microorganisms are cultured in a suitable nutrient solution, harvested (for
example by
centrifuging) and suspended in a smaller volume (in salt or buffer solutions,
such as
physiological sodium chloride solution or aqueous solutions of potassium
phosphate,
sodium acetate, sodium maleate, magnesium sulfate, or simply in tap water,
distilled water
or nutrient solutions). The substrate is then added to a cell suspension of
this type and the
oxidation reaction according to the invention is carried out under the
conditions described.
The conditions for oxidizing a substrate in growing microorganism cultures
or fractionated cell extracts are advantageous for carrying out the process
according to the
invention with concentrated cell suspensions. In particular the temperature
range is from
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about 0 °C to about 45 °C and the pH range is from about 2 to
about 10. There are no special
nutrients necessary in the process of the invention. More importantly, washed
or
immobilized cells can simply be added to a solution of substrate, without any
nutrient
medium present.
It is also possible to carry out the process according to the invention with
polypeptide extracts or polypeptide extract fractions prepared from cells. The
extracts can
be crude extracts, such as obtained by conventional digestion of microorganism
cells.
Methods to break up cells include, but are not limited to, mechanical
disruption, physical
disruption, chemical disruption, and enzymatic disruption. Such means to break
up cells
include ultrasonic treatments, passages through French pressure cells,
grindings with quartz
sand, autolysis, heating, osmotic shock, alkali treatment, detergents, or
repeated freezing
and thawing.
If the process according to the invention is to be carried out with partially
purified polypeptide extract preparations, the methods of protein chemistry,
such as
ultracentrifuging, precipitation reactions, ion exchange chromatography or
adsorption
chromatography, gel filtration or electrophoretic methods, can be employed to
obtain such
preparations. In order to carry out the reaction according to the invention
with fractionated
cell extracts, it may be necessary to add to the assay system additional
reactants such as,
physiological or synthetic electron acceptors, like NAD+, NADP+, methylene
blue,
dichlorophenolindophenol, tetrazolium salts and the like. When these reactants
are used,
they can be employed either in equimolar amounts (concentrations which
correspond to that
of the substrate employed) or in catalytic amounts (concentrations which are
markedly
below the chosen concentration of substrate). If, when using catalytic
amounts, it is to be
ensured that the process according to the invention is carried out
approximately
quantitatively, a system which continuously regenerates the reactant which is
present only
in a catalytic amount must also be added to the reaction mixture. This system
can be, for
example, a polypeptide which ensures reoxidation (in the presence of oxygen or
other
oxidizing agents) of an electron acceptor which is reduced in the course of
the reaction
according to the invention.
If nutrient media is used with intact microorganisms in a growing culture,
nutrient media can be solid, semi-solid or liquid. Aqueous-liquid nutrient
media are
preferably employed when media is used. Suitable media and suitable conditions
for
cultivation include known media and known conditions to which substrate can be
added.
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The substrate to be oxidized in the process of the invention can be added to
the base nutrient medium either on its own or as a mixture with one or more
oxidizable
compounds. Additional oxidizable compounds which can be used include polyols,
such as
sorbitol or glycerol.
If one or more oxidizable compounds are added to the nutrient solution, the
substrate to be oxidized can be added either prior to inoculation or at any
desired subsequent
time (between the early log phase and the late stationary growth phase). In
such a case the
oxidizing organism is preferably pre-cultured with the oxidizable compounds.
The
inoculation of the nutrient media is effected by a variety of methods
including slanted tube
cultures and flask cultures.
Contamination of the reaction solution should be avoided. To avoid
contamination, sterilization of the nutrient media, sterilization of the
reaction vessels and
sterilization of the air required for aeration is preferably undertaken. It is
possible to use,
for example, steam sterilization or dry sterilization for sterilization of the
reaction vessels.
The air and the nutrient media can likewise be sterilized by steam or by
filtration. Heat
sterilization of the reaction solution containing the substrate is also
possible.
The process of the invention can be carried out under aerobic conditions
using shake flasks or aerated and agitated tanks. Preferably, the process is
carried out by
the aerobic submersion procedure in tanks, for example in conventional
fermentors. It is
possible to carry out the process continuously or with batch or fed batch
modes, preferably
the batch mode.
It is advantageous to ensure that the microorganisms are adequately brought
into contact with oxygen and the substrate. This can be effected by several
methods
including shaking, stirring and aerating.
If foam occurs in an undesired amount during the process, chemical foam
control agents, such as liquid fats and oils, oil-in-water emulsions,
paraffins, higher alcohols
(such as octadecanol), silicone oils, polyoxyethylene compounds and
polyoxypropylene
compounds, can be added. Foam can also be suppressed or eliminated with the
aid of
mechanical devices.
H. Example of Dioxygenase Shuffling
The toluene dioxygenase genes todCIC2BA (Zylstra G. J. and D. T. Gibson.
(1989)
J. Biol. Chem. 264: 14940-14946) were used as a substrate for shuffling a
family of genes.
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The length of this four gene operon is 3.591 kb. The tetrachlorobenzene
dioxygenase genes
tecAlA2A3A4 (Biel et al. (1997) Eur. J. Biochem. 247:190-199) were also used
as a
substrate for shuffling a family of genes. The length of this four gene operon
is 3.587 kb.
After shuffling the family of genes, the shuffled sequences were cloned into
the expression
vector, pTrc99a (Pharmacia) and transformed into E. coli cells. The
transformed cells were
plated onto LB (Luria Bertani) agar plates containing ampicillin and incubated
at 37°C for
20 hr. Restriction digests were used to determine that 23 out of the 24
randomly selected
library clones contained sequences that were chimeras of the two parental
sequences.
TIER 1 ASSAY
The library was screened for active clones using a plate assay which detects
the
oxidation of indole to indigo by an active dioxygenase enzyme (Ensley et al.
(1983) Science
222: 167-169). The library was plated onto LB agar plates containing
ampicillin and
incubated at 37°C for 20 hr after which the plates are placed at
23°C and incubated for an
additional 24-48 hr. Clones expressing active dioxygenases produced colored
colonies from
accumulation of indigo. The color ranged from blue to blue-grey and the
intensity of the
color varied from clone to clone depending on the level of enzyme activity.
The library was
determined to be 70% active. The colored colonies were picked using an Q-bot
(Genetix)
into 384-well plates containing LB ampicillin and incubated at 37°C
with shaking for 20 hr.
The library plates were stored after the addition of sterile glycerol (10%
final concentration)
at -20°C until further screening.
TIER II ASSAY
The following high-throughput (HTP) assay was developed and used to identify
shuffled dioxygenases with improved oxidation of p-xylene to p-xylene diol.
The assay is
not limited to the substrate, p-xylene, and can be used for any volatile or
toxic (to the test
organism) substrate.
Library clones are grown to saturation in a 96-well plates) with 250u12xYT
containing
ampicillin and 0.2%glucose. The plates) are inoculated with 3 ul/well
inoculum. The plates) are
incubated at 250 rpm, 37°C, with 85-90% humidity for 20 hr. The
cultures are subcultured into
deep 96-well induction plates) containing 0.5 ml 2xYT containing ampicillin
and 1mM IPTG/well.
The plates) are incubated at 37°C, 250 rpm, with 85-90% humidity, 6 hr.
The induced cells are
harvested by centrifugation at 3000 rpm, 10 min., 10°C. The cells are
washed once with minimal
media and resuspended to a final volume of 0.4 ml. The cells are transferred
to an assay plates)
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containing the volatile or toxic substrate to be tested embedded in solidified
1% agarose on the
bottom of each well of the plate(s). The assay plates) are sealed and
incubated at 23°C for 1 hr
with vigorous shaking. The cell suspension is transferred to a clean
polypropylene 96-well plates)
and the cells pelleted by centrifugation at 3000 rpm, 10 min, at 10°C.
The cell-free supernatants are
transferred to a 96-well plates) and the diol product analyzed by
spectrophotometric methods,
HPLC, or GC-MS. Potential positive clones are those clones with improved
properties when
compared to the best parent.
Using the HTP assay described above, 1080 library clones were screened and
compared to the parental dioxygenases for the ability to oxidize p-xylene to p-
xylene diol.
Nine clones were identified as potential positives and retested to confirm the
improvements.
See table 1 for levels of improvement and Figure 16 to illustrate crossovers
in these nine
clones. The nine clones have 2.2 to 3.7-fold improved activity when compared
to the best
parent. In addition, the two parent sequences and the nine potential positive
clones were
transformed into an alternative E. coli host strain and tested for oxidation
of p-xylene to p-
xylene diol using the H'TP plate assay. The nine clones and the parents were
tested after
growth in two types of media; 2xYT and minimal media with fructose as the
carbon source.
Of the nine clones, six were reconfirmed as 1.7-2.8-fold improved over the
best parent.
_1 -fold ave.
Positivesst im rov. 2nd -fold ave. readstdev im rov.
Screen screen im rov.
Clone 2.99 1.6 3.853.172.5,2.6 3.336667 0.4535782.2+/-.44
1
Clone 3.25 1.7 3.893.072.5,2.6 3.403333 0.4309682.3+/-.40
2
Clone 6.53 3.5 + 5.144.973.4,4.2 5.546667 0.8558233.7+/-.35
3
Clone 3.47 2.6 + 4.154.782.6 4.2 4.133333 0.6551593.1 +/-.75
4
Clone 4.22 2.5 + 3.843.492.5,2.9 3.85 0.3651032.6+/-.18
5
Clone 5.2 3.8 4.583.642.8,3.2 4.473333 0.7854513.3+/-.41
6
Clone 4.14 3.6 + 3.843.012.5,2.6 3.663333 0.5853492.9+/-.49
7
Clone 2.47 1.6 + 4.054.222.5,3.7 3.58 0.9650392.6+/-.86
8
Clone 5.01 3.6 + 4.053.752.5,3.3 4.27 0.6581793.1 +/-.46
9
I. Kits
Also provided is a kit or system utilizing any one of the selection
strategies,
materials, components, methods or substrates herein before described. Kits
will optionally
additionally include instructions for performing methods or assays, packaging
materials,
one or more containers which contain assay, device or system components, or
the like.
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In an additional aspect, the present invention provides kits embodying the
methods and apparatus herein. Kits of the invention optionally include one or
more of the
following: (1) a shuffled component as described herein; (2) instructions for
practicing the
methods described herein, and/or for operating the selection procedure herein;
(3) one or
more dioxygenase assay component; (4) a container for holding dioxygenase
nucleic acids
or polypeptides, other nucleic acids, transgenic plants, animals, cells, or
the like and, (5)
packaging materials.
In a further embodiment, the present invention provides for the use of any
composition or kit herein, for the practice of any method or assay herein,
and/or for the use
of any apparatus or kit to practice any assay or method herein.
In yet another embodiment, the kit of the invention includes one or more
improved dioxygenase polypeptides of the invention. In a preferred embodiment,
the kit
includes a library of improved dioxygenase polypeptides.
While the foregoing invention has been described in some detail for purposes
of clarity and understanding, it will be clear to one skilled in the art from
a reading of this
disclosure that various changes in form and detail can be made without
departing from the
true scope of the invention. For example, all the techniques and apparatus
described above
may be used in various combinations. All publications, patent applications,
patents, and
other documents cited in this application are incorporated by reference in
their entirety for
all purposes to the same extent as if each individual publication or patent
document were
individually so denoted.
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