Note: Descriptions are shown in the official language in which they were submitted.
2 1 73793
PENTOSE FERMENTATION BY RECOMBINANT ZYMOMONAS
FlFLD OF THE INVENTION
This invention relates to recombinant Zymomonas mobilis strains, metabolizing
xylose and arabinose and bearing xylose and arabinose utilization and pentose phosphate
pathway genes, useful for the fermentation of the xylose and arabinose components in
cellulosic biomass to ethanol. This invention also relates to the process of using these
strains for the rapid and efficient fermentation of the xylose and arabinose components in
cellulosic biomass to ethanol.
21 73793
BACKGROUND OF THE TNVENTION
Cellulosic biomass is a favorable feedstock for fuel ethanol production because it is
both readily available and less expensive than either corn or sugarcane. However,
substantial hurdles must be overcome before a typical cellulosic feedstock can be utilized
S effectively as a substrate for the fermentative production of ethanol. The typical feedstock
is comprised of approximately 35-45% cellulose, 30-40% hemicellulose, 15% lignin and
10% of other components. The cellulose fraction is comprised of polymers of the hexose
sugar, glucose. The hemicellulose fraction is comprised mostly of pentose sugars,
including xylose and arabinose.
Whereas microorganisms are known that can efficiently ferment the glucose
component in cellulose, conversion of the xylose and arabinose in the hemicellulose
fraction to ethanol has been difficult and this remains to be one of the economic
bottlenecks in a biomass to ethanol conversion scheme. The rapid and efficient utilization
of the xylose and arabinose components in cellulosic biomass is desirable in thedevelopment of a commercial process.
Zymomonas mobilis is a bacterium that has been utilized as a natural fermentative
agent in the production of alcoholic beverages, such as pulque and palm wines produced
from plant saps. Comparative performance trials have suggested that Zymomonas may
become an important industrial ethanol-producing microorganism because of its 5-10%
higher yield and up to S-fold higher productivity compared to traditional yeast
fermentations. Because of its potential value, several processes based on the use of
21 73793
Zymomonas for the production of industrial ethanol from glucose-based feedstocks have
been disclosed in US Patent Nos. 4,731,329, 4,812,410, 4,816,399, and 4,876,196.While Zymomonas may become an important fuel ethanol-producing
microorganism from glucose-based feedstocks, its substrate utilization range is restricted
to fermentation of glucose, sucrose and fructose and, is not naturally suited for
fermentation of the pentose component in cellulosic feedstocks. Zymomonas contains the
Entner-Douderoff pathway that allows it to ferment glucose very efficiently to ethanol as
the sole fermentation product. However, Zymomonas is naturally unable to ferment the
pentose sugars in cellulosic biomass because it lacks the essential pentose assimilation and
metabolism pathways. Thus, an opportunity exists to genetically engineer this organism
for the fermentation of pentose sugars, such as xylose and arabinose to ethanol.Genetic engineering attempts have been made to enhance ethanol production by
fermentation by transferring genes from one species to another. For example, see U.S.
Patents 5,000,000 and 5,028,539. Gene cloning and expression of various enzymes
including enzymes for creating a new metabolic pathway are also known. For example see
U.S. Patents 5,272,073, 5,041,378, 5,168,056 and 5,266,475. However, none of these
discoveries has successfully broadened the r~lnl~ll~ble substrate range of a microorganism
which could not previously ferment pentose sugars to ethanol.
Previous attempts to introduce a pentose catabolic pathway from either
Xanthomonas or Klebsiella into Zymomonas have been unsuccessful and the recombinant
strains were incapable of growth on xylose as the sole carbon source (Feldmann et al.,
1992. Appl. Microbiol.Biotechnol. 38:354-361; Liu et al., 1988. J. Biotechnol. 7: 61-77).
2 1 73793
SUMMARY OF THE INVENTION
The present invention successfully introduces a catabolic pathway for fermentation
of pentose sugars, such as xylose or arabinose, into a microorganism, such as Zymomonas,
which previously did not have the ability to ferment pentose sugars into ethanol. For the
first time, such microorganisms are capable of growing on xylose or arabinose as a sole
carbon source and fermenting either of these pentoses directly eO ethanol. One
embodiment introduces the genes encoding xylose isomerase and xylolukinase, xylose can
be converted to xylulose-5-P. Another embodiment introduces the genes encoding
L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase, which
allow the conversion of L-arabinose to D-xylulose-5-P. Then, by introducing two more
genes encoding enzymes in the pentose phosphate pathway, transaldolase and
transketolase, xylulose-5-P can be further converted to the key intermediates that couple
pentose metabolism to the glycolytic Entner-Douderoff pathway, and consequently, permit
the microorganism to metabolize pentose to ethanol. Any pentose sugar, not just
arabinose and xylose, which can be converted to xylulose-5-P can be coupled to the
glycolytic Enter-Douderoff pathway, and consequently to ethanol production, by the
introduction of these two genes which encode transaldolase and transketolase.
Accordingly, another embodiment of the present invention provides a process for
fermenting any pentose sugar which can be converted to xylulose-5-P to ethanol.
One aspect of the present invention provides compositions of Zymomonas mobilis
containing the genes encoding L-arabinose isomerase, L-ribulokinase, L-ribulose
5-phosphate 4-epimerase, transaldolase and transketolase cloned under the control of one
2 1 73793
or more Z. mobilis promoters such that said genes are coordinately expressed in said cells
of Z. mobilis and confer upon said cells the ability to grow on and ferment arabinose
directly to ethanol. In particular, compositions of Z. mobilis are provided which contain
the L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase genes
from Escherichia coli cloned precisely under the control of the Z. mobilis glyceraldehyde-
3-phosphate dehydrogenase (GAP) promoter and the transaldolase and transketolasegenes from Escherichia coli cloned precisely under the control of the Z. mobilis enolase
(ENO) promoter, such that all five said genes are contained on a single plasmid vector and
are coordinately expressed in said cells of Z. mobilis, conferring upon said cells the ability
to grow on and ferment arabinose directly to ethanol.
Another aspect of the present invention provides a process for producing ethanolfrom arabinose, or cellulosic feedstocks containing arabinose, by culturing the above
mentioned genetically-engineered strains of Z. mobilis in a culture medium containing
arabinose as a carbon source and along with an additional nitrogen source.
A further aspect of the present invention provides compositions of Zymomonas
mobilis containing the genes encoding xylose isomerase, xylulokinase, transaldolase and
transketolase which are under the control of one or more promoters recognized by Z.
mobilis, such that these genes are expressed in Z. mobilis. The genes confer upon
Zymomonas the ability to grow on and r~"ne-lt xylose directly to ethanol upon these cells.
In particular, compositions of Z. mobilis are provided which contain the xylose
isomerase and xylulokinase genes from Escherichia coli which are cloned precisely under
the control of the Z. mobilis glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter.
2 1 73793
The transaldolase and transketolase genes from Escherichia coli which are clonedprecisely under the control of the Z. mobilis enolase (ENO) promoter, are also provided
to Z. mobilis. All four of these genes are expressed in the cells of Z. mobilis conferring
upon these cells the ability to grow on and ferment xylose directly to ethanol. The cloned
genes may be provided on any number of vectors but preferably are contained on a single
plasmid vector. More preferably, the genes are integrated into the host genome.
Another aspect of the present invention is cultures of microorganisms with the
above described abilities. The cultures may be biologically pure, mixed together, or mixed
with other strains or different organisms to aid in the metabolism of the substrates or a
mixture of substrates into ethanol. A related aspect of the present invention is the culture
broth per se which may tolerate a small amount of contamination.
Yet another aspect of the present invention is a process for producing ethanol from
a pentose sugar, such as xylose or arabinose, mixtures thereof, or cellulosic feedstocks
containing hemicellulose, by culturing the above mentioned genetically-engineered
microorganisms in a culture medium containing the pentose sugars. An additional aspect
of the present invention is the modification of the catabolic pathway of a microorganism,
such as Zymomonas, which previously did not have the ability to ferment pentose sugars
to ethanol. Such microorganisms are capable of growing on arabinose or xylose as a sole
carbon source and fermenting arabinose or xylose directly to ethanol. By introducing the
genes for converting arabinose into ethanol, a microorganism without arabinose
ferrnentation ability may be converted into a microorganism capable of fermenting
arabinose into ethanol. Similarly, by introducing the genes for converting xylose into
21 73793
ethanol, a microorganism without xylose fermentation ability may be converted into a
microorganism capable of fermenting xylose into ethanol.
The introduction of the genes for L-arabinose isomerase, L-ribulokinase, and L-
ribulose 5-phosphate 4-epimerase in addition to transaldolase and transketolase allow a
microbe, such as Zymomonas, to metabolize arabinose to ethanol. The introduction of the
genes for xylose isomerase and xylolukinase, in addition to transaldolase and transketolase
allow a microbe, such as Zymomonas, to metabolize xylose to ethanol.
BRIEF DESCRIPTION OF THE DRAMNGS
Figure 1 shows a schematic of a process for producing the recombinant plasrnid
pZB5.
Figure 2 shows the comparative yield of ethanol using a control Zymomonas
mobilis and the present recombinant strain containing pZB5 when grown on glucose,
xylose or a mixture of the two sugars of sugars as the carbon source.
Figure 3 shows a schematic of a process for producing the recombinant plasmid,
pZB206.
Figure 4 shows the comparative yield of ethanol using a control Zymomonas
mobilis and the present recombinant strain containing pZB206 when grown on glucose,
arabinose or a mixture of the two sugars as the carbon source.
Figure S shows the cofermentation of a ~ of xylose and glucose by the
microorganism of the present invention.
2 1 737~3
Figure 6 shows the cofermentation of a mixture of xylose, glucose and cellulose by
cellulase and the rnicroorganism of the present invention.
Figure 7 shows the cofermentation of a mixture of xylose and cellulose by cellulase
and the microorganism of the present invention.
S
DESCRTPTION OF THE PREFERRED EMBODIMENTS
The invention is the development of recombinant Zymomonas and other microbial
strains with an expanded substrate utilization range and which are capable of growth on
and/or efficient ethanol production from xylose, arabinose or other pentose sugars, alone
or in combination, as the sole carbon source.
The microorganisms used to prepare the present invention are those which are
capable of being genetically altered to produce the necessary enzymes to form a metabolic
pathway for catabolizing pentose sugars, particularly xylose and arabinose. The
microorganism may naturally have some enzymes in the pathway but is not able to ferment
xylose or arabinose into ethanol until it has been genetically altered.
The manner of genetic alteration may use any combination of known genetic
engineering techniques such as mutation and addition of foreign DNA, provided that the
microorganism is able to ferment a pentose sugar to ethanol after tre~tmt-nt Foreign
DNA may be introduced into the microorganism by any conventional technique such as
conjugation, transformation, transduction or electroporation.
Many microorg~ni~ms which are capable of fermenting sugars to ethanol lack at
least one of the genes for the enzymes which make up a metabolic pathway for converting
2 1 73793
xylose, arabinose and other pentose sugars into ethanol. Exogenous genes may be added
to complete a metabolic pathway. One need not add genes necessary for every step if the
host microorganism already produces an enzyme in the pathway. The number of genes to
be added will depend on the starting microorganism. In the situation of imparting xylose
fermentation capability to naturally occurring Zymomonas mobilis, four genes arenecessary to produce enzymes to enable the pathway for metabolizing xylose to anintermediate which is further metabolized to ethanol using the glycolytic Entner-Douderoff
pathway. In the situation of imparting arabinose fermentation capability to naturally
occurring Zymomonas mobilis, five genes are necessary to produce enzymes to enable the
pathway for metabolizing arabinose to an intermediate which is further metabolized to
ethanol using the glycolytic Entner-Douderoff pathway.
The indigenous Zymomonas genes may be altered by any known genetic
manipulation technique to provide a protein with the necessary enzyme activity to produce
the desired metabolic pathway. The altered genes may complement one or more of the
introduced genes from another host to complete the metabolic pathway. This procedure
may be advantageous by reducing the number of genes one needs to add to the host cell.
For example, Zymomonas's native transketolase may be used to substitute for a foreign
transketolase gene, such as the one disclosed from E. coli.
Sufficient genes may be added so that the recipient microorganism may ferment
xylose, arabinose or other pentose sugars as the sole carbon source. The microorganism
may or may not be able to grow and multiply using xylose, arabinose, or combinations of
21 73793
both xylose and arabinose, as the sole carbon source, but may be capable of fermenting
xylose, arabinose, or combinations of both xylose and arabinose, to ethanol.
A gene may be added to a cell by way of a vector. The vector may be in the form
of a plasmid, cosmid or virus which is compatible to the cell's DNA and any resident
plasmids. Generally; vectors either integrate into the recipient microorganism's DNA or
the vector has an origin of replication to stably maintain the vector throughout many
microbial generations. The origin of replication may code for replication under a wide
range of stringency conditions.
To express the gene(s), a structural gene is generally placed downstream from a
promotor region on the DNA. The promotor must be recognized by the recipient
microorganism. In addition to the promotor, one may include regulatory sequences to
increase or control expression. Expression may be controlled by an inducer or a repressor
so that the recipient microorganism expresses the gene(s) only when desired.
In a preferred embodiment of the invention, xylose, arabinose or other pentose
lS sugar metabolic pathway genes are obtained from pentose metabolizing microorgani~ms
and added to Zymomonas which does not otherwise ferment pentose sugars to ethanol.
Especially preferred is Zymomonas mobilis, which historically has been used for
ft-rm~n~ing liquids containing sugar, such as plant sap for example, into alcoholic
beverages. Certain strains of Zymomonas are tolerant of up to 1.5% sodium chloride and
other mutants are tolerant to acetic acid, other microbial inhibitors, high temperatures
and/or high ethanol concentrations. The selection of host strain will depend on the
substrate being used.
-10-
2 1 73793
In another embodiment of the invention, the source for the genes encoding pentose
metabolism enzymes is selected from the group consisting of: Xanthomonas, Klebsiella,
E. coli, Rhodobacter, Flavobacterium, Acetobacter, Gluconobacter, Rhizobium,
Agrobacterium, Salmonella, Pseudomonads and Zymomonas. In general the source of
S the genes for pentose sugar metabolism is any Gram-negative bacterium capable of
ntili7ing pentose sugars for growth. A preferred organism for the source of genes is E.
coli. The preferred genes encode L-arabinose isomerase, L-ribulokinase, L-ribulose 5-
phosphate 4-epimerase, xylose isomerase, xylulokinase, transaldolase and transketolase.
Expression of these genes is under the control of promoters that function in Zymomonas.
Strong glycolytic promoters are preferred. The promoters for glyceraldehyde-3-phosphate
dehydrogenase and enolase are particularly pler~llcd. Dirr~rellt genes may be under the
control of different promoters or other expression altering sequences.
Some or all of the genes may be located together in the same vector or they may
be on different vectors or integrated into the genome. Their expression may be such that
the newly formed metabolic pathway is formed to enable the microorganism to ferment
xylose, arabinose or other pentoses to ethanol. Preferably, the genes for L-arabinose
isomerase, L-ribulokinase, L-ribulose S-phosphate 4-epimerase, xylose isomerase,xylulokinase, transaldolase and transketolase are under the control of one or more
functional promoters when in Zymomonas. The genes on a vector may be in any order,
grouping or orientation relative to each other, providing that, if more than one promotor is
present on the vector, the direction of transcription from one promotor does not adversely
affect expression of the genes.
2 1 73793
In other preferred embodiments of the present invention, a genetic element
comprising any two or more of the above described genes may be placed on the same
vector. Particularly pl~r~lled is a plasmid containing both the transaldolase and the
transketolase genes. These vectors preferably have the genes under the control of a
S promotor recognized by Zymomonas. The Examples below show plasmids pZBET,
pZB4, pZBS and pZB206, all of which are examples of vectors carrying DNA encoding
two or more of the above described genes.
The expression of the genes and the resulting functional activity of their
corresponding gene products represent a new biochemical pathway that links pentose
metabolism to the central Entner-Douderoff pathway in Zymomonas, conferring uponthese cells, for the first time, the ability to grow on and ferment pentose directly to
ethanol. The genes on a vector may be in any orientation relative to the direction of
transcription of these genes provided that they do not interfere with each other. The
examples below have shown that the genes perform in essentially the same way regardless
of orientation.
The microorganism(s) according to the present invention may be used alone or
together to ferment xylose, arabinose and other pentose sugars contained in a medium to
produce ethanol. The medium may include other ferment~ble sugars, such as glucose. If
microbial growth is desired, other nutrients n~cess~ry for microbial growth may be added
and the microorganism(s) allowed to reproduce.
Transaldolase and transketolase are key enzymes of the pentose phosphate
pathway and are required for ferrnentation by Zymomonas of any pentose sugar which can
-12-
21 73793
be converted to xylulose -5-P to ethanol. A preferred embodiment of the present
invention is the expression of the genes for transaldolase and transketolase in Zymomonas
in conjunction with any other set of genes that convert pentose sugar to xylulose-5-P.
Pentose sugars suitable for fermentation by the present invention include, but are not
limited to xylose and arabinose. An example of added genes needed for fermentation of
arabinose are L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-
epimerase genes in addition to transaldolase and transketolase genes. An example of
added genes needed for fermentation of xylose are xylose isomerase and xylolukinase
genes in addition to transaldolase and transketolase genes.
In an especially pl~relled embodiment of the invention, genes for xylose, arabinose
and other pentose utilization, and genes for transaldolase and transketolase are obtained
from organisms cont:lining them, and are expressed in Zymomonas. Efficient transport of
the pentoses into Zymomonas may be through native Zymomonas transport proteins,
mutated Zymomonas transport proteins, or through the addition of new facilitatedtransporters introduced by cloning new transport genes into Zymomonas with or without
mutagenesis of the cloned transport genes.
The step of microbial growth may be separate from fermentation. Xylose,
arabinose, and other pentoses, or l~ s thereof may be used as a carbon source for
microbial growth or one can separately culture the microorganisms on any medium (with
or without a pentose) until sufficient numbers of microorg~ni~m~ are present as a first
step, and then add a medium containing a pentose for fermentation in a second step. If a
-13-
2 1 73793
two step method is used, one may control expression of the genes in the new metabolic
pathway so that greater expression occurs during the second step.
The choice of substrates will depend on cost and supply of the substrate to be
fermented to ethanol. A typical low-cost supply of pentoses is from hemicellulose.
Xylose, arabinose and other pentoses are liberated from hemicellulosic materials by steam
and/or an acid or alkali pretreatment. Smaller amounts of other sugars such as glucose are
also separated during this pretreatment and are also fermented by Zymomonas to ethanol.
When the substrate is cellulosic m:lten~l, the cellulose may be hydrolyzed to sugars
simultaneously or separately and also fermented to ethanol. Since hemicellulose is
O generally easier to hydrolyze to sugars than cellulose, it is preferable to prehydrolyze the
cellulosic material, separate the pentoses and then hydrolyze the cellulose by treatment
with steam, acid, aL~ali, cellulases or combinations thereof to fonn glucose. Hexoses and
pentoses may be fermented to ethanol simultaneously, sequentially, separately or together
using the microorganisms of the present invention. If so desired, the hexoses may be
fermented to ethanol by a different microorganism than the pentoses, such as yeast, natural
Zymomonas, etc.
Many fermentation conditions are known per se as shown by the references
mentioned in the Background of the Invention section above. Zymomonas mobilis is a
facultative anaerobic bacterium. It has theoretical yields of ethanol from sugar of up to
97% which provides for little microbial growth, if so desired. The opl.imLIlll pH conditions
range from about 3.5 to about 7.5. Substrate concentrations of up to about 25% (based
on glucose), and under some conditions even higher, may be used. Unlike other ethanol
21 73793
producing microorganisms, no oxygen is needed at any stage for microorganism survival.
Also unlike yeast, oxygen does not drastically reduce ethanol production or greatly
increase cell growth. Agitation is not necessary but may enhance availability of substrate
and diffusion of ethanol. Accordingly, the range of fermentation conditions may be quite
S broad. Likewise, any of the many known types of apparata may be used for the present
invention.
The microorganisms according to the present invention may be used as a
biologically pure culture or may be used with other ethanol producing microorganisms in
mixed culture. Microorganisms able to ferment xylose can be mixed with microorganisms
able to ferment arabinose. This mixed pentose fermenting culture can be cultured itself or
can then be mixed with microorganisms able to ferment glucose. Biologically purecultures are generally easier to optimize but mixed cultures may be able to maximize
substrate utilization. One may also add enzyme to the f~rment~r to aid in the degradation
of substrates or to enhance ethanol production. For example, cellulase may be added to
degrade cellulose to glucose sirnultaneously with the fermentation of glucose to ethanol by
microorganisms in the same fermenter. Likewise, a hemicellulase may be added to
degrade hemicellulose.
In the ~l~f~ led embodiment using genetically engineered Zymomonas, cultures arefound to be relatively resistant to cont~min:~tion by other microorg~nism~. Nonetheless, it
is prt;r;;lled to eliminate or disable preexisting deleterious microorganisms in the substrate
before adding the Zymomonas culture.
2 1 73793
After fermentation, the ethanol, which may achieve concentrations of up to about13% (w/v), is separated from the fermentation broth by any of the many conventional
techniques known to separate ethanol from aqueous solutions. These methods include
evaporation, distillation, solvent extraction and membrane separation. Particles of
S substrate or microorganisms may be removed before ethanol separation to enhance
separation efficiency.
Once the fermentation is complete, excess microorganisms and unfermented
substrate may be either recycled or removed in whole or in part. If removed, themicroorganisms may be killed, dried or otherwise treated. This mixture may be used as
animal feed, fertilizer, burnt as fuel or discarded.
While the discussion of the fermentation in this specification generally refers to a
batch process, parts or all of the entire process may be performed continuously. To retain
the microorganisms in the fermenter, one may separate solid particles from the fluids. This
may be performed by centrifugation, flocculation, sedimentation, filtration, etc.
Alternatively, the microorganisms may be immobilized before retention in the fermenter or
to provide easier separation.
Unless specifically defined otherwise, all technical or scientific terms used herein
have the same meaning as commonly understood by one of ordinary skill in the art to
which this invention belongs. Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of the present invention, the
-16-
2 1 73793
preferred methods and materials are better illustrated by the use of the following non-
limiting examples. The following examples are offered by way of illustration and not by
way of limitation.
FXAMPLE 1
Isolation of the Xy~ose Isomerase and Xylulokinase
Genes and Fusion to a Zymomonas GAP Promoter
The Escherichia coli xylose isomerase and xylulokinase genes were initially
obtained on a 7 kb HpaI/EcoRI restriction fragment from plasmid pLC 1-3 (Clarke, L. and
J. Carbon, 1977. Cell. 9:91-99). This DNA fragment was recovered from an agarose gel
and subcloned into the SmaVEcoRI sites in a pBlueScript plasmid ~Stratagene, LaJolla,
CA), which had been dephosphorylated with calf intestinal phosphatase, to generate the
plasmid designated pBSX.
To remove excess DNA, pBSX was digested either with NsiI and HindIII or with
NsiI and SmaI. After treatment with T4 DNA polymerase, the digested DNAs were
separately ligated under dilute conditions favoring intramolecular ligation and were then
transformed into E. coli HB101. Restriction analyses of the plasmid DNA from
ampicillin-resistant transfo~ anl~ confirmed the presence of the expected deletion
derivatives. The plasmid with the expected 587 bp NsiIlHindIII deletion was designated
pXKH and contains the xylose isomerase and xylulokinase genes with the 3'-flanking
xylose operon transcriptional terminator. The plasmid with the approximately 900 bp
2 1 73793
NsiVSmaI deletion was designated pXKS and contains the xylose isomerase and
xylulokinase genes without the 3'-flanking xylose operon transcriptional terminator.
To express the xylose isomerase and xylulokinase genes in Zymomonas, they were
precisely fused to a Zymomonas glyceraldehyde-3-phosphate dehydrogenase (GAP)
promoter using a polymerase chain reaction (PCR)-mediated overlap extension technique.
This approach allowed precise fusion of the GAP promoter containing a ribosome binding
site to the translational start codon of the xylose isomerase gene, thus ensuring that the
expression of the xylose isomerase and xylulokinase genes would be regulated solely by
the GAP promoter.
To accomplish this precise fusion, 308 bp of 5'-flanking DNA upstream of the
GAP structural gene comprising the GAP promoter and the first 893 bp of the xylose
isomerase structural gene were separately synthesized in a PCR using a common linking
oligonucleotide primer. The individual DNA fragments were recovered from an agarose
gel and combined in a second PCR in which the complementary ends at the 3'-end of the
GAP promoter and the 5'-end of the xylose isomerase gene were annealed. The addition of
the 5'-GAP and 3'-xylA primers then allowed the synthesis of a 1213 bp DNA fragment
comprising a precise fusion of the GAP promoter to the 5'-end of the xylose isomerase
gene.
The primers used to synthesize the 308 bp DNA fragment comprising the GAP
promoter were based on the known DNA sequence of the 5'-flanking region of the GAP
gene (Conway et al., 1987. J. Bacteriol. 169: 5653-5662) and included:
-18-
21 73793
NotI
5'-PRIMER: 5'- CCCTCGAGCGGCCGCGTTCGATCAACAACCCGAATCCTATCG-3'
(SEQ ID NO:l)
XhoI
3'-PRIMER: 5'-GGTCAAAATAGGCTTGCATGTTTATTCTCCTAACTTATTAA
GTAGCTATTATATTCC-3'(SEQ ID NO:2)
A 15 bp DNA sequence, comprising restriction sites for the restriction enzymes XhoI and
NotI, was incorporated at the 5'-end of the synthesized GAP promoter. A 19 bp DNA
sequence (BOLD), corresponding to the 5'-end of the xylose isomerase structural gene,
was added to the 3'-end of the synthesized GAP promoter.
The primers used to synthesize the DNA fragment comprising the first 893 bp of
the xylose isomerase structural gene were based on its known DNA sequence (Lawlis et
al., 1984, Appl. Environ. Microbiol. 47: 15-21) and included:
5'-PRIMER: 5'-
GTTAGGAGAATAAACATGCAAGCCTA'l l l lGACCAGCTCGATCG
CG-3'(SEQ ID NO:3)
3'-PRIMER: 5'-GGTTGGCGTCGACAGAAC-3'(SEQ ID NO:4)
SalI
An 18 bp DNA sequence (BOLD), corresponding to the 3'-end of the GAP promoter was
added to the 5'-end of the synthesized xylose isomerase structural gene fragment.
The 1213 bp DNA fragment, comprising a precise fusion of the GAP promoter to
the 5'-end of the xylose isomerase gene was used to replace a 2.5 kb XhoVSalI restriction
fragment containing the native xylose isomerase promoter and 5'-end of the xylose
isomerase gene in plasmids pXKH and pXKS. The 1213 bp DNA fragment was digested
with SalI and XhoI restriction endonucleases and ligated separately to the larger of the
-19-
2 1 73793
two Sall/XhoI restriction fragments from plasmids pXKH and pXKS, previously purified
by preparative agarose gel electrophoresis. The ligated DNA was used to transform E.
coli HB101 and restriction analyses of the plasmid DNA from ampicillin-resistanttransforrnants confirmed the presence of the expected plasmids, which have been
designated as pGapXKH and pGapXKS. Digestion of either plasmid with the NotI
restriction enzyme liberates the approximately 4.1 kb and 4.4 kb restriction fragments,
respectively, containing the xylose isomerase and xylulokinase genes under the control of
the GAP promoter, hereafter referred to as the GAP-xylA/xylB operon. This construct is
shown in Figure 1.
EXAMPLE 2
Isolation and Linkage of the Transaldolase and
Transketolase Genes in a Synthetic Operon Under
Control of a Zymomonas ENO Promoter.
The Escherichia coli transaldolase and transketolase genes were isolated
separately, synthetically linked and precisely fused to the Zymomonas enolase (ENO)
promoter by PCR-mediated overlap extension. The transaldolase gene, localized within 0'-
2.5' minutes of the Escherichia coli genome, was obtained by PCR synthesis from total
genomic DNA. The primers used to synthesize the 954 bp DNA fragment comprising the
transaldolase gene were based on its known DNA sequence (Yura et al., 1992. Nucleic
Acids Res. 20: 3305-3308) and included:
-20-
21 73793
5'-PRIMER: 5'-CGTCTAAAAGATTTTAAGAAAGGTTTCGATATGACGGACAA
ATTGACC-3'(SEQ ID NO:5)
3'-PRIMER:
5'CATl~TGACTCCAGATCTAGATTACAGCAGATCGCCGATCA'l'l"l'l'l'
TCC-3'(SEQ ID NO:6) XbaI
A 33 bp DNA sequence (BOLD), corresponding to the 3'-end of the ENO promoter wasadded to the 5'-end of the synthesized transaldolase gene. A 21 bp DNA sequence
comprising a restriction site for the restriction enzyme XbaI was incorporated at the 3'-end
of the synthesized transaldolase gene to facilitate its subsequent subcloning.
The primers used to synthesize the 196 bp DNA fragment comprising the ENO
promoter were based on the known DNA sequence of the 5'-flanking region of the ENO
gene (Burnett et al., 1992. J. Bacteriol. 174: 6548-6553) and included:
5'-PRIMER: 5'-CCAGATCTCCAGTTACTCAATACG-3'(SEQ ID NO:7)
BglII
3'-PRIMER: 5'-
GGTCAATTTGTCCGTCATATCGAAA'l'l'l'l'l~l'l'AAAAT~l-l'l'l'AG
ACG-3'(SEQ ID NO:8)
A 6 bp DNA sequence comprising a restriction site for the restriction enzyme BglII was
incorporated at the 5'-end of the synthesized ENO promoter to facilitate its subsequent
subcloning. An 18 bp DNA sequence (BOLD), corresponding to the 5'-end of the
transaldolase gene was added to the 3'-end of the synthesized ENO promoter.
The transaldolase gene (tal) was then precisely fused to the ENO promoter by
PCR-mediated overlap extension. To accomplish this precise fusion, the 196 bp of 5'-
fl~nking DNA upstream of the ENO structural gene comprising the ENO promoter and
21 73793
the 954 bp DNA fragment comprising the transaldolase gene were separately synthesized
in a PCR using a common linking oligonucleotide primer. The individual DNA fragments
were recovered from an agarose gel and then combined in a second PCR in which the
complementary ends at the 3'-end of the ENO promoter and the 5'-end of the transaldolase
S gene were annealed. The addition of the 5'-ENO and 3'-tal primers then allowed the
synthesis of a 1174 bp DNA fragment comprising a precise fusion of the ENO promoter to
the transaldolase gene. This 1174 bp DNA fragment was digested with the XbaI
restriction enzyme and then ligated to plasmid pUC18 that had been sequentially digested
with the SmaI restriction enzyme, treated with Taq polymerase in the presence of dTTP
and finally digested with XbaI. The ligated DNA was used to transform E. coli DHSa and
restriction analyses of the plasmid DNA from ampicillin-resistant transformants confirmçd
the presence of the expected plasmid, which has been designated as pEnoTAL.
The transketolase gene (tktA) was obtained by PCR synthesis from E. coli W3110
genomic DNA. The primers used to synthesize the 2077 bp DNA fragment comprising the
transketolase gene were based on its known DNA sequence (Sprenger, 1992. J. Bacteriol.
174: 1707-1708) and included:
5'-PRIMER: 5'-GCTCTAGACGATCTGGAGTCAAAATGTCC-3'(SEQ ID NO:9)
XbaI
3'-PRIMER: 5'-AGATCTGCGCAAACGGACATTATCAAGG-3'(SEQ ID NO: 10)
BglII
A 8 bp DNA sequence comprising a restriction site for the restriction enzyme XbaI was
incorporated at the 5'-end of the tktA gene and a 7 bp DNA sequence comprising a
21 73793
restriction site for the restriction enzyme Bg'lII was incorporated at the 3'-end of the tktA
gene to facilitate its subsequent subcloning. Following PCR synthesis, the 2077 bp DNA
fragment comprising the transketolase gene was purified by preparative agarose gel
electrophoresis, digested with the XbaI restriction enzyme and ligated to plasmid pUC18
that had been sequentially digested with the HincII restriction enzyme, treated with Taq
polymerase in the presence of dTTP and finally digested with XbaI. The ligated DNA was
used to transform E. coli DH5 a and restriction analyses of the plasmid DNA fromampicillin-resistant transformants confirmed the presence of the expected plasmid, which
has been designated as pUC-TKT.
The transketolase gene was then subcloned downstream of the ENO-transaldolase
fusion to create a synthetic operon comprised of the transaldolase and transketolase genes
both under the control of the ENO promoter. To do this, plasmid pUC-TKT was digested
with the XbaI and SphI restriction enzymes and the approximately 2 kb restriction
fragment containing the transketolase gene was purified by preparative agarose gel
electrophoresis and ligated to plasmid pEno-TAL that had been previously digested with
the same restriction enzymes. The ligated DNA was used to transform E. coli DH5a and
restriction analyses of the plasmid DNA from ampicillin-resistant transformants confirmed
the presence of the expected plasmid, which has been designated as pEnoTAL/TKT.
Digestion of this plasmid with the BglII restriction enzyme liberates an approximately 3 kb
restriction fragment containing the transaldolase and transketolase operon under the
control of the ENO promoter, hereafter referred to as the ENO-tal/tktA operon. This
construct is also shown in Figure 1.
21 73793
EXAMPLE 3
Construction of a Shuttle Vector and Transfer
of the Xylose Metabolism and Pentose Phosphate
Pathway Genes into Zymomonas
A shuttle vector was constructed to permit the simultaneous transfer of the xylose
metabolism and pentose phosphate pathway genes into Zymomonas. A small native 2.7 kb
plasmid from Z. mobilis ATCC 10988 was purified by preparative agarose gel
electrophoresis, linearized by digestion with the AvaI restriction enzyme and ligated to the
similarly digested plasmid pACYC184 (New England BioLabs, Beverly, MA) which hadbeen dephosphorylated by treatment with calf intestinal phosphatase. The ligated DNA
was used to transform E. coli HB101 and restriction analyses of the plasmid DNA from
tetracycline-resistant transformants confirrned the presence of the expected plasmid, which
has been designated as pZB 186.
This plasmid was then modified to accept the xylose metabolism genes on a singleNotI restriction fragment. Plasmid pZB 186 was linearized with the EcoRI restriction
enzyme and the cohesive ends were filled-in by treatment with the Klenow fragment of
DNA polymerase. NotI linkers were added according to standard methodology and then
the plasmid was digested with the NotI restriction enzyme and ligated under dilute
conditions favoring intramolecular ligation. The ligated DNA was used to transform E.
coli DH5 a and restriction analyses of the plasmid DNA from tetracycline-resistant
-24-
21 73793
transformants confirmed the presence of the added NotI restriction site in pZB186. The
modified plasmid has been designated pZB188.
To introduce the ENO-taVtkt operon into this shuttle vector, the approximately 3kb BglII restriction fragment from plasmid pEnoTAL/TKT was purified by preparative
agarose gel electrophoresis and ligated to pZB188 that had been sequentially passaged
through E. coli JMl 10, linearized by digestion with the BclI restriction enzyme and
dephosphorylated by treatment with calf intestinal phosphatase. The ligated DNA was
used to transform E. coli DH5 a and restriction analyses of the plasmid DNA fromtetracycline-resistant transformants confirmed the presence of the expected plasmid, which
has been designated as pZBET.
To also introduce the GAP-xylA/xylB operon into this plasmid, the approximately
4.1 kb and 4.4 kb NotI restriction fragments from plasmids pGapXKH and pGapXKS,
respectively, were purified by preparative agarose gel electrophoresis and separately
ligated to NotI linearized pZBET. The ligated DNA was used to transform E. coli HB101
and restriction analyses of the plasmid DNA from tetracycline-resistant transformants
confirmed the presence of the expected plasmids. The plasmid cont~ining the GAP-xylA/xylB operon from pGapXKH in clockwise orientation and the ENO-tal/tkt operon
from pEnoTAL/TKT in counterclockwise orientation has been designated pZB4. The
plasmid containing the GAP-xylA/xylB operon from pGapXKS in clockwise orientation
and the ENO-taVtkt operon from pEnoTAL/TKT in counterclockwise orientation has
been de~ign~ted pZB5. The orientation of pZB4 and pZB5 may be viewed in Figure 1.
-25-
21 73793
Plasmids pZB4 and pZB5 were separately transformed into Z. mobilis CP4 by
electroporation of approximately 109 cells/ml with 4 ,ug DNA in 40 ,ul of 10% (w/v)
glycerol at 16 kv/cm, 200Q and 25yF. After electroporation, the cells were allowed to
recover at 30C for 3-16 hours in a liquid medium comprised of 5% glucose, 10% yeast
extract (Difco), 5% Tryptone (Difco), 0.25% ammonium sulfate, 0.02% potassium
phosphate, dibasic and lmM magnesium sulfate. Transformants containing pZB4 and
pZB5 were isolated following anaerobic incubation at 30C for 2 or more days in the same
medium additionally containing 1.5% agar and tetracycline (20 ,ug/ml) and were
subsequently confirmed by restriction analyses of the plasmid DNA from tetracycline-
resistant transformants.
Enzymatic analyses of Z. mobilis CP4 (pZB4) demonstrated the presence of xylose
isomerase (0.35 U/min/mg), xylulokinase (1.4 U/min/mg), transaldolase (1.9 U/min/mg)
and transketolase (0.27 U/min/mg) activities and thus confirmed the expression of all four
genes. These enzymatic activities were either undetectable or significantly lower (xylose
isomerase, 0.008/min/mg; xylulokinase, undetectable; transaldolase, 0.014 U/min/mg; and
transketolase, 0.032 U/min/mg) in the control strain containing the shuttle vector alone
(CP4 [pZB186]).
FXAMPLE 4
F~lTn~nt~tion Performance of Recombinant Zyrnomonas
Containing the Xylose Metabolism and
Pentose Phosphate Pathway Genes
-26-
21 73793
The fermentation performance of the recombinant zymomonas containing the
xylose isomerase, xylulokinase, transaldolase and transketolase genes was evaluated in a
medium comprising 1% (w/v) yeast extract (Difco), 0.2% potassium phosphate, dibasic
S and either 5% glucose, or 5% xylose, or 2.5% glucose and 2.5% xylose.
The recombinant zymomonas strains were first propagated at 30C in the above
medium cont~ining 5% glucose or xylose in a bottle with 80 ml of working volume
without agitation until late log-phase. The cells were then inoculated to 200 ml of the
above fermentation medium in a 250 ml flask at an initial OD600=0.05-0. 1. The cultures
were grown at 30C under anaerobic conditions using CO2-traps with gentle shaking (150
rpm) for mixing. The cell growth was monitored as optical density at 600 nm. Theresidual sugars as well as ethanol concentrations were determined on HPLC (HP 1090L)
(Hewlett Packard, Wilmington, DE) using a Bio-Rad Aminex HPX-97H column.
The results presented in Figure 2 show that in contrast to the control strain
containing the shuttle vector alone (CP4[pZB 186]), the recombinant containing the added
xylose isomerase, xylulokinase, transaldolase and transketolase genes demonstrated
growth and ethanol production from xylose as a carbon source. The recombinant strain
produces ethanol from glucose as efficiently as the control strain at 94% of theoretical
yield. The recombinant strain additionally produces ethanol from xylose at 84% of
theoretical yield in 79 hours. Furthermore, in the combined presence of glucose and
xylose, the recombinant strain ferments both sugars simultaneously to ethanol at 88% of
2 1 73793
theoretical yield within 48 hours, thus providing the foundation for advanced process
designs with cofermentation of mixed-sugar feedstocks.
EXAMPLE S
Isolation of the L-Arabinose Isomerase, L-Ribulokinase,
and L-Ribulose 5-Phosphate 4-Epimerase Genes
and Fusion to a Zymomonas GAP Promoter
The L-arabinose isomerase (araA), L-ribulokinase (araB), and L-ribulose 5-
phosphate 4-epimerase (araD) genes were isolated separately from the native araBAD
operon of Escherichia coli B/r (Lee et al., 1986. Gene 47: 231-244) using polymerase
chain reaction (PCR) synthesis, and synthetically linked to form a new araBAD operon.
To express the L-ribulokinase, L-arabinose isomerase, and L-ribulose 5-phosphate 4-
epimerase (araBAD) genes in Zymomonas, the genes were precisely fused to a
Zymomonas glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter using a PCR-
m~i;lted overlap extension technique. This approach allowed precise fusion of the GAP
promoter containing a ribosome binding site to the translational start codon of the L-
ribulokinase gene, thus ensuring that the expression of the araBAD genes would be
regulated solely by the GAP promoter. To accomplish this precise fusion, 308 bp of 5'-
flanking DNA upstream of the GAP structural gene comprising the GAP promoter and the
first 582 bp of the araB structural gene were separately synthesized in a PCR using a
common linking oligonucleotide primer. The individual DNA fragments were recovered
-28-
2 1 73793
from an agarose gel and combined in a second PCR in which the complementary ends at
the 3'-end of the GAP promoter and the 5'-end of the araB gene were annealed. The
addition of the 5'-GAP and 3'-araB primers then allowed the synthesis of a 902 bp DNA
fragment comprising a precise fusion of the GAP promoter to araB gene.
The primers used to synthesize the 308 bp DNA fragment comprising the GAP
promoter were based on the known DNA sequence of the 5'-flanking region of the GAP
gene (Conway et al., 1987. J. Bacteriol. 169: 5653-5662) and included:
5'-PRIMER: 5'- GG.A~TTCGCGGCCGCGTTCGATCAACAACCCGAATCC-3'(SEQ
ID NO: 11) EcoRI
3'-PRIMER: 5'-CAATTGCAATCGCCATGTl lATTCTCCTAACTTATTAA
GTAGCTATTATATTCC-3'(SEQ ID NO:12)
A 15 bp DNA sequence, comprising restriction sites for the restriction enzymes
EcoRI and NotI, was incorporated at the 5'-end of the synthesized GAP promoter. A 16
bp DNA sequence (BOLD), corresponding to the 5'-end of araB gene, was added to the
3'-end of the synthesized GAP promoter.
The primers used to synthesi7e the DNA fragment comprising the first 582 bp of
the araB gene were based on its known DNA sequence (Lee et al., 1986, Gene 47: 231-
244) and included:
5'-PRIMER: 5'-
GTTAGGAGAAACATGGCGATTGCAATTGGCCTCGATTTTGGC-3 '
(SEQ ID NO:13)
-29-
21 73793
3'-PRIMER: 5'-CGGGCGGGTGGTACCGGAAAG-3' (SEQ ID NO:14)
KpnI
A 15 bp DNA sequence (BOLD), corresponding to the 3'-end of the GAP
5 promoter was added to the 5'-end of the synthesized araB gene fragment.
Following the second PCR synthesis, the 902 bp PCR fragment was purified by
preparative agarose gel electrophoresis and digested with EcoRI and KpnI to generate the
891 bp EcoRI-KpnI DNA fragment, comprising a precise fusion of the GAP promoter to
the araB gene.
The 2679 bp DNA fragment, comprising the 3'-end of the araB and araA genes
was obtained by PCR synthesis from the Escherichia coli B/r chromosome. The primers
used to synthesize this DNA fragment were based on its known DNA sequence (Lee et al.,
1986, Gene 47: 231-244) and included:
5'-PRIMER: 5'-C l-l l CCGGTACCACCCGCCCG-3' (SEQ ID NO: 15)
KpnI
3'-PRIMER: 5'-
CTAACATGTTGACTC( l l CTCTAGACTTAGCGACGAAATCCGTAATACAC-3'
(SEQ ID NO: 16) XbaI
A 26 bp DNA sequence, comprising restriction site for XbaI, was incorporated at
the 3'-end of the araA gene. Following PCR synthesis, the 2679 bp PCR fragment was
purified by preparative agarose gel electrophoresis, digested with KpnI and XbaI to
generate the 2652 bp KpnI-XbaI DNA fragment, comprising the 3'-end of the araB and
the araA genes.
-30-
2 1 73793
To remove the repetitive extragenic palindromic sequences between araA and
araD in the native araBAD operon, the araD gene encoding L-ribulose 5-phosphate 4-
epimerase was isolated separately from the Escherichia coli B/r chromosome using PCR
synthesis, then linked to 3'- end of araA to form a new araBAD operon. The primers
5 used to synthesize the 916 bp DNA fragment comprising the araD gene were based on its
known DNA sequence (Lee et al., 1986, Gene 47: 231-244) and included:
5'-PRIMER: 5'-
CGGATl-rCGTCGCTAAGTCTAGAGAAGGAGTCAACATGTTAGAAGATCTC-3 '
(SEQ ID NO:17) XbaI
3'-PRIMER: 5'-CCCCCAAGCTTGCGGCCGCGGCCCGTTGTCCGTCGCCAG-3'
(SEQIDNO:18) HindIII NotI
A 23 bp DNA sequence, comprising a restriction site for XbaI, was incorporated
at the 5'-end of the araD gene and a 19 bp DNA sequence, comprising restriction sites for
HindIII and NotI, was incorporated at the 3'-end of the araD gene to facilitate its
subsequent subcloning. Following PCR synthesis, the 916 bp PCR fragment was purified
by plepa,~ /e agarose gel electrophoresis and digested with the XbaI and Hindm to
generate the 892 bp DNA fragment, comprising the araD gene, that was ligated to
plasmid pUC18 that had been digested with the same restriction enzymes. The ligated
DNA was used to transform E. coli DHSa and restriction analyses of the plasmid DNA
from ampicillin-resistant transformants co,lrlnled the presence of the expected plasmid,
which has been dçsign~ted as pUC-araD.
21 73793
To construct a new araBAD operon, araD was linked to the 3'-end of the araA.
To do this, the 2652 bp KpnI-XbaI DNA fragment, comprising the 3'-end of the araB and
the araA genes was ligated to pUC-araD that had been digested with KpnI and XbaIrestriction enzymes. The ligated DNA was used to transform E. coli DH5a and restriction
S analyses of the plasmid DNA from ampicillin-resistant transformants conflrmed the
presence of the expected plasmid, which has been designated as pUC-araB'AD. The
plasmid pUC-araB'AD contains the partial new araBAD operon.
The plasmid pBRMCS which was constructed by inserting the EcoRI-Hindm
multiple cloning site fragment of pUC18 into the EcoRI and Hindm sites in pBR322, was
used to subclone the new Pgap-araBAD operon (see below). The 3544 bp ara-B'AD
fragment was isolated by preparative agarose gel electrophoresis following digestion of
pUC-araB'AD with KpnI and HindIII, and ligated to pBRMCS that had been digested
with the same restriction enzymes. The ligated DNA was used to transform E. coli DHSa
and restriction analyses of the plasmid DNA from ampicillin-resistant transformants
confirmed the presence of the expected plasmid, which has been designated as pBRMCS-
araB'AD. The previously obtained 891 bp EcoRI-KpnI DNA fr~gm~nt comprising a
precise fusion of the GAP promoter to the araB gene, was then ligated to pBRMCS-araB'AD that had been digested with KpnI and EcoRI restriction enzymes. The ligated
DNA was used to transform E. coli DHSa and restriction analyses of the plasmid DNA
from ampicillin-resistant transformants confirmed the presence of the expected plasmid,
which has been designated as pBR gap-araBAD. Digestion of this plasmid with the NotI
restriction enzyme liberates an approximately 4.4 kb restriction fragment containing the
-32-
21 737~3
L-arabinose isomerase, L-ribulokinaset and L-ribulose 5-phosphate 4-epimerase operon
under the control of the GAP promoter, hereafter referred to as the P8ap-araBAD operon
(Figure 3).
FXAl~IPLF 6
S Construction of a Recombinant Plasmid Containing Arabinose Metabolism and Pentose
Phosphate Pathway Genes and Transfer into Zymomonas
The plasmids pZBET containing the PenO-talltkt A operon comprising the
transaldolase and transketolase genes from Escherichia coli cloned precisely under the
control of the Z. mobilis enolase (ENO) promoter in both clockwise and counterclockwise
orientations were previously constructed in Example 2. To introduce the P8ap-araBAD
operon into this plasmid, the approximately 4.4 kb NotI restriction fragment from
plasmids pBR gap-araBAD was purified by preparative agarose gel electrophoresis and
separately ligated to NotI line~ri7e~ pZBET. The ligated DNA was used to transform
E. coli DH5a and restriction analyses of the plasmid DNA from tetracycline-resistant
transforrnants confirmed the presence of the expected plasrnids. The plasmid cont~ining
the P~nO-talltkt A operon and the P8ap-araBAD operon in clockwise orientations has been
de.sign~ted pZB200. The plasmid containing the PenO-talltkt A operon in clockwise
orientation and the Pgap-araBAD operon in counterclockwise orientation has been
desi~n~te(l pZB202. The plasmid containing the PenO-talltkt A operon in counter-clockwise orientation and the P8ap-araBAD operon in clockwise orientation has been
2 1 73793
designated pZB204. The plasmid containing the PenO-talltkt A operon and the P8a~-
araBAD operon in counterclockwise orientations has been designated pZB206 (Figure 3).
Plasmids pZB200, pZB202, pZB204 and pZB206 were separately transformed
into Z. mobilis ATCC 39676 by electroporation of approximately 109 cells/ml with 1. 2 to
3.0 pg DNA in 40 pl of 10% (w/v) glycerol at 16 kv/cm, 200Q and 25,uF. After
electroporation, the cells were allowed to recover at 30C for 3- 16 hours in a liquid
medium comprised of 5% glucose, 10% yeast extract (Difco), 5% Tryptone (Difco),
0.25% ammonium sulfate, 0.02% potassium phosphate, dibasic and lmM magnesium
sulfate. Transformants containing pZB200, pZB202, pZB204 and pZB206 were isolated
following anaerobic incubation at 30C for 2 or more days in the same medium
additionally containing 1.5% agar and tetracycline (20 ,ug/ml) and were subsequently
confirmed by restriction analyses of the plasmid DNA frorn tetracycline-resistant
transformants.
EXAMPI_E 7
Fermentation Performance of Recombinant Zymomonas Containing the Arabinose
Metabolism and Pentose Phosphate Pathway Genes
The fermentation performance of the recombinant Zymomonas cont~ining the
L-arabinose isomerase, L-ribulokinase, L-ribulose 5-phosphate 4-epimerase, transaldolase
and transketolase genes was evaluated in a medium comprised of 1% (w/v) yeast extract
(Difco), 0.2% potassium phosphate, dibasic and either 2.5% arabinose or 2.5% arabinose
and 2.5% glucose. The recombinant Zymomonas strains were first propagated at 30 C in
-34-
2 1 73793
above medium containing 5% glucose till late logphase. The cells were then inoculated to
95 ml of fermentation medium in a 100 ml bottle at an initial OD6oo=o 15 at 600 nm. The
culture was grown at 30 C or 37 C without shaking.
The results presented in Figure 4 show that, in contrast to the control strain
S cont~ining the shuttle vector alone (pZB186), the recombinant strain containing the added
L-arabinose isomerase, L-ribulokinase, L-ribulose 5-phosphate 4-epimerase, transaldolase
and transketolase genes (pZB206) demonstrates growth on and ethanol production from
arabinose as a sole carbon source. The recombinant strain of the present invention
produces ethanol from arabinose at 91% or 96% of theoretical consumed sugar yield in 96
hours at 30C or 37C, respectively. Furthermore, in the combined presence of glucose
and arabinose, the recombinant strain ferments both sugars to ethanol at 89% or 96% of
theoretical consumed sugar yield in 96 hours at 30C or 37C, respectively, thus providing
the foundation for advanced process designs requiring cofermentation of mixed-sugar
feedstocks.
EXAMPLE 8
Using Recombinant Zymomonas Containing the Xylose Metabolism and Pentose
Phosphate Pathway Genes to Coferment Glucose and Xylose
The r~lnlen~tion performance of the recombinant Zymomonas containing the D-
xylose isomerase, D-xylulokinase, transaldolase, and transketolase genes was evaluated on
a ~ ue of 3.5% (w/v) D-xylose and 6% (w/v) D-glucose. Fermentation was carried out
in an unsparged 500 mL working volume fermenter operating at a temperature of 37C, an
-35-
21 73793
agitation rate of l S0 rpm, and was inoculated with approximately 0.6 g of dry cell mass
per liter (g DCM/L). The fermentation pH was controlled at 5.2 by the automatic addition
of concentrated potassium hydroxide. The fermentation medium comprised 1% (w/v)
yeast extract (Difco) and 0.2% (w/v) dibasic potassium phosphate; tetracycline was added
at a level of 10 mg/L to ensure plasmid retention.
The recombinant strain from Example 3 containing the added genes encoding
enzymes for xylose utilization reln~llted the mixture of 6% (w/v) glucose and 3.5% (w/v)
xylose to about 42 g/L ethanol in 48 hours to achieve an overall (net) yield of available
sugars of 86% of theoretical. See Figure 5.
EXAMPT F 9
Using Recombinant Zymomonas Containing the Xylose Metabolism and Pentose
Phosphate Pathway Genes to Coferment Cellulose, Glucose and Xylose
The fermentation performance of the recombinant Zymomonas cont~ining the
D-xylose isomerase, D-xylulokinase, transaldolase, and transketolase genes as produced in
Example 3 was evaluated on a mixture of 3.5% (w/v) D-xylose, 3% (w/v) D-glucose and
3% (w/v) Sigmacell-S0 microcrystalline cellulose (Sigma). CPN cellulase enzyme
complex (Iogen) was added at a loading of 25 filter paper units per gram of cellulose
(FPU/g cellulose) to hydrolyze the cellulose. Fermentation was carried out in anunsparged S00 rnL working volume fermenter at a temperature of 37C and an agitation
rate of 150 rpm, and was inoculated with approximately 0.6 g of dry cell mass per liter
-36-
21 73793
(g DCM/L). The fermentation pH was controlled at 5.2 by the automatic addition of
concentrated potassium hydroxide. The fermentation medium comprised 1% (w/v) yeast
extract (Difco) and 0.2% (w/v) dibasic potassium phosphate; tetracycline was added at a
level of 10 mg/L to ensure plasmid retention.
In the presence of exogenous cellulase, the recombinant strain produced by
Example 3 above, containing the added genes encoding for xylose utilization fermented
the mixture of 3% (w/v) cellulose, 3% (w/v) glucose and 3.5% (w/v) xylose to about
40 g/L ethanol in 120 hours to achieve an overall (net) yield on all potentially available
sugars above 80% of theoretical. See Figure 6.
EXAMPT F 10
Using Recombinant Zymomonas Containing the Xylose Metabolism and Pentose
Phosphate Pathway Genes to Coferment Cellulose and Xylose
The fermentation performance of the recombinant Zymomonas cont~ining the
D-xylose isomerase, D-xylulokinase, transaldolase, and transketolase genes was evaluated
on a mixture of 3.5% (w/v) D-xylose and 6% (w/v) Sigmacell-50 microcrystalline
cellulose (Sigma). CPN cellulase enzyme complex (Iogen) was added at a loading of 25
filter paper units per gram of cellulose (FPU/g cellulose) to hydrolyze the cellulose.
Fermentation was carried out in an unsparged 500 mL working volume fermenter
operating at a temperature of 37C and an agitation rate of 150 rpm, and was inoculated
with approximately 0.6 g of dry cell mass per liter (g DCM/L). The fermentation pH was
-37-
2~ 73793
controlled at 5.2 by the automatic addition of concentrated potassium hydroxide. The
fermentation medium comprised 1% (w/v) yeast extract (Difco) and 0.2% (w/v) dibasic
potassium phosphate; tetracycline was added at a level of 10 mg/L to ensure plasmid
retention.
As shown in Figure 7, in the presence of exogenous cellulase, the recombinant
strain produced by Example 3 above, containing the added genes encoding for xylose
utilization fermented the mixture of 6% (w/v) cellulose and 3.5% (w/v) xylose to about
38 g/L ethanol in 120 hours to achieve an overall (net) yield on all potentially available
sugars above 72% of theoretical.
EXAMPLE 1 1
Using Recombinant Zymomonas Containing the Arabinose Metabolism and Pentose
Phosphate Pathway Genes to Coferment
Cellulose, Glucose and Arabinose
Fermentation of mixtures of L-arabinose, D-glucose, and cellulose can be carriedout using the recombinant Zymomonas containing the L-arabinose isomerase,
L-ribulokinase, L-ribulose 5-phosphate 4-epimerase, transaldolase, and transketolase
genes in a manner similar to that described in Example 7 above. Using this approach,
yields based on total potentially available sugars (D-glucose + L-arabinose) of greater
than 75% could be achieved. For example, lllixL~ues of 2.5% (w/v) L-arabinose, 2.5%
(w/v) D-glucose, and 2.5% (w/v) Sigmacell-50 microcrystalline cellulose (Sigma) could be
2 1 73793
fermented in an unsparged 500 mL working volume fermenter operating at a temperature
of 37C, an agitation rate of 150 rpm, using an inoculum loading of approximately 0.6 g
of dry cell mass per liter (g DCMIL). In this case, a cellulase enzyme complex such as
CPN cellulase (Iogen) would be added at an appropliate loading, such as 25 filter paper
units per gram of cellulose (FPU/g cellulose), to hydrolyze the cellulose. Fermentation pH
would be controlled at an appropliate level to uncouple fermentation from growth, such as
pH 5.2 by the automatic addition of concentrated potassium hydroxide. The fermentation
medium would be comprised of 1% (w/V) yeast extract (Difco) and 0.2% (w/v) dibasic
potassium phosphate, and tetracycline added at a level of approximately 10 mg/L to
ensure plasmid retention.
EXAMPLE 12
Using Recombinant Zymomonas Containing the Arabinose Metabolism and Pentose
Phosphate Pathway Genes to Coferment
Cellulose and Arabinose
Fermentation of mixtures of L-arabinose and cellulose can be carried out using the
recombinant Zymomonas conLaining the L-arabinose isomerase, L-ribulokinase, L-ribulose
5-phosphate 4-epimerase, transaldolase, and transketolase genes in a manner similar to
that described in Example 10 above. Using this approach, yields based on total potentially
available sugars (D-glucose + L-arabinose) of greater than 70% could be achieved. For
example, mixtures of 2.5% (w/v) L-arabinose and 5% (w/v) Sigmacell-50 microcrystalline
-39-
21 737~3
cellulose (Sigma) could be fermented in an unsparged 500 mL working volume fermenter
operating at a temperature of 37C, an agitation rate of 150 rpm, using an inoculum
loading of approximately 0.6 g of dry cell mass per liter (g DCM/L). In this case, a
cellulase enzyme complex such as CPN cellulase (Iogen) would be added at an al~pl-Jpliate
loading, such as 25 filter paper units per gram of cellulose (FPU/g cellulose), to hydrolyze
the cellulose. Fermentation pH would be controlled at an appropriate level such as pH 5.2
by the automatic addition of concentrated potassium hydroxide. The fermentation
medium would be comprised of 1% (w/v) yeast extract (Difco) and 2% (w/v) dibasicpotassium phosphate, and tetracycline added at a level of approximately 10 mg/L to
ensure plasmid retention.
EXAMPLF 13
Using Mixed Cultures of the Recombinant Zymomonas Containing the Xylose Metabolism
and Pentose Phosphate Pathway Genes in Combination with the Recombinant Zymomonas
Containing the Arabinose Metabolism and Pentose Phosphate Pathway Genes to
Coferment Mixtures of Xylose and Arabinose and Glucose, Mixtures of Xylose and
Arabinose and Cellulose, or Mixtures of Xylose and Arabinose and Glucose and Cellulose
Fermentation of mixtures of L-arabinose, D-xylose, D-glucose, and cellulose can
be carried out by using a mixed cultured comprised of the recombinant Zymomonas
containing the L-arabinose isomerase, L-ribulokinase, L-ribulose S-phosphate
4-epimerase, transaldolase, and transketolase genes in combination with the recombinant
-40-
2~ 73793
zymomonas containing the D-xylose isomerase, D-xylulokinase, transaldolase, and
transketolase genes. Using this approach, yields based on total potentially available sugars
(D-glucose + D-xylose + L-arabinose) of greater than 70% could be achieved. For
example, mixtures of 2% (w/v) L-arabinose, 2% (w/v) D-xylose, 2% (w/v) D-glucose, and
2% (w/v) Sigmacell-50 microcrystalline cellulose (Sigma) could be fermented in an
unsparged 500 mL working volume fermenter operating at a temperature of 37C, anagitation rate of 150 rpm, using an inoculum loading of approximately 0.3 g of dry cell
mass per liter (g DCM/L) of the arabinose-fermenting strain in combination with
approximately 0.3 g of dry cell mass per liter (g DCM/L) of the xylose-fermenting strain.
Inoculum ratios of the two recombinant strains can be varied from 1:1, as recited herein,
to equal the proportion of the arabinose:xylose ratio in the mixture. In this particular
example, since cellulose is present, a cellulase enzyme complex such as 25 filter paper
units per gram of cellulose (FPU/g) is added to hydrolyze the cellulose. If a mixture of
only L-arabinose, D-xylose, and D-glucose were to be fermented, it would not be
necessary to add cellulase enzyme complex. Fermentation pH would be controlled at an
appropliate level such as pH 5.2 by the automatic addition of concentrated potassium
hydroxide. The fermentation medium would be comprised of 1% (w/v) yeast extract
(Difco) and 0.2% (w/v) dibasic potassium phosphate, and tetracycline added at a level of
approximately 10 mg/L to ensure retention of the plasmids by both of the strains. Since
growth would be minimi7ed by operating at 37C, one of the strains would not
outcompete or overtake the other.
-41-
2 1 73793
It is to be understood that the phraseology or terminology employed herein is for
the purpose of description and not of limitation.
All references mentioned in this application are incorporated by reference.
-42-
2 1 737~3
SEQUENCE LISTING (42(a) - 42( i
(l) GENERAL INFORMATION:
(i) INV~IIORS: Picataggio, Stephen
Zhang, Min
Eddy, Christina
Deanda, Kristine
Finkelstein, Mark
Mohagheghi, Ali
Newman, Mildred
McMillan, James
(ii) TITLE OF INVENTION: Pentose Fermentation by Recombinant Zymomonas
(iii) NUMBER OF SEQUENCES: 18
(iv) CORRESPONDENCE ADDRESS FOR APPLICP.NT:
(A) ADDRESSEE: MIDWEST RESEARCH INSTITUTE
(B) STREET: 425 Volker Blvd.
(C) CITY: Kansas City
(D) STATE: MISSOURI
(E) COUNTRY: USA
(F)ZIP: 64110
( v ) ATTORNEY/AGENT INFORMATION: u . s .
(A) NAME: O'CONNOR, EDNA
(B) REGISTRATION NUMBER: 29,252
(C) REFERENCE/DOCKET NUMBER: NREL 95-26 and 27
(D) TELEPHONE: (303)384-7573
( ~ ~ TELEFAX: (303)384-7499
- 4 2 ( a ) -
- 21 73793
(2) INFORMATION FOR SEQ ID NO:I:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(~i) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
CCCTCGAGCG GCCGCGTTCG ATCAACAACC CGAATCCTAT CG
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GGTCAAAATA GGCTTGCATG TTTATTCTCC TAACTTATTA AGTAGCTATT
ATATTCC
- 4 2 ( b ) -
21 737~3
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genornic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GTTAGGAGAA TAAACATGCA AGCCTATTTT GACCAGCTCG ATCGCG
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GGTTGGCGTC GACAGAAC
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 bases
(B) TYPE: nucleic acid
-42 ( c)--
21 73793
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CGTCTAAAAG ATTTTAAGAA AGGTTTCGAT ATGACGGACA AATTGACC
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CATTTTGACT CCAGATCTAG ATTACAGCAG ATCGCCGATC A'l"l''l"l''l''l'CC
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
- 4 2 ( d ) -
2 ~ 73793
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CCAGATCTCC AGTTACTCAA TACG
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GGTCAATTTG TCCGTCATAT CGAAATTTTC TTAAAATCTT TTAGACG
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
-42( e) -
21 737q3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GCTCTAGACG ATCTGGAGTC AAAATGTCC
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
AGATCTGCGC AAACGGACAT TATCMGG
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
GGAATTCGCG GCCGCGTTCG ATCAACMCC CGAATCC
(2) INFORMATION FOR SEQ ID NO: 12:
-42( f ) -
21 73793
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CAATTGCAAT CGCCATGTTT ATTCTCCTAA CTTATTAAGT AGCTATTATA TTCC
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GTTAGGAGAA ACATGGCGAT TGCAATTGGC CTCGATTTTG GC
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- 4 2 ( g ) -
` 21 737~3
(ii) MOLECULE TYPE: DNA (genornic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
CGGGCGGGTG GTACCGGAAA G
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE. DNA (genornic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CTTTCCGGTA CCACCCGCCC G
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genornic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
-42 ( h) -
- 2 1 73793
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
CTAACATGTT GACTCCTTCT CTAGACTTAG CGACGAAATC CGTAATACAC
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1~:
CGGATTTCGT CGCTAAGTCT AGAGAAGGAG TCAACATGTT AGAAGATCTC
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
CCCCCAAGCT TGCGGCCGCG GCCCGTTGTC CGTCGCCAG
-42( i)-
