Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Fatty Acid Hydroperoxide Lyase Nucleic Acid Sequences
INTRODUCTION
This application claims the benefit of U.S. Provisional Application Number
60/090,924 filed June 26, 1998, U.S. Provisional Application Number 60/121,965
filed
February 26, 1999, and U.S. Provisional Application Number 60/121,968 filed
February 26,
1999.
Technical Field
This invention relates to the application of genetic engineering techniques to
plants.
More specifically, the invention relates to plant hydroperoxide lyase
sequences and methods
for the use of such sequences.
Back rg ound
With the development of genetic engineering techniques, it is now possible to
transfer
genes from a variety of organism into the genome of a large number of
different plant species.
2 0 This process has many advantages over plant breeding techniques, as genes
may now be
transferred from one plant species to another plant species, rather than
simply from a plant to
the same, or different, but closely related, species.
Degradation of polyunsaturated fatty acids starts by the oxygenation atcis-cis
double
bonds of polyunsaturated fatty acids. This reaction is catalyzed by
lipoxygenase (EC
2 5 1.13.11.12) enzymes which are present in plants, animals and
microorganisms. The
oxygenated products, called fatty acid hydroperoxides, are precursors for many
important
hormones (e.g. lipoxins, jasmonic acid, traumatic acid) and flavor/fragrance
molecules (e.g.
cis-3-hexenol, 1-octen-3-ol) in plants.
Compounds, such as jasmonic acid, are produced from hydroperoxides, such as 13-
3 0 hydroperoxylinolenic acid, via an allene oxide synthase (referred to as
AOS) and an allene
oxide cyclase (referred to as ACS)-dependent pathway. Jasmonic acid is
involved in stress
and disease resistance signaling responses via the octadecanoid pathway. 13-
hydroperoxylinolenic can also be catabolized by peroxygenases to form cutin
monomers.
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Alternatively, 13-hydroperoxylinolenic can be catabolyzed by hydroperoxide
lyase eventually
forming volatile aldehydes and traumatic acid.
Fatty acid hydroperoxide lyase (HPO lyase) catalyzes the cleavage of carbon-
carbon
bonds in polyunsaturated fatty acid hyroperoxides to produce short-chain
aldehydes and w-
oxo-acids (Vick, et al. ( 1976) Plant Physiol. 57:780-788). The products of
lysis of fatty acid
hydroperoxides, such as short-chain volatile aldehydes are common in plant
species. The
short-chain volatile aldehydes contribute to the "green notes" in a wide
variety of plant
leaves, vegetables and fruits. "Green notes" are volatile molecules that
contribute to the
organoleptic qualities of flavor and fragrance of edible plant tissues. These
qualities are often
referred to as grassy, or "green" characteristics. Other short-chain volatile
aldehydes, such as
(3Z, 6Z)-nonadienol produced by the lysis of fatty acid 9-hydroperoxide by a
fatty acid 9-
hydroperoxide lyase (9-HPO lyase or 9-HPOL), contribute a melon aroma and/or a
melon
flavor, or sometimes referred to as a "melon" or "fresh" characteristic, to
fruits and
vegetables. Such characteristics are important to industries concerned with
fragrances and
flavorings.
Furthermore, short-chain aldehydes are also thought to be involved in disease
resistance. For example, Croft, et al (( 1993) Plant Physiol. 101:13-24)
recently reported that
(32)-hexenol and (2E~-hexenal levels increased during a hypersensitive-
response in kidney
bean plants. In addition, they also demonstrated that (2E~-hexenal is an
effective antibacterial
2 0 agent.
The characterization of hydroperoxide (also referred to as HPO lyase or HPOL)
is
useful for the further study of plant fatty acid metabolism systems and for
the development of
transgenic plant with increased organoleptic properties, including aromas and
flavors.
Studies of plant mechanisms may provide means to further enhance, control,
modify, or
2 5 otherwise alter the organoleptic qualities of edible plant tissues.
Furthermore, the elucidation
of the physiological roles of HPO lyase and it's products may be useful for
the further study
of disease resistance responses, such as the HR response. Of particular
interest are the nucleic
acid sequences of genes encoding proteins which may be useful for applications
in genetic
engineering.
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SUMMARY OF THE INVENTION
The present invention is directed to hydroperoxide lyase (also referred to
herein as
HPO lyase and HPOL), and in particular to HPO lyase polynucleotides. The
polynucleotides
of the present invention include those derived from plant sources.
In one aspect of the present invention, polynucloetides are provided which
encode
HPO lyase polypeptides. In particular, polynucleoddes are provided which
encode 13-HPO
lyase polypeptides, and polynucleotides are provided encoding 9-HPO lyase
polypeptides.
One aspect of the present invention relates to oligonucleotides which include
partial or
complete HPO lyase encoding sequences.
It is also an aspect of the present invention to provide recombinant DNA
constructs
which can be used for transcription or transcription and translation
(expression) of HPO
lyase. In particular, constructs are provided which are capable of
transcription or
transcription and translation in host cells. Particularly preferred constructs
are those capable
of transcription or transcription and translation in plant cells.
In another aspect of the present invention, methods are provided for
production of
HPO lyase in a host cell or progeny thereof. In particular, host cells are
transformed or
transfected with a DNA construct which can be used for transcription or
transcription and
translation of HPO lyase. The recombinant cells which contain HPO lyase are
also part of the
2 0 present invention. Particularly preferred host cells include yeast,
bacterial, insect, and plant
cells.
In a further aspect, the present invention relates to methods of using
polynucleotide
and polypeptide sequences to modify the volatile content of host cells.
Preferred host cells of
the present invention include bacterial, yeast, insect and plant host cells.
Host cells having
2 5 such a modified volatile content are also contemplated herein.
In yet a further aspect of the present invention, methods of using
polynucleotide and
polypeptide sequences of the present invention to produce host plants having
an altered
response to diseases are provided.
The modified host cells and oils obtained by the expression or suppression of
the HPO
3 0 lyase proteins are also considered part of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
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Figure 1 shows the complete nucleotide sequence of the Arabidopsis HPO lyase.
Figure 2 shows a comparison of the amino acid sequences of the bell pepper HPO
lyase and the Arabidopsis HPO lyase-like sequence.
Figure 3 shows a comparison of the amino acid sequences of the Arabidopsis
allene
oxide synthase and the Arabidopsis HPO lyase-like sequence.
Figure 4 shows the complete nucleotide sequence of the tomato HPO lyase.
Figure 5 shows the complete nucleotide sequence of the cucumber allene oxide
synthase.
Figure 6 shows the complete nucleotide sequence of the cucumber 9-
Hydroperoxide
Lyase.
Figure 7 shows the amino acid sequence alignment between the bell pepper,
banana,
and Arabidopsis HPO lyase, with the highly conserved peptide sequences
highlighted.
Figure 8 Provides the percent similarity in the upper right corner and the
percent
divergence in the lower right corner for the nucleotide sequences (Figure 8A)
and amino acid
sequences (Figure 8B) of the bell pepper HPOL (CaHPOL), tomato fruit HPOL
(LeHPOL),
cucumber hypocotyl HPOL (CsCI7HPOL, pseudogene), Arabidopsis inflorecence HPOL
(AtHPOL), banana leaf HPOL (MsHPOL), cucumber hypocotyl 9-HPOL (Cs 15HPOL),
Guayule AOS (GuAOS), flaxseed AOS (LiAOS), and the Arabidopsis AOS (AtAOS).
Figure 9 shows the gas chromatography (GC) analysis of the cucumber 9-HPO
lyase
2 0 using linoleic acid 13-hydroperoxide (Figure 9A) and linoleic acid 9-
hydroperoxide (figure
9B) substrates.
Figure 10 provides the results of the spectrophotometric assay of the cucumber
9-
HPO lyase expressed from E. toll using linoleic acid 13-hydroperoxide and
linoleic acid 9-
hydroperoxide substrates.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to hydroperoxide lyase (also referred to herein
as HPO
lyase and HPOL), particularly the isolated HPO lyase nucleic acid sequences
encoding the
3 0 HPO lyase protein from plant sources. A hydroperoxide lyase of this
invention includes any
nucleic acid sequence encoding amino acids from a host cell source, such as a
polypeptide,
obtainable from a cell source, which demonstrates the ability to form short-
chain aldehydes
and oxo-acids from fatty acid hydroperoxides under plant enzyme reactive
conditions. By
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"enzyme reactive conditions" is meant that any necessary conditions are
available in an
environment (i.e., such factors as temperature, pH, lack of inhibiting
substances) which will
permit the enzyme to function: By "enzyme reactive conditions" is meant that
any necessary
conditions are available in an environment {i.e., such factors as temperature,
pH, lack of
inhibiting substances) which will permit the enzyme to function.
The term HPO lyase encoding sequence as applied herein refers to any
polynucleotide
sequence which encodes a hydroperoxide lyase polypeptide which is capable of
producing
short-chain aldehydes and oxo-acids from fatty acid hydroperoxides. HPO lyase
encoding
sequence can encode polypeptides having preferential activity towards
particular fatty acid
hydroperoxides. Particular fatty acid hydroperoxides include, but are not
limited to fatty acid
13-hydroperoxide, and fatty acid 9-hydroperoxide.
As used herein, the term 13-hydroperoxide lyase ( 13-HPO lyase or 13-HPOL)
refers
to any enzyme which forms short chain aldehydes andoxo-acids from fatty acid
13-
hydroperoxides. Examples of short chain aldehydes include, but are not limited
to cis 3-
Hexenal and examples of oxo-acids include, but are not limited to 12-oxo-
(9Z)dodecenoic
acid. An example of fatty acid 13-hydroperoxide include, but is not limited to
Linolenic acid
13-hydroperoxide.
As used herein, the term 9-hydroperoxide lyase (9-HPO lyase or 9-HPOL) refers
to
any enzyme which forms short chain aldehydes and oxo-acids from fatty acid 9-
2 0 hydroperoxides. Examples of short chain aldehydes include, but are not
limited to (3Z,6Z)-
nonadienal and examples of oxo-acids include, but are not limited to 9-oxo-
nonanoic acid.
An example of fatty acid 9-hydroperoxide include, but is not limited to
Linolenic acid 9-
hydroperoxide or 9-hydroperoxy-( 10E, 12Z, 15Z)~ctadecadienoic acid.
2 5 Isolated proteins, Polypeptides and Polynucleotides
A first aspect of the present invention relates to isolated HPO lyase
polynucleotides.
The polynucleotide sequences of the present invention include isolated
polynucleotides that
encode the polypeptides of the invention having a deduced amino acid sequence
selected
from the group of sequences set forth in the Sequence Listing and to other
polynucleotide
3 0 sequences closely related to such sequences and variants thereof.
The invention provides a polynucleotide sequence identical over its entire
length to
each coding sequence as set forth in the Sequence Listing. The invention also
provides the
coding sequence for the mature polypeptide or a fragment thereof, as well as
the coding
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sequence for the mature polypeptide or a fragment thereof in a reading frame
with other
coding sequences, such as those encoding a leader or secretory sequence, a pre-
, pro-, or
prepro- protein sequence. The polynucleotide can also include non-coding
sequences,
including for example, but not limited to, non-coding 5' and 3' sequences,
such as the
transcribed, untranslated sequences, termination signals, ribosome binding
sites, sequences
that stabilize mRNA, introns, polyadenylation signals, and additional coding
sequence that
encodes additional amino acids. For example, a marker sequence can be included
to facilitate
the purification of the fused polypeptide. Polynucleotides of the present
invention also
include polynucleotides comprising a structural gene and the naturally
associated sequences
that control gene expression.
The invention also includes polynucleotides of the formula:
X-(RO~ (RZ)-(Rs)n Y
wherein, at the 5' end, X is hydrogen, and at the 3' end, Y is hydrogen or a
metal, R, and R3
are any nucleic acid residue, n is an integer between 1 and 3000, preferably
between 1 and
1000 and R2 is a nucleic acid sequence of the invention, particularly a
nucleic acid sequence
selected from the group set forth in the Sequence Listing. In the formula, R2
is oriented so
that its 5' end residue is at the left, bound to R,, and its 3' end residue is
at the right, bound to
R3. Any stretch of nucleic acid residues denoted by either R group, where R is
greater than 1,
may be either a heteropolymer or a homopolyrner, preferably a heteropolymer.
2 0 The invention also relates to variants of the polynucleotides described
herein that
encode for variants of the polypeptides of the invention. Variants that are
fragments of the
polynucleotides of the invention can be used to synthesize full-length
polynucleotides of the
invention. Preferred embodiments are polynucleotides encoding polypeptide
variants wherein
5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues of a polypeptide
sequence of the
2 5 invention are substituted, added or deleted, in any combination.
Particularly preferred are
substitutions, additions, and deletions that are silent such that they do not
alter the properties
or activities of the polynucleotide or polypeptide.
Further preferred embodiments of the invention that are at least 50%, 60%, or
70%
identical over their entire length to a polynucleotide encoding a polypeptide
of the invention,
3 0 and polynucleotides that are complementary to such polynucleotides. More
preferable are
polynucleotides that comprise a region that is at least 80% identical over its
entire length to a
polynucleotide encoding a polypepdde of the invention and polynucleotides that
are
complementary thereto. In this regard, polynucleotides at least 90% identical
over their entire
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length are particularly preferred, those at least 95% identical are especially
preferred. Further,
those with at least 97% identity are highly preferred and those with at least
98% and 99%
identity are particularly highly preferred, with those at least 99% being the
most highly
preferred.
Preferred embodiments are polynucleotides that encode polypeptides that retain
substantially the same biological function or activity as the mature
polypeptides encoded by
the polynucleotides set forth in the Sequence Listing.
The invention further relates to polynucleotides that hybridize to the above-
described
sequences. In particular, the invention relates to polynucleotides that
hybridize under
stringent conditions to the above-described polynucieotides. As used herein,
the terms
"stringent conditions" and "stringent hybridization conditions" mean that
hybridization will
generally occur if there is at least 95% and preferably at least 97% identity
between the
sequences. An example of stringent hybridization conditions is overnight
incubation at 42°C
in a solution comprising 50% formamide, 5x SSC ( 150 mM NaCl, 15 mM trisodium
citrate),
50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate,
and 20
micrograms/milliliter denatured, sheared salmon sperm DNA, followed by washing
the
hybridization support in O.lx SSC at approximately 65°C. Other
hybridization and wash
conditions are well known and are exemplified in Sambrook, et al., Molecular
Cloning: A
Laboratory Manual, Second Edition, cold Spring Harbor, NY ( 1989),
particularly Chapter 11.
2 0 The invention also provides a polynucleotide consisting essentially of a
polynucleotide sequence obtainable by screening an appropriate library
containing the
complete gene for a polynucleotide sequence set for in the Sequence Listing
under stringent
hybridization conditions with a probe having the sequence of said
polynucleotide sequence or
a fragment thereof; and isolating said polynucleotide sequence. Fragments
useful for
2 5 obtaining such a polynucleotide include, for example, probes and primers
as described herein.
As discussed herein regarding polynucleotide assays of the invention, for
example,
polynucleotides of the invention can be used as a hybridization probe for RNA,
cDNA, or
genomic DNA to isolate full length cDNAs or genomic clones encoding a
polypeptide and to
isolate cDNA or genomic clones of other genes that have a high sequence
similarity to a
3 0 polynucleotide set forth in the Sequence Listing. Such probes will
generally comprise at least
15 bases. Preferably such probes will have at least 30 bases and can have at
least 50 bases.
Particularly preferred probes will have between 30 bases and 50 bases,
inclusive.
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The coding region of each gene that comprises or is comprised by a
polynucleotide
sequence set forth in the Sequence Listing may be isolated by screening using
a DNA
sequence provided in the Sequence Listing to synthesize an oligonucleotide
probe. A labeled
oligonucleotide having a sequence complementary to that of a gene of the
invention is then
used to screen a library of cDNA, genomic DNA or mRNA to identify members of
the library
which hybridize to the probe. For example, synthetic oligonucleotides are
prepared which
correspond to the HPO lyase EST sequences. The oligonucleotides are used as
primers in
polymerase chain reaction (PCR) techniques to obtain S' and 3' terminal
sequence of HPO
lyase genes. Alternatively, where oligonucleotides of low degeneracy can be
prepared from
particular HPO lyase peptides, such probes may be used directly to screen gene
libraries for
HPO lyase gene sequences. In particular, screening of cDNA libraries inphage
vectors is
useful in such methods due to lower levels of background hybridization.
Typically, a HPO lyase sequence obtainable from the use of nucleic acid probes
will
show 60-70% sequence identity between the target HPO lyase sequence and the
encoding
sequence used as a probe. However, lengthy sequences with as little as 50-60%
sequence
identity may also be obtained. The nucleic acid probes may be a lengthy
fragment of the
nucleic acid sequence, or may also be a shorter, oligonucleotide probe. When
longer nucleic
acid fragments are employed as probes (greater than about 100 bp), one may
screen at lower
stringencies in order to obtain sequences from the target sample which have 20-
50%
2 0 deviation (i.e., 50-80% sequence homology) from the sequences used as
probe.
Oligonucleotide probes can be considerably shorter than the entire nucleic
acid sequence
encoding an HPO lyase enzyme, but should be at least about 10, preferably at
least about 15,
and more preferably at least about 20 nucleotides. A higher degree of sequence
identity is
desired when shorter regions are used as opposed to longer regions. It may
thus be desirable
2 5 to identify regions of highly conserved amino acid sequence to design
oligonucleotide probes
for detecting and recovering other related HPO lyase genes. Shorter probes are
often
particularly useful for polymerase chain reactions (PCR), especially when
highly conserved
sequences can be identified. (See, Gould, et al., PNAS USA (1989) 86:1934-
1938.).
Another aspect of the present invention relates to HPO lyase polypeptides.
Such
3 0 polypeptides include isolated polypeptides set forth in the Sequence
Listing, as well as
poiypeptides and fragments thereof, particularly those polypeptides which
exhibit HPO lyase
activity and also those polypeptides which have at least 50%, 60% or 70%
identity, preferably
at least 80°!o identity, more preferably at least 90% identity, and
most preferably at least 95%
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identity to a polypeptide sequence selected from the group of sequences set
forth in the
Sequence Listing, and also include portions of such polypeptides, wherein such
portion of the
polypeptide preferably includes at least 30 amino acids and more preferably
includes at least
50 amino acids.
"Identity", as is well understood in the art, is a relationship between two or
more
polypeptide sequences or two or more polynucleotide sequences, as determined
by comparing
the sequences. In the art, "identity" also means the degree of sequence
relatedness between
polypeptide or polynucleotide sequences, as determined by the match between
strings of such
sequences. "Identity" can be readily calculated by known methods including,
but not limited
to, those described in Computational Molecular Biology, Lesk, A.M., ed.,
Oxford University
Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith,
D.W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part l,
Griffin,
A.M. and Griffin, H.G., eds., Humana Press, New Jersey ( 1994); Sequence
Analysis in
Molecular Biology, von Heinje, G., Academic Press (1987); Sequence Analysis
Primer,
Gribskov, M. and Devereux, J., eds., Stockton Press, New York ( 1991 ); and
Carillo, H., and
Lipman, D., SIAM J Applied Marh, 48:1073 (1988). Methods to determine identity
are
designed to give the largest match between the sequences tested. Moreover,
methods to
determine identity are codified in publicly available programs. Computer
programs which
can be used to determine identity between two sequences include, but are not
limited to, GCG
2 0 (Devereux, J., et al., Nucleic Acids Research 12( 1):387 ( 1984); suite of
five BLAST
programs, three designed for nucleotide sequences queries (BLASTN, BLASTX, and
TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN)
(Coulson, Trends in Biotechnology, 12: 76-80 ( 1994); Birren, et al., Genome
Analysis, 1:
543-559 ( 1997)). The BLAST X program is publicly available from NCBI and
other sources
2 5 (BLAST Manual, Altschul, S., et al., NCBI NLM NIH, Bethesda, MD 20894;
Altschul, S., et
al., J. Mol. Biol., 215:403-410 ( 1990)). The well known Smith Waterman
algorithm can also
be used to determine identity.
Parameters for polypeptide sequence comparison typically include the
following:
Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 ( 1970)
3 0 Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.
Acad.
Sci USA 89:10915-10919 (1992)
Gap Penalty: 12
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Gap Length Penalty: 4
A program which can be used with these parameters is publicly available as the
"gap"
program from Genetics Computer Group, Madison Wisconsin. The above parameters
along
with no penalty for end gap are the default parameters for peptide
comparisons.
Parameters for polynucleotide sequence comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 ( 1970)
Comparison matrix: matches = +10; mismatches = 0
Gap Penalty: 50
Gap Length Penalty: 3
A program which can be used with these parameters is publicly available as the
"gap"
program from Genetics Computer Group, Madison Wisconsin. The above parameters
are the
default parameters for nucleic acid comparisons.
The invention also includes polypeptides of the formula:
X-(R~)n-(R2)-(R3)n-Y
wherein, at the amino terminus, X is hydrogen, and at the carboxyl terminus, Y
is hydrogen or
a metal; R, and R3 are any amino acid residue, n is an integer between 1 and
1000, and R2 is
an amino acid sequence of the invention, particularly an amino acid sequence
selected from
the group set forth in the Sequence Listing. In the formula, R2 is oriented so
that its amino
terminal residue is at the left, bound to Rl, and its carboxy terminal residue
is at the right,
2 0 bound to R3. Any stretch of amino acid residues denoted by either R group,
where R is
greater than 1, may be either a heteropolymer or a homopolymer, preferably a
heteropolymer.
The polypeptides of the present invention can be mature protein or can be part
of a
fusion protein.
Fragments and variants of the polypeptides are also considered to be a part of
the
2 5 invention. A fragment is a variant polypeptide which has an amino acid
sequence that is
entirely the same as part but not all of the amino acid sequence of the
previously described
polypeptides. The fragments can be "free-standing" or comprised within a
larger polypeptide
of which the fragment forms a part or a region, most preferably as a single
continuous region.
Preferred fragments are biologically active fragments which are those
fragments that mediate
3 0 activities of the polypeptides of the invention, including those with
similar activity or
improved activity or with a decreased activity. Also included are those
fragments that
antigenic or immunogenic in an animal, particularly a human.
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Variants of the polypeptide also include polypeptides that vary from the
sequences set
forth in the Sequence Listing by conservative amino acid substitutions,
substitution of a
residue by another with like characteristics. In general, such substitutions
are among Ala,
Val, Leu and Ile; between Ser and Thr; between Asp and Glu; between Asn and
Gln; between
Lys and Arg; or between Phe and Tyr. Particularly preferred are variants in
which 5 to 10; 1
to S; 1 to 3 or one amino acids) are substituted, deleted, or added, in any
combination.
Variants that are fragments of the polypeptides of the invention can be used
to
produce the corresponding full length polypeptide by peptide synthesis.
Therefore, these
variants can be used as intermediates for producing the full-length
polypeptides of the
invention.
The poiynucleotides and polypeptides of the invention can be used, for
example, in
the transformation of host cells, such as plant host cells, as further
discussed herein.
The invention also provides polynucleotides that encode a polypeptide that is
a mature
protein plus additional amino or carboxyl-terminal amino acids, or amino acids
within the
mature polypeptide (for example, when the mature form of the protein has more
than one
polypeptide chain). Such sequences can, for example, play a role in the
processing of a
protein from a precursor to a mature form, allow protein transport, shorten or
lengthen protein
half life, or facilitate manipulation of the protein in assays or production.
It is contemplated
that cellular enzymes can be used to remove any additional amino acids from
the mature
2 0 protein.
A precursor protein, having the mature form of the polypeptide fused to one or
more
prosequences may be an inactive form of the polypeptide. The inactive
precursors generally
are activated when the prosequences are removed. Some or ail of the
prosequences may be
removed prior to activation. Such precursor protein are generally called
proproteins.
In the examples provided below, a nucleic acid sequence fromArabidopsis is
identified from Genbank which is highly homologous to a bell pepper HPO lyase.
The
Arabidopsis sequence had been previously reported to encode allene oxide
synthase.
Nucleic acid sequence comparisons between the bell pepper HPO lyase, an
Arabidopsis allene oxide synthase (Laudert, et al. (1996) Plant Mol Biol
31{2)323-335), and
3 0 the Arabidopsis sequence from Genbank indicates that the Arabidopsis
sequence is more
similar to the bell pepper HPO lyase than to the allene oxide synthase.
Also provided in the examples below, a nucleic acid sequence from cucumber
(Cucumis sativus) is identified from cDNA libraries made from total RNA
isolated from
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cucumber hypocotyls. A full length coding sequence is obtained, and the
product encoded by
the full length sequence demonstrates activity towards the substrate linolenic
acid 9-
hydroperoxide to produce (3Z, 6Z)-nonadienal and 9-oxo-nonanoic acid.
Plant Constructs and Methods of Use
Of interest is the use of the nucleotide sequences in recombinant DNA
constructs to
direct the transcription or transcription and translation (expression) of the
HPO lyase
sequences of the present invention in a host cell. Of particular interest is
the use of the
polynucleotide sequences of the present invention in recombinant DNA
constructs to direct
the transcription or transcription and translation (expression) of the HPO
lyase sequences of
the present invention in a host plant cell.
The expression constructs generally comprise a promoter functional in a host
cell
operably linked to a nucleic acid sequence encoding an HPO lyase of the
present invention
and a transcriptional termination region functional in a host plant cell. Of
particular interest
is the use of promoters (also referred to as transcriptional initiation
regions) functional in
plant host cells.
Those skilled in the art will recognize that there are a number of promoters
which are
functional in plant cells, and have been described in the literature.
Chloroplast and plastid
2 0 specific promoters, chloroplast or plastid functional promoters, and
chloroplast or plastid
operable promoters are also envisioned.
One set of promoters functional in plant cells are constitutive promoters such
as the
CaMV35S or FMV35S promoters that yield high levels of expression in most plant
organs.
Enhanced or duplicated versions of the CaMV35S and FMV35S promoters are useful
in the
2 5 practice of this invention (Odell, et al. ( 1985) Nature 313:810-812;
Rogers, U.S. Patent
Number 5,378, 619). In addition, it may also be preferred to bring about
expression of the
HPO lyase gene in specific tissues of the plant, such as leaf, stem, root,
tuber, seed, fruit, etc.,
and the promoter chosen should have the desired tissue and developmental
specificity.
Of particular interest is the expression of the nucleic acid sequences of the
present
3 0 invention from transcription initiation regions which are preferentially
expressed in a plant
seed tissue. Examples of such seed preferential transcription initiation
sequences include
those sequences derived from sequences encoding plant storage protein genes or
from genes
involved in fatty acid biosynthesis in oilseeds. Examples of such promoters
include the 5'
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regulatory regions from such genes as napin (Kridl et al., Seed Sci. Res.
1:209;219 ( 1991)),
phaseolin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase,
soybean a' subunit
of (3-conglycinin (soy 7s, (Chen et al., Proc. Natl. Acad. Sci., 83:8560-8564
(1986))) and
oleosin.
It may be advantageous to direct the localization of proteins conferring HPO
lyase to a
particular subcellular compartment, for example, to the mitochondrion,
endoplasmic
reticulum, vacuoles, chloroplast or other plastidic compartment. For example,
where the
genes of interest of the present invention will be targeted to plastids, such
as chloroplasts, for
expression, the constructs will also employ the use of sequences to direct the
gene to the
plastid. Such sequences are referred to herein as chloroplast transit peptides
{CTP) or plastid
transit peptides (PTP). In this manner, where the gene of interest is not
directly inserted into
the plastid, the expression construct will additionally contain a gene
encoding a transit
peptide to direct the gene of interest to the plastid. The chloroplast transit
peptides may be
derived from the gene of interest, or may be derived from a heterologous
sequence having a
CTP. Such transit peptides are known in the art. See, for example, Von Heijne
et al. (1991)
Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-
17550; della-
Cioppa et al. ( 1987) Plant Physiol. 84:965-968; Romer et al. ( 1993) Biochem.
Biophys. Res
Commun. 196:1414-1421; and, Shah et al. (1986) Science 233:478-4.81.
Depending upon the intended use, the constructs may contain the nucleic acid
2 0 sequence which encodes the entire HPO lyase protein, or a portion thereof.
For example,
where antisense inhibition of a given HPO lyase protein is desired, the entire
HPO lyase
sequence is not required. Furthermore, where HPO lyase sequences used in
constructs are
intended for use as probes, it may be advantageous to prepare constructs
containing only a
particular portion of a HPO lyase encoding sequence, for example a sequence
which is
2 5 discovered to encode a highly conserved HPO lyase region.
The skilled artisan will recognize that there are various methods for the
inhibition of
expression of endogenous sequences in a host cell. Such methods include, but
are not limited
to antisense suppression (Smith, et al. ( 1988) Nature 334:724-726) , co-
suppression (Napoli,
et al. (1989) Plant Cell 2:279-289), ribozymes (PCT Publication WO 97/10328),
and
3 0 combinations of sense and antisense Waterhouse, et al. ( 1998) Proc. Natl.
Acad. Sci. USA
95:13959-13964. Methods for the suppression of endogenous sequences in a host
cell
typically employ the transcription or transcription and translation of at
least a portion of the
13
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WO 00/00627 PCTNS99/14777
sequence to be suppressed. Such sequences may be homologous to coding as well
as non-
coding regions of the endogenous sequence.
Regulatory transcript termination regions may be provided in plant expression
constructs of this invention as well. Transcript termination regions may be
provided by the
DNA sequence encoding the HPO lyase or a convenient transcription termination
region
derived from a different gene source, for example, the transcript termination
region which is
naturally associated with the transcript initiation region. The skilled
artisan will recognize
that any convenient transcript termination region which is capable of
terminating transcription
in a plant cell may be employed in the constructs of the present invention.
Alternatively, constructs may be prepared to direct the expression of the HPO
lyase
sequences directly from the host plant cell plastid. Such constructs and
methods are known in
the art and are generally described, for example, in Svab, et al. ( 1990)
Proc. Natl. Acad. Sci.
USA 87:8526-8530 and Svab and Maliga ( 1993) Proc. Natl. Acad. Sci. USA 90:913-
917 and
in U.S. Patent Number 5,693,507.
A plant cell, tissue, organ, or plant into which the recombinant DNA
constructs
containing the expression constructs have been introduced is considered
transformed,
transfected, or transgenic. A transgenic or transformed cell or plant also
includes progeny of
the cell or plant and progeny produced from a breeding program employing such
a transgenic
plant as a parent in a cross and exhibiting an altered phenotype resulting
from the presence of
2 0 a HPO lyase nucleic acid sequence.
Plant expression or transcription constructs having an HPO lyase as the DNA
sequence of interest for increased or decreased expression thereof may be
employed with a
wide variety of plant life, particularly, plant life involved in the
production of vegetable oils
for edible and industrial uses. Most especially preferred are seed, fruit,
vegetable and leaf
2 5 crops. Plants of interest include, but are not limited to, rapeseed
(Canola and High Erucic
Acid varieties), sunflower, safflower, cotton, soybean, peanut, coconut and
oil palms, corn
tomato, strawberry, bell pepper and melon.. Depending on the method for
introducing the
recombinant constructs into the host cell, other DNA sequences may be
required.
Importantly, this invention is applicable to dicotyledyons and monocotyledons
species alike
3 0 and will be readily applicable to new and/or improved transformation and
regulation
techniques.
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WO 00/00627 PCT/US99/14777
Of particular interest, is the use of HPO lyase constructs in plants to
produce plants or
plant parts, including, but not limited to leaves, stems, roots, reproductive,
and seed, with a
modified volatilecontent.
It is contemplated that the gene sequences may be synthesized, either
completely or in
part, especially where it is desirable to provide plant-preferred sequences.
Thus, all or a
portion of the desired structural gene (that portion of the gene which encodes
the HPO lyase
protein) may be synthesized using codons preferred by a selected host. Host-
preferred codons
may be determined, for example, from the codons used most frequently in the
proteins
expressed in a desired host species.
One skilled in the art will readily recognize that antibody preparations,
nucleic acid
probes (DNA and RNA) and the like may be prepared and used to screen and
recover
"homologous" or "related" sequences from a variety of plant sources.
Homologous sequences
are found when there is an identity of sequence, which may be determined upon
comparison
of sequence information, nucleic acid or amino acid, or through hybridization
reactions
between a known HPO LYASE and a candidate source. Conservative changes, such
as
Glu/Asp, Val/Ile, Ser/Thr, Arg/Lys and Gln/Asn may also be considered in
determining
sequence homology. Amino acid sequences are considered homologous by as little
as 25%
sequence identity between the two complete mature proteins. (See generally,
Doolittle, R.F.,
OF URFS and ORFS (University Science Books, CA, 1986.)
2 0 Thus, other HPO lyase may be obtained from the specific sequences provided
herein.
Furthermore, it will be apparent that one can obtain natural and synthetic
sequences,
including modified amino acid sequences and starting materials for synthetic-
protein
modeling from the exemplified HPO lyase sequences and from sequences which are
obtained
through the use of such exemplified sequences. Modified amino acid sequences
include
2 5 sequences which have been mutated, truncated, increased and the like,
whether such
sequences were partially or wholly synthesized. Sequences which are actually
purified from
plant preparations or are identical or encode identical proteins thereto,
regardless of the
method used to obtain the protein or sequence, are equally considered
naturally derived.
For immunological screening, antibodies to the protein can be prepared by
injecting
3 0 rabbits or mice with the purified protein or portion thereof, such methods
of preparing
antibodies being well known to those in the art. Either monoclonal or
polyclonal antibodies
can be produced, although typically polyclonal antibodies are more useful for
gene isolation.
Western analysis may be conducted to determine that a related protein is
present in a crude
CA 02301856 2000-02-24
WO 00/00627 PCTNS99/14777
extract of the desired plant species, as determined by cross-reaction with the
antibodies to the
encoded proteins. When cross-reactivity is observed, genes encoding the
related proteins are
isolated by screening expression libraries representing the desired plant
species. Expression
libraries can be constructed in a variety of commercially available vectors,
including lambda
gtl l, as described in Sambrook, et al. (Molecular Cloning: A Laboratory
Manual, Second
Edition ( 1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
Potential host cells include both prokaryotic and eukaryotic cells. A host
cell may be
unicellular or found in a multicellar differentiated or undifferentiated
organism depending
upon the intended use. Cells of this invention may be distinguished by having
an HPO lyase
foreign to the wild-type cell present therein, for example, by having a
recombinant nucleic
acid construct encoding a HPO lyase therein not native to the host species.
Preferred host
cells include bacterial, yeast, insect, mammalian, and plant host cells.
Particularly preferred
host cells include yeast and plant host cells.
Prokaryotic cells include gram negative as well as gram positive bacteria, for
example
E. coli, and B. subtilis strains. Suitable examples are well known to the
skilled artisan. As
described in more detail in the examples that follow, an HPO lyase isolated
from cucumber
hypocotyl is expressed in E coli, strain M 15. The protein expressed from the
E coli is capable
of producing the aldehyde 3(Z)-nonenal and 2(E)-nonenal from linoleic acid 9-
hydroperoxide.
Thus, the HPO lyase isolated from the cucumber hypocotyl encodes a 9-HPO
lyase.
2 0 Eukaryotic host cells include fungi, including yeasts, insect cells, and
plant cells.
Methods for the expression of DNA sequences of interest in yeast cells are
known in the art
and are generally described in "Guide to yeast genetics and molecular
biology",Guthrie and
Fink, eds. Methods in enzymology , Academic Press, Inc. Vol 194 (1991) and
Gene
expression technology", Goeddel ed, Methods in Enzymology, Academic Press,
Inc., Vol 185
(1991). In addition, methods for the expression of HPO lyase genes are
described in
European patent Application EP 0 801 133 A2, the entirety of which is
incorporated herein by
reference.
The fungal recombinant vector may be any vector which can be conveniently
subjected to recombinant DNA procedures. The choice of a vector will typically
depend on
3 0 the compatibility of the vector with the fungal host cell into which the
vector is to be
introduced. The vector may be a linear or a closed circular plasmid. The
vector system may
be a single vector or piasmid or two or more vectors or plasmids which
together contain the
total DNA to be introduced into the genome of the fungal host.
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WO 00/00627 PCT/US99/14777
The fungal vector may be an autonomously replicating vector, i,e., a vector
which
exists as an extrachromosomal entity, the replication of which is independent
of chromosomal
replication; e.g., a plasmid, an extrachromosomal element, a minichromosome,
or an artificial
chromosome. The vector may contain any means for assuring self replication.
Alternatively,
the vector may be one which, when introduced into the fungal cell, is
integrated into the
genome and replicated together with the chromosomes) into which it has been
integrated.
For integration, the vector may rely on the nucleic acid sequence of the
vector for stable
integration of the vector into the genome by homologous or nonhomologous
recombination.
Alternatively, the vector may contain additional nucleic acid sequences for
directing
integration by homologous recombination into the genome of the fungal host.
The additional
nucleic acid sequences enable the vector to be integrated into the host cell
genome at a precise
locations) in the chromosome(s). To increase the likelihood of integration at
a precise
location, there should be preferably two nucleic acid sequences which
individually contain a
sufficient number of nucleic acids, preferably 400bp to 1500bp, more
preferably 800bp to
1000bp, which are highly homologous with the corresponding target sequence to
enhance the
probability of homologous recombination. These nucleic acid sequences may be
any
sequence that is homologous with a target sequence in the genome of the fungal
host cell and,
furthermore, may be non-encoding or encoding sequences.
For autonomous replication, the vector may further comprise an origin of
replication
2 0 enabling the vector to replicate autonomously in the host cell in
question. Examples of origin
of replications for use in a yeast host cell are the 2 micron origin of
replication and the
combination of CEN3 and ARS 1. Any origin of replication may be used which is
compatible with the fungal host cell of choice.
The fungal vectors of the present invention preferably contain one or more
selectable
2 5 markers which permit easy selection of transformed cells. A selectable
marker is a gene the
product of which provides, for example biocide or viral resistance, resistance
to heavy metals,
prototrophy to auxotrophs and the like. The selectable marker may be selected
from the
group including, but not limited to, amdS (acetamidase), argB (ornithine
carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB
(hygromycin
3 0 phosphotransferase), niaD (nitrate reductase), pyre (orotidine-5'-
phosphatedecarboxylase)
and sC (sulfate adenyltransferase) and trpC (anthranilate synthase). Preferred
for use in an
Aspergillus cell are the amdS and pyre markers of Aspergillus nidulans or
Aspergillus oryzae
and the bar marker of Streptomyces hygroscopicus. Furthermore, selection may
be
17
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WO 00/00627 PCT/US99/14777
accomplished by co-transformation, e.g., as described in WO 91/17243, the
entirety of which
is herein incorporated by reference. A nucleic acid sequence of the present
invention may be
operably linked to a suitable promoter sequence. The promoter sequence is a
nucleic acid
sequence which is recognized by the fungal host cell for expression of the
nucleic acid
sequence. The promoter sequence contains transcription and translation control
sequences
which mediate the expression of the protein or fragment thereof.
A promoter may be any nucleic acid sequence which shows transcriptional
activity in
the fungal host cell of choice and maybe obtained from genes
encodingpolypeptides either
homologous or heterologous to the host cell. Examples of suitable promoters
for directing
the transcription of a nucleic acid construct of the invention in a
filamentous fungal host are
promoters obtained from the genes encodingAspergillus oryzae TAKA amylase,
Rhizomucor
miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase,
Aspergidlus niger acid
stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase
(glaA),
Rhizomucor miehei lipase, Aspergildus oryzae alkaline protease, Aspergillus
oryzae triose
phosphate isomerase, Aspergildus nidulans acetamidase and hybrids thereof. In
a yeast host, a
useful promoter is the Saccharomyces cerevisiae enolase (eno-1) promoter.
Particularly
preferred promoters are the TAKA amylase, NA2-tpi (a hybrid of the promoters
from the
genes encoding Aspergillus niger neutral alpha -amylase and Aspergillus oryzae
triose
phosphate isomerase) and glaA promoters.
2 0 A protein or fragment thereof encoding nucleic acid molecule of the
present invention
may also be operably linked to a terminator sequence at its 3' terminus. The
terminator
sequence may be native to the nucleic acid sequence encoding the protein or
fragment thereof
or may be obtained from foreign sources. Any terminator which is functional in
the fungal
host cell of choice may be used in the present invention, but particularly
preferred terminators
2 5 are obtained from the genes encoding Aspergillus oryzae TAKA amylase,
Aspergillus niger
glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger
alpha-
glucosidase and Saccharomyces cerevisiae enolase.
A protein or fragment thereof encoding nucleic acid molecule of the present
invention
may also be operably linked to a suitable leader sequence. A leader sequence
is a
3 0 nontranslated region of a mRNA which is important for translation by the
fungal host. The
leader sequence is operably linked to the 5' terminus of the nucleic acid
sequence encoding
the protein or fragment thereof. The leader sequence may be native to the
nucleic acid
sequence encoding the protein or fragment thereof or may be obtained from
foreign sources.
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WO 00/00627 PCT/US99/14777
Any leader sequence which is functional in the fungal host cell of choice may
be used in the
present invention, but particularly preferred leaders are obtained from the
genes encoding
Aspergillus oryzae TAKA amylase andAspergillus oryzae triose phosphate
isomerase.
A polyadenylation sequence may also be operably linked to the 3' terminus of
the
nucleic acid sequence of the present invention. The polyadenylation sequence
is a sequence
which when transcribed is recognized by the fungal host to add polyadenosine
residues to
transcribed mRNA. The polyadenylation sequence may be native to the nucleic
acid
sequence encoding the protein or fragment thereof or may be obtained from
foreign sources.
Any polyadenylation sequence which is functional in the fungal host of choice
may be used in
the present invention, but particularly preferred polyadenylation sequences
are obtained from
the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger
glucoamylase,
Aspergillus nidulans anthranilate synthase and Aspergillus niger alpha-
glucosidase.
To avoid the necessity of disrupting the cell to obtain the protein or
fragment thereof
and to minimize the amount of possible degradation of the expressed protein or
fragment
thereof within the cell, it is preferred that expression of the protein or
fragment thereof gives
rise to a product secreted outside the cell. To this end, a protein or
fragment thereof of the
present invention may be linked to a signal peptide linked to the amino
terminus of the
protein or fragment thereof. A signal peptide is an amino acid sequence which
permits the
secretion of the protein or fragment thereof from the fungal host into the
culture medium.
2 0 The signal peptide may be native to the protein or fragment thereof of the
invention or may be
obtained from foreign sources. The 5' end of the coding sequence of the
nucleic acid
sequence of the present invention may inherently contain a signal peptide
coding region
naturally linked in translation reading frame with the segment of the coding
region which
encodes the secreted protein or fragment thereof. Alternatively, the 5' end of
the coding
2 5 sequence may contain a signal peptide coding region which is foreign to
that portion of the
coding sequence which encodes the secreted protein or fragment thereof. The
foreign signal
peptide may be required where the coding sequence does not normally contain a
signal
peptide coding region. Alternatively, the foreign signal peptide may simply
replace the
natural signal peptide to obtain enhanced secretion of the desired protein or
fragment thereof.
3 0 The foreign signal peptide coding region may be obtained from a
glucoamyiase or an amylase
gene from an Aspergillus species, a lipase or proteinase gene from Rhizomucor
miehei, the
gene for the alpha-factor from Saccharomyces cerevisiae, or the calf
preprochymosin gene.
An effective signal peptide for fungal host cells is the Aspergillus oryzae
TAKA amylase
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WO 00/00627 PCT/US99/14777
signal, Aspergillus niger neutral amylase signal, the Rhizomucor miehei
aspartic proteinase
signal, the Humicola lanuginosus cellulase signal, or the Rhizomucor miehei
lipase signal.
However, any signal peptide capable of permitting secretion of the protein or
fragment
thereof in a fungal host of choice may be used in the present invention.
A protein or fragment thereof encoding nucleic acid molecule of the present
invention
may also be linked to a propeptide coding region. A propeptide is an amino
acid sequence
found at the amino terminus of aproprotein or proenzyme. Cleavage of the
propeptide from
the proprotein yields a mature biochemically active protein. The resulting
polypeptide is
known as a propolypeptide or proenzyme (or a zymogen in some cases).
Propolypeptides are
generally inactive and can be converted to mature active polypeptides by
catalytic or
autocatalytic cleavage of the propeptide from the propolypeptide or proenzyme.
The
propeptide coding region may be native to the protein or fragment thereof or
may be obtained
from foreign sources. The foreign propeptide coding region may be obtained
from the
Saccharomyces cerevisiae alpha-factor gene or Myceliophthora thermophila
laccase gene
(WO 95/33836, the entirety of which is herein incorporated by reference).
The procedures used to ligate the elements described above to construct the
recombinant expression vector of the present invention are well known to one
skilled in the
art (see, for example, Sambrook et al., Molecular Cloning, A Laboratory
Manual, 2nd ed.,
Cold Spring Harbor, N.Y., (1989)).
2 0 The present invention also relates to recombinant fungal host cells
produced by the
methods of the present invention which are advantageously used with the
recombinant vector
of the present invention. The cell is preferably transformed with a vector
comprising a
nucleic acid sequence of the invention followed by integration of the vector
into the host
chromosome. The choice of fungal host cells will to a large extent depend upon
the gene
2 5 encoding the protein or fragment thereof and its source. The fungal host
cell may, for
example, be a yeast cell or a filamentous fungal cell.
"Yeast" as used herein includes Ascosporogenous yeast (Endomycetales),
Basidiosporogenous yeast and yeast belonging to the Fungi Imperfecti
(Blastomycetes). The
Ascosporogenous yeasts are divided into the families Spermophthoraceae and
3 0 Saccharomycetaceae. The latter is comprised of four subfamilies,
Schizosaccharomycoideae
(for example, genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae and
Saccharomycoideae (for example, genera Pichia, Kluyveromyces and
Saccharomyces). The
Basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium,
CA 02301856 2000-02-24
WO 00/00627 PCT/US99/14777
Sporidiobolus, Filobasidium and Filobasidiella. Yeast belonging to the Fungi
Imperfecti are
divided into two families, Sporobolomycetaceae (for example, genera
Sorobolomyces and
Bullera) and Cryptococcaceae (for example, genus Candida). Since the
classification of
yeast may change in the future, for the purposes of this invention, yeast
shall be defined as
described in Biology and Activities of Yeast (Skinner et al., Soc. App.
Bacteriol. Symposium
Series No. 9, ( 1980), the entirety of which is herein incorporated by
reference). The biology
of yeast and manipulation of yeast genetics are well known in the art (see,
for example,
Biochemistry and Genetics of Yeast, Baeil et al. (ed.), 2nd edition, 1987; The
Yeasts, Rose
and Harrison (eds.), 2nd ed., ( 1987); and The Molecular Biology of the Yeast
Saccharomyces,
Strathern et al. (eds.), ( 1981 ), all of which are herein incorporated by
reference in their
entirety).
The recombinant fungal host cells of the present invention may further
comprise one
or more sequences which encode one or more factors that are advantageous in
the expression
of the protein or fragment thereof, for example, an activator (e.g., a trans-
acting factor), a
chaperone and a processing protease. The nucleic acids encoding one or more of
these factors
are preferably not operably linked to the nucleic acid encoding the protein or
fragment
thereof. An activator is a protein which activates transcription of a nucleic
acid sequence
encoding a polypeptide (Kudla et al., EMBO 9:1355-1364(1990); Jarai and
Buxton, Current
Genetics 26:2238-244(i994); Verdier, Yeast 6:271-297(1990), all of which are
herein
2 0 incorporated by reference in their entirety). The nucleic acid sequence
encoding an activator
may be obtained from the genes encoding Saccharomyces cerevisiae heme
activator protein 1
(hap 1 ), Saccharomyces cerevisiae galactose metabolizing protein 4 (gal4) and
Aspergillus
nidulans ammonia regulation protein (areA). For further examples, see Verdier,
Yeast 6:271- ..-
297 ( 1990); MacKenzie et al., Journal of Gen. Microbiol. 139:2295-2307 (
1993), both of
2 5 which are herein incorporated by reference in their entirety). A chaperone
is a protein which
assists another protein in folding properly (Hard et al., TIBS 19:20-25 (
1994); Bergeron et al.,
TIBS 19:124-128 ( 1994); Demolder et al., J. Biotechnology 32:179-189 ( 1994);
Craig,
Science 260:1902-1903(1993); Gething and Sambrook, Nature 355:33-45 (1992);
Puig and
Gilbert, J Biol. Chem. 269:7764-7771 ( 1994); Wang and Tsou, FASEB Journal
7:1515-11157
3 0 (1993); Robinson et al., Biollechnology 1:381-384 (1994), all of which are
herein
incorporated by reference in their entirety). The nucleic acid sequence
encoding a chaperone
may be obtained from the genes encodingAspergillus oryzae protein disulphide
isomerase,
Saccharomyces cerevisiae calnexin, Saccharomyces cerevisiae BiP/GRP78 and
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CA 02301856 2000-02-24
WO 00/00627 PCT/US99/14777
Saccharomyces cerevisiae Hsp70. For further examples, see Gething and
Sambrook, Nature
355:33-45 (1992); Hard et al., TIBS 19:20-25 ( 1994). A processing protease is
a protease that
cleaves a propeptide to generate a mature biochemically active polypeptide
(Enderlin and
Ogrydziak, Yeast 10:67-79 (1994); Fuller et al., Proc. Natl. Acad. Sci.
(U.S.A.) 86:1434-1438
(1989); Julius et al., Cell 37:1075-1089 (1984); Julius et al., Cell 32:839-
852 {1983}, all of
which are incorporated by reference in their entirety). The nucleic acid
sequence encoding a
processing protease may be obtained from the genes encodingAspergillus niger
Kex2,
Saccharomyces cerevisiae dipeptidylaminopeptidase, Saccharomyces cerevisiae
Kex2 and
Yarrowia lipolytica dibasic processing endoprotease (xpr6). Any factor that is
functional in
the fungal host cell of choice may be used in the present invention.
Fungal cells may be transformed by a process involving protoplast formation,
transformation of the protoplasts and regeneration of the cell wall in a
manner known per se.
Suitable procedures for transformation of Aspergillus host cells are described
in EP 238 023
and Yelton et al., Proc. Natl. Acad. Sci. (U.S.A.) 81:1470-1474 (1984), both
of which are
herein incorporated by reference in their entirety. A suitable method of
transforming
Fusarium species is described by Malardier et al., Gene 78:147-156 (1989), the
entirety of
which is herein incorporated by reference. Yeast may be transformed using the
procedures
described by Becker and Guarente, In: Abelson and Simon, (eds.), Guide to
Yeast Genetics
and Molecular Biology, Methods Enzymol. Volume 194, pp 182-187, Academic
Press, Inc.,
2 0 New York; Ito et al., J. Bacteriology 153:163 ( 1983); Hinnen et al.,
Proc. Natl. Acad. Sci.
(U.S.A.) 75:1920 (1978), all of which are herein incorporated by reference in
their entirety.
The present invention also relates to methods of producing the protein or
fragment
thereof comprising culturing the recombinant fungal host cells under
conditions conducive for
expression of the protein or fragment thereof. The fungal cells of the present
invention are
2 5 cultivated in a nutrient medium suitable for production of the protein or
fragment thereof
using methods known in the art. For example, the cell may be cultivated by
shake flask
cultivation, small-scale or large-scale fermentation (including continuous,
batch, fed-batch, or
solid state fermentations) in laboratory or industrial fermentors performed in
a suitable
medium and under conditions allowing the protein or fragment thereof to be
expressed and/or
3 0 isolated. The cultivation takes place in a suitable nutrient medium
comprising carbon and
nitrogen sources and inorganic salts, using procedures known in the art (see,
e.g., Bennett and
LaSure (eds.), More Gene Manipulations in Fungi, Academic Press, CA, (1991),
the entirety
of which is herein incorporated by reference). Suitable media are available
from commercial
22
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WO 00/00627 PCT/US99/14777
suppliers or may be prepared according to published compositions (e.g., in
catalogues of the
American Type Culture Collection, Manassas, VA). If the protein or fragment
thereof is
secreted into the nutrient medium, a protein or fragment thereof can be
recovered directly
from the medium. If the protein or fragment thereof is not secreted, it is
recovered from cell
lysates.
The expressed protein or fragment thereof. may be detected using methods known
in
the art that are specific for the particular protein or fragment. These
detection methods may
include the use of specific antibodies, formation of an enzyme product, or
disappearance of an
enzyme substrate. For example, if the protein or fragment thereof has
enzymatic activity, an
enzyme assay may be used. Alternatively, ifpolyclonal or monoclonal antibodies
specific to
the protein or fragment thereof are available, immunoassays may be employed
using the
antibodies to the protein or fragment thereof. The techniques of enzyme assay
and
immunoassay are well known to those skilled in the art.
The resulting protein or fragment thereof may be recovered by methods known in
the
arts. For example, the protein or fragment thereof may be recovered from the
nutrient
medium by conventional procedures including, but not limited to,
centrifugation, filtration,
extraction, spray-drying, evaporation, or precipitation. The recovered protein
or fragment
thereof may then be further purified by a variety of chromatographic
procedures, e.g., ion
exchange chromatography, gel filtration chromatography, affinity
chromatography, or the
2 0 like.
Methods for the expression of DNA sequences of interest in insect host cells
are also
well known in the art, and are reviewed by Lucow and Summers, ( 1988)
Bioltechnology 6:47-
55, the entirety of which is incorporated herein by reference.
To confirm the activity and specificity of the proteins encoded by the
identified
2 5 nucleic acid sequences as HPO lyase enzymes, in vitro assays can be
performed in insect cell
cultures using bacuiovirus expression systems. Such baculovirus expression
systems are
known in the art and are described by Lee, et al. U.S. Patent Number
5,348,886, the entirety
of which is herein incorporated by reference.
In addition, other expression constructs may be prepared to assay for protein
activity
3 0 utilizing different expression systems. Such expression constructs are
transformed into yeast
or prokaryotic host and assayed for HPO lyase activity. Such expression
systems are known
in the art and are readily available through commercial sources.
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The method of transformation in obtaining such transgenic plants is not
critical to the
instant invention, and various methods of plant transformation are currently
available.
Furthermore, as newer methods become available to transform crops, they may
also be
directly applied hereunder. For example, many plant species naturally
susceptible to
Agrobacterium infection may be successfully transformed via tripartite or
binary vector
methods of Agrobacterium mediated transformation. In many instances, it will
be desirable
to have the construct bordered on one or both sides by T-DNA, particularly
having the left
and right borders, more particularly the right border. This is particularly
useful when the
construct uses A. tumefaciens or A. rhizogenes as a mode for transformation,
although the T-
DNA borders may find use with other modes of transformation. In addition,
techniques of
microinjection, DNA particle bombardment, and electroporation have been
developed which
allow for the transformation of various monocot and dicot plant species.
Normally, included with the DNA construct will be a structural gene having the
necessary regulatory regions for expression in a host and providing for
selection of
transformant cells. The gene may provide for resistance to a cytotoxic agent,
e.g. antibiotic,
heavy metal, toxin, etc., complementation providing prototrophy to an
auxotrophic host, viral
immunity or the like. Depending upon the number of different host species the
expression
conswct or components thereof are introduced, one or more markers may be
employed,
where different conditions for selection are used for the different hosts.
2 0 Where Agrobacterium is used for plant cell transformation, a vector may be
used
which may be introduced into the Agrobacterium host for homologous
recombination with T-
DNA or the Ti- or Ri-plasmid present in the Agrobacterium host. The Ti- or Ri-
plasmid
containing the T-DNA for recombination may be armed (capable of causing gall
formation)
or disarmed (incapable of causing gall formation), the latter being
permissible, so long as the
2 5 vir genes are present in the transformed Agrobacterium host. The armed
plasmid can give a
mixture of normal plant cells and gall.
In some instances where Agrobacterium is used as the vehicle for transforming
host
plant cells, the expression or transcription construct bordered by the T-DNA
border regions)
will be inserted into a broad host range vector capable of replication in E.
codi and
3 0 Agrobacterium, there being broad host range vectors described in the
literature. Commonly
used is pRK2 or derivatives thereof. See, for example, Ditta, et al., (Proc.
Nat. Acad. Sci.,
U.S.A. ( 1980) 77:7347-735 I ) and EPA 0 120 515, which are incorporated
herein by reference.
Alternatively, one may insert the sequences to be expressed in plant cells
into a vector
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WO 00/00627 PCT/US99/14777
containing separate replication sequences, one of which stabilizes the vector
in E. coli, and
the other in Agrobacterium. See, for example, McBride and Summerfelt (Plant
Mol. Biol.
( 1990) 14:269-276), wherein the pRiHRI (Jouanin, et al., Mol. Gen. Genet. (
1985) 201:370-
374) origin of replication is utilized and provides for added stability of the
plant expression
vectors in host Agrobacterium cells.
Included with the expression construct and the T-DNA will be one or more
markers,
which allow for selection of transformed Agrobacterium and transformed plant
cells. A
number of markers have been developed for use with plant cells, such as
resistance to
chloramphenicol, kanamycin, the aminoglycoside 6418, hygromycin, or the like.
The
particular marker employed is not essential to this invention, one or another
marker being
preferred depending on the particular host and the manner of construction.
For transformation of plant cells using Agrobacterium, explants may be
combined and
incubated with the transformed Agrobacterium for sufficient time for
transformation, the
bacteria killed, and the plant cells cultured in an appropriate selective
medium. Once callus
forms, shoot formation can be encouraged by employing the appropriate plant
hormones in
accordance with known methods and the shoots transferred to rooting medium for
regeneration of plants. The plants may then be grown to seed and the seed used
to establish
repetitive generations and for isolation of vegetable oils.
Thus, in another aspect of the present invention, methods for modifying the
volatile
2 0 composition of a host cell. In general the methods involve either
increasing or decreasing the
levels of volatile compounds in a host cell. The method generally comprises
the use of
expression constructs to direct the expression of the polynucleotides of the
present invention
in a host cell.
Of particular interest is the use of expression constructs to modify the
levels of
2 5 volatile compounds in a host plant cell. Most particular, the methods find
use in modifying
the levels of volatile compounds in plant parts including, but not limited to,
leaves, roots,
stems, flowers, tuber, fruits, legumes, seeds, and seed oils obtained from
plant seeds.
In another embodiment of the present invention, expression constructs are
provided
which direct the expression of nucleic acid sequences encoding HPO lyase
fromArabidopsis
3 0 in bacterial and plant tissues.
Of particular interest in the present invention, is the use of such expression
constructs
to produce transgenic plants with increased production of short-chain volatile
aldehydes in
plant fruits and tissues. Such volatile aldehydes are important constituents
of the
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characteristic flavors of fruits, vegetables and green leaves (also referred
to as "green notes").
Thus, the HPO lyase sequence of the present invention may be used in
expression constructs
to produce transgenic plants with improved green note flavor characteristics.
In order to increase lipid peroxidation, and thereby increasing "green note"
and/or
"melon" flavors/fragrances, in a plant tissue, coexpression of a plant or
other 9-HPO lyase
and/or 13-HPO lyase in a plant tissue with a second gene involved in lipid
peroxidation may
also find use in the present invention. For example, coexpression of a 13-HPO
lyase andlor
9-HPO lyase sequence in a plant tissue with a DNA sequence encoding for
another protein
involved in lipid peroxidation, such as a lipoxygenase may increase lipid
peroxidation and
increase the total short-chain aldehydes produced in the plant tissue. .Such
an increase in
short-chain aldehydes may increase the "green note" and/or "melon" flavor in
an edible plant
tissue.
Host cells expressing the 9-HPO lyases of the present invention provide a
novel
source of volatile aldehydes contributing to "green" and/or "melon" notes for
use in various
applications. Furthermore, the host cells may also contain constructs
providing for a
increased production of enzymes involved in lipid peroxidation, for example
lipoxygenase.
In addition, the host cells may also produce an increased amount of a
particular fatty acid, or
have a general increase in fatty acids. Such host cells may be obtained using
traditional
breeding techniques, including mutagenesis, as well as hosts genetically
engineered with such
2 0 an altered fatty acid composition.
Furthermore, plant host cells containing a construct providing for the
expression of
the HPO lyase sequences of the present invention find use as a source for
aldehydes in
reactions for the production of alcohols for use in flavorings and aromatic
products. Such
methods are known in the art and are described for example in U.S. Patent
Number 5,695,973
2 5 and in PCT Publication WO 95/26413 the entireties of which are
incorporated herein by
reference. Generally, a mixture of aldehydes and alcohols are obtained from
such methods.
The methods generally involve a reaction mixture containing at least one
unsaturated fatty
acid, a plant material having a relatively high amount of enzyme activity of
lipoxygenase and
hydroperoxide lyase, and a source of alcohol dehydrogenase.
3 0 The unsaturated fatty acid may vary and include a single unsaturated fatty
acid species
as well as mixtures of several unsaturated fatty acids. The fatty acids are
provided in a free
acid form, and examples include, but are not limited to oleic acid, linoleic
acid, linolenic acid
(alpha and gamma forms), arachidonic acid, eicosapentaenoic acid, and
ricinoleic acid.
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Sources of the alcohol dehydrogenase include yeasts, as well as non-yeast
molds. The
alcohol dehydrogenase has the ability to convert an aldehyde to an alcohol.
The yeast and
non-yeast molds further provide a source of nicotine adenine dinucleotide
(NADH) as a
reducing agent.
The nucleic acid sequences of the present invention may also find use in
expression
constructs for the production of transgenic plants with increased resistance
to various
pathogens. Transgenic plants expressing the HPO lyase sequence of the present
invention
may exhibit an enhanced hypersensitive-reaction (HR response) in response to
pathogen
attack due to the increased production of aldehydes involved in the HR
response, such as
(32)-hexenal and (2E)-hexenal (Croft, et al. (1993) Plant Physiol. 101:13-24).
Aldehydes,
such as {2E)-hexenal, have also been shown to be effective anti-bacterial
agents, further
contributing to enhanced disease resistance (Croft, et al. (1993), supra).
Furthermore, these
compounds may be involved in a general wounding response in plants.
Also of particular interest in the present invention is the use of 9-HPO lyase
nucleic
acid sequences in constructs to direct the expression of 9-HPO lyase in a
prokaryotic and/or
eukaryotic host cells for the production of flavorings and aromas.
The invention now being generally described, it will be more readily
understood by
reference to the following examples which are included for purposes of
illustration only and
are not intended to limit the present invention.
EXAMPLES
Example 1 Identification of Arabidopsis HPO Lyase Sequences
A nucleic acid fragment encoding hydroperoxide lyase from Bell pepper has been
previously cloned and sequenced (Matsui, et al. ( 1996) FEES Letters 394:21-
24). The
2 5 nucleotide sequence was used to search Genbank for HPO lyase related
sequences. One
accession identified from Genbank (Accession Number 297339,
(http://www.ncbi.nlm.nih.gov/web/Genbank/Index.html)) containing a genomic
sequence
from Arabidopsis was reported to encode an allene oxide synthase (Laudert, et
al. ( 1996)
Plant Mol. Baol. 31:323-335).
3 0 Sequence comparisons between the bell pepper HPO lyase, Arabidopsis allene
oxide
synthase (Laudert, et al. ( 1996) supra) and the Arabidopsis HPO lyase-like
sequence from
Genbank using Genetyx Mac (Software Development Co. Ltd.) indicated that
theArabidopsis
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WO 00/00627 PCTNS99/14777
HPO lyase-like sequence is more similar to the bell pepper HPO lyase (57%
identity) (see
Figure 2) than to the allene oxide synthase sequence (39% identity) (see
Figure 3).
Example 2 Construction of Arabidopsis cDNA libraries
Total RNA from seedling, inflorescence, and silique tissues of Arabidopsis
thaliana is
isolated for use in construction of complementary (cDNA) libraries. The
procedure is an
adaptation of the DNA isolation protocol of Webb and Knapp (D.M. Webb and S.J.
Knapp,
(1990) Plant Molec. Reporter, 8, 180-185). The following description assumes
the use of lg
fresh weight of tissue. Frozen seed tissue is powdered by grinding under
liquid nitrogen. The
powder is added to lOml REC buffer (SOmM Tris-HCI, pH 9, 0.8M NaCi, lOmM EDTA,
0.5% w/v CTAB (cetyltrimethyl-ammonium bromide)) along with 0.2g insoluble
polyvinylpolypyrrolidone, and ground at room temperature. The homogenate is
centrifuged
for 5 minutes at 12,000 xg to pellet insoluble material. The resulting
supernatant fraction is
extracted with chloroform, and the top phase is recovered.
The RNA is then precipitated by addition of 1 volume RecP (SOmM Tris-HCL pH9,
lOmM EDTA and 0.5% (w/v) CTAB) and collected by brief centrifugation as
before. The
RNA pellet is redissolved in 0.4 ml of 1M NaCI. The RNA pellet is redissolved
in water and
extracted with phenol/chloroform. Sufficient 3M potassium acetate (pH 5) is
added to make
the mixture 0.3M in acetate, followed by addition of two volumes of ethanol to
precipitate the
2 0 RNA. After washing with ethanol, this final RNA precipitate is dissolved
in water and stored
frozen.
Alternatively, total RNAs may be obtained using TRIzoI reagent (BRL Life
Technologies, Gaithersburg, MD) following the manufacturers protocol.
Complementary DNAs (cDNA) are obtained from the RNAs using the Marathon
2 5 cDNA Amplification Kit (Clontech, Palo Alto, CA) following the
manufacturers directions.
Example 3 Cloning of HPO Lyase Sequences
In order to characterize the protein encoded by theArabidopsis cDNA GenBank
sequence, the entire coding region corresponding to the Arabidopsis HPO lyase-
like cDNA
3 0 was obtained. (Figure 1 ) Synthetic oligo-nucleotide primers are designed
to amplify the 5'
and 3' ends from the HPO lyase-like sequence from RNA obtained in Example 2.
Primers
are designed according to the Arabidopsis HPO lyase-like sequence and are used
in Rapid
Amplification of cDNA Ends (RACE) reactions (Frohman et al. ( 1988) Proc.
Natl. Acad. Sci.
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WO 00/00627 PCT/US99/14777
USA 85:8998-9002). Amplification of flanking sequences from cDNA clones are
performed
using the Marathon cDNA Amplification kit (Clontech) according to the
manufacturers
protocol.
A pair of primers were designed to amplify the 5' and 3' regions from
theArabidopsis
HPO lyase-like cDNA from the libraries described in example 2 above. These two
primers,
HPOL28 (for 3' RACE, 5'-CGGTTCCTCTGCGCCTCTCTCGCCGGCG-3') and HPOL21
(for 5' RACE, 5'-GCGGAACCGGAGGACTAAAACGCAGC-3') are used in PCR reactions
with Adapter specific primers (AP15'-CCATCCTAATACGACTCACTATAGGGC-3')
provided in the Marathon cDNA Amplification Kit. For amplification of the 5'
region of the
HPO lyase-like cDNA the primers AP1 and HPOL 21 were used, and for the
amplification of
the 3' region the AP 1 primer was used in a reaction with the primer HPOL28.
The cycle
conditions used are: 94°C for 1 minute followed by 5 rounds of
94°C for 5 seconds, 72°C for
4 minutes, followed by 5 rounds of amplification using 94°C for 5
seconds, 70°C for 4
minutes, and finally 25 cycles of 94°C for 5 seconds, and 68°C
for 4 minutes
A single fragment of 1100 by was obtained from the 3'RACE reaction with RNA
obtained from the silique tissue described above. To confirm that the PCR
product contained
sequence corresponding to the HPO lyase-like sequence, a second round of PCR
reactions
using the same conditions described above was performed with the gel purified
1100 by
fragment. A reaction was performed with the primers HPOL13 (5'-
2 0 CTTGGCGTAGTTCCTCAGCCTCTTG-3') and AP2 (5'-
ACTCACTATAGGGCTCGAGCGGC-3' ) to amplify an approximately 1000 by fragment as
a confirmation of the HPO lyase-like sequence. The reamplified 1000 by
fragment was gel
purified and cloned into the pCR2.1 TOPO vector (Invitrogen, Carlsbad, CA) to
create the
plasmid pCGN8094.
2 5 The 5' RACE reaction produced many non-specific fragments. A 1000 by
fragment
was excised from the gel and cloned into the pCR2.1 TOPO(Invitrogen) cloning
vector to
create the plasmid pCGN8091.
Figure 1 discloses the complete nucleotide sequence of the Arabidopsis HPO
lyase.
The cDNA has a sequence of 1687 bp, and 47 and 137 by of 5'- and 3'-noncoding
regions,
3 0 respectively. There is a stop codon in the 5'-noncoding region in-frame
with the
initiation codon. The longest ORF encodes a polypeptide of 492 amino acids
with a
calculated molecular mass of 54851 Da.
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In order to determine the expression pattern of the Arabidopsis HPO lyase
sequence,
Northern blot analysis of total RNA isolated, from various organs, as well as
wounded, methyl
jasmonate, and pathogen challenged leaves, of Arabidopsis is performed.
Total RNA is isolated from rosetta leaves, leaves on stems, stems,
inflorescence,
green buds, closed flowers, open flowers, siliques (5-10 mm), sffiques (<5
mm), and 2, 3, 4,
5, and 6 day after germination seedlings using TRIzoI reagent (Life
Technologies,
Gaithersburg, MD). The isolated RNA samples ( 10-20 ~,g) are separated on a
formaldehyde-
agarose gel and transferred to Hybond-N (Amersham). The transferred RNA is
hybridized
overnight with a probe corresponding to the 5' half of the Arabidopsis HPO
lyase cDNA at
65°C in 6x SSC, 5x Denhardt's solution, 0.2% SDS, 20~.g/ml salmon sperm
DNA, 20 mM
sodium phosphate buffer, pH 7Ø The hybridized membranes are washed once with
2x SSC,
0.1% SDS at 60°C for 20 minutes, and twice with 0.25x SSC, 0.1% SDS at
60° C for 20
minutes each. A 1.6 kb transcript corresponding to the HPO lyase gene is
observed in RNA
isolated from all the tissue sources examined. The highest level of expression
is observed in
the inflorescence. Additional bands of approximately 3.0 and 3.3 kb are
observed possibly
due to read-through of the intron-exon junction during transcription. In
addition, Rojo, et al.
(1998) Plant Journal 13:153-165 reports the hybridization of two mRNA bands of
different
size on Northern blots using a probe of HPO lyase.
To determine if the HPO lyase sequence is expressed in response to wounding
and
methyl jasmonate RNA is isolated from wounded leaves and leaves treated with
methyl
jasmonate (MJ). HPO lyase expression is also examined for induction by fungal
attack.
Arabidopsis thaliana ecotype No-O is grown in soil under 16 hour light at
22° C at
65% relative humidity for three weeks. For wounding, each leaf is wounded once
with a
hemostat on the upper third of the leaf in line with the midvein. In each
rosette, half of the
2 5 leaves are wounded and half are not. These are referred to as local and
systemic leaves
respectively.
For treatment with MJ, plants are enclosed in airtight 9.25 L jars. Neat MJ (
10 or 50
~,1, Aldrich Co. Milwaukee, WI) is applied onto four cotton swabs and placed
in the jars
without directly touching the plants. Fresh MJ treated cotton swabs are
replaced in the jar
3 0 each time the jar is opened. For pathogen induction, spores of the fungus
Botrytis cinerea are
sprayed on the rosette leaves of 3 week old plants at a concentration of 1 (~
spores per ml in
1 % glucose.
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Total RNA is isolated from the tissues using TRIzoI reagent (Life
Technologies) as
described by the manufacturer and transferred to membrane as described above.
Expression of the HPO lyase mRNA is observed at 6 and 24 hours after wounding.
High levels of HPO lyase induction are seen in leaves wounded with a hemostat.
The
induction is distinct after 6 hours of the treatment and the amount of HPO
lyase mRNA
increased at least until 24 hours. In the systemic leaves, induction of the
HPO lyase mRNA is
also evident. After 6 hours of treatment, the level of HPO lyase mRNA is
almost the same as
that of the local leaves, however, the amount increases only slightly
afterwards and at 24
hours after treatment, the level is observably lower than that of the local
leaves.
Plants treated with methyl jasrnonate demonstrated a low expression of HPO
lyase are
observed in both 10~,i and 50p,1 treatments at all time points examined.
Infection with the fungal pathogen Botrytis cinerea did not induce HPO lyase
expression. However, necrotic lesions are observed on the leaf surfaces of
treated plants 5 to
6 days post inoculation.
Example 4 Preparation HPO Lyase Expression Constructs
A set of constructs are prepared for transformation into either plant or
bacterial hosts
to further characterize the Arabidopsis HPO lyase-like sequence. The 5' RACE
product in
pCGN8091 was PCR amplified using the primers Alex2 (5'-
CGGGATCCATGTTGTTGAGAACGATGGCGGCG-3') and Alex4 (5'-
CAATCTCCGGCGTTCTCGTCG-3'). The Alex2 primer contains the restriction
endonuclease site BamHI for the convenient cloning of the PCR product into the
pQE30
expression vector (Qiagen, Hilden, Germany) in frame with the ATG start codon
of the
vector. In addition to the oligonucleotide primers (0.2N.M each), the PCR
reaction mix
2 5 contained 0.2 mM each of dATP, dCTP, dGTP and dTTP, 1.0% glycerol, 0.2 mM
Tris-HCl
(pH 8.3), 4.6 mM KCI, 1.5 mM EDTA, 15 l.~M dithiothreitol, 7.3 pgm/ml BSA, 1.1
mM
KOAc and 0.1 units Pfu DNA polymerase (BRL Life Technologies, Gaithersburg,
MD). The
mixtures were amplified using the following conditions: 1 cycle of 95°C
for 10 minutes; 30
cycles of 94°C for 20 seconds, 60°C for 30 seconds, and
72°C for 1.5 minutes; and, 1 cycle of
3 0 72°C for 7 minutes in a Perkin-Elmer 9800 thermocycler. The
resulting PCR product was
digested with BamHI and HindIII and ligated into the vector pQE30 to create
the vector
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WO 00/00627 PCT/US99/14777
pCGN8099. The 3' terminus of the Arabidopsis HPO lyase was cloned into the
HindITI site
of pCGN8099 from pCGN8094 to create the E. coli expression vector pCGN8100
A binary vector for plant transformation, pCGN5138, was constructed from
pCGN 1558 (McBride and Summerfelt, ( 1990) Plant Molecular Biology, 14:269-
276). The
polylinker of pCGN 1558 was replaced as an HindIIIlEcoRI fragment with a
polylinker
containing unique restriction endonuclease sites, HindIII, SseIlPstI, NotI,
BamHI, Swal, XbaI,
PacI, AscI, and Asp718.
An antisense construct of the Arabidopsis HPO lyase-like nucleotide sequence
was
prepared for transformation of Arabidopsis. The nucleic acid sequence encoding
the 5' 1000
by nucleotides from pCGN8091 were cloned as an EcoRI fragment into the
plasrnid
pBluescript II SK (Stratagene, La Jolla, CA) to create the vector pCGN8093.
The 3' RACE
product from pCGN8090 was cloned as a HindIII fragment into pCGN8093 to create
a full
length HPO lyase coding sequence in the plasmid pCGN8094. The KpnI site of
pCGN8094
was removed by digesting with KpnI and filling in the site with Klenow
fragment, and the
HPO lyase coding sequence was cloned from this plasmid as a SmaI fragment into
the StuI
site of pCGN8059. This yields the plasmid pCGN8101. The plasmid pCGN8059
contains a
multiple cloning site downstream of the 35S promoter and the hsp70 leader
sequence to allow
for the cloning of sequences for expression from the 35S promoter sequence.
This vector also
contains the nopaline synthase transcription termination (nos 3') sequences
(Fraley et al.,
2 0 Proc. Natl. Acad. Sci ( 1983) 80:4803-4807 and Depicker et al., J. Molec.
Appl. Genet. ( 1982)
l: 562-573). The fragment containing the 35S promoter/hsp70 leader,
antisenseArabidopsis
HPO lyase sequence, and nos3' termination sequence was cloned from pCGN8101 as
a NotI
fragment into the same site of pCGN5138 to create the antisense expression
construct
pCGN8102.
Example 5 E coli Expression
The expression vector pCGN8100 was transformed into E coli (strain M15,
Qiagen,
Hilden, Germany) using a calcium chloride procedure described in Maniatis, et
al. (( 1989)
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor
Laboratory,
3 0 Cold Spring Harbor, New York). Transformed colonies were screened by
Western
immunoblot analysis for expression of the HPO Lyase protein using antibodies
raised to the
bell pepper HPO lyase as described in Shibata, et al. (1995) Plant Cell
Physiol. 97:1059-
1072.
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Hydroperoxide lyase activity was determined by gaschromotography (GC) methods
described by Matsui, et al. (1991), Phytochemistry, 30:2109-2113.
TABLE 1
Sample Area nmole nmole/l Omin/mg
glpp 24677 130 153
Control 4089 28 24
The results of the GC analysis shown in Table 1, demonstrates that the
Arabidopsis
HPO lyase-like sequence encodes a HPO lyase enzyme.
Example b Transformation of Arabidopsis with Antisense HPO Lyase Constructs
A variety of methods have been developed to insert a DNA sequence of interest
into the
genome of a plant host to obtain the transcription or transcription and
translation of the sequence
to effect phenotypic changes.
The plant binary constructs pCGN8101 are used in plant transformation to
direct the
expression of the antisense nucleic acid sequence of the Arabidopsis HPO lyase-
like sequence
from plant tissues.
Transgenic Arabidopsis thaliana plants may be obtained by Agrobacterium-
mediated
transformation as described by Valverkens et al., (Proc. Nat. Acad. Sci.
(1988) 85:5536-
5540), or as described by Bent et al. ((1994), Science 265:1856-1860), or
Bechtold et al.
((1993), C.R.Acad.Sci, Life Sciences 316:1194-1199).
Example 7 Analysis of Transgenic Plants
Transgenic Arabidopsis plants containing pCGN8101 are analyzed for the
decreased
production of Hexenal by High Pressure Liquied Chromotography (HPLC) analysis
of protein
extracts as described in Shibata et al. (1995) Plant Cell Physiol. 36:147-156.
2 5 Transgenic plants overexpressing the Arabidopsis HPO lyase of the present
invention
may be screened using a photometric assay or by the HPLC assay which are both
described in
Shibata, et al. (1995) supra.
Example 8 Identification of Additional HPO lyase Sequences
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Additional HPO lyase-like sequences are obtained from tomato and cucumber
tissues.
Total RNA was isolated from cucumber hypocotyls and tomato immature fruit
tissue using
TRIzoI reagent (Gibco-BRL Life Technologies, Gaithersburg, MD) following the
manufacturers protocol.
Complementary DNAs (cDNA) are obtained using the Marathon cDNA Amplification
Kit (Clontech, Palo Alto, CA) following the manufacturers directions.
The sequences of HPO iyase from bell pepper (Matsui, et al. ( 1996) supra),
banana
(European Patent Application, Publication Number EP 0 801 133 A2) and
Arabidopsis were
aligned using ClustalW (http://www.clustalw.genome.ad.jp/) and seven conserved
peptide
sequences were identified (see Figure 7 for positions, Table 2 for a listing
Table 2
Peptide Sequence Primer Name Oligonucleotide Sequence
1 PGSYG HPOL1S 5'-ATNCCNGGNWSNTAYGG-3'
2 QPLEEI HPOL2S 5'-CARCCNYTNGARGARAT-3'
HPOL2AS 5'-ATYTCYTCNARNGGYTG-3'
3 GFNAYGG HPOL3S 5'- GGNTTYAAYGCNTWYGGNGG-3'
HPOL3AS 5'-CCNCCRSANGCRTTRA.ANCC-3'
4 YQPLVM HPOL4S 5'-TAYCARCCNYTNGTNATG-3'
HPOL4AS 5'-CATNACNARNGGYTGRTA-3'
5 VFDEPE HPOLSS 5'-GTNTT'YGAYGANCCNGA-3'
HPOLSAS 5'-TCNGGNTCRTCRAANAC-3'
6 NGPQTG HPOL6AS 5'-CCNGTYTWNGGNCCRTT-3'
7 NKQCAAKD HPOL7AS 5'-CYTTNGCNGCRCAYTGYTTRTT-3'
A set of synthetic oligonucleotides (Table 2) are synthesized for use in
polymerase
chain reactions with the cDNAs obtained above to identify sequences which are
homologous
to HPO lyase sequences. The PCR reactions are carried out using Advantage cDNA
Polymerase Mix (Clonctech, Palo AIto,CA) using the reaction conditions
according to the
manufacturers protocol. The letter "S" in the oligonucleotide name designates
a PCR primer
designed to amplify the sense strand, or forward reaction primer. The letters
"AS" designates
a PCR primer designed to amplify the antisense strand, or reverse reaction
primer. In the
2 0 oligonucleotide sequence. the letters "N" represents an A, C> G, or a T,
the letter "S"
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WO 00/00627 PCT/US99/14777
represents a C or a G in that position, the letter "Y" represents a C or a T,
and the letter "R"
represents an A or a G in that position.
A single PCR product, of approximately 475 bp, was amplifed in reactions
containing
the primers 4HPOL3S and 11HPOL7AS, with the cDNAs obtained from both cucumber
and
tomato (described above). The 475 by PCR product from tomato and cucumber were
cloned
into the plasmid pCR2.1TOP0 (invitrogen) to yield the plasmids T15 and C15
respectively.
In PCR reactions with 6HPOL4S and 11HPOL7AS, a single product, of
approximately 200
bp, was obtained from amplification reactions with cDNA obtained from cucumber
hypocotyl
tissue. The 200 by product was cloned into pCR2.1 TOPO (Invitrogen), to create
the plasmid
C17.
The nucleotide sequence of each PCR product was determined by automated
sequencing. The sequences obtained are compared to nucleic acid and amino acid
sequences
of HPO lyase sequences from bell pepper, Arabidopsis, and banana leaf, as well
as to DNA
and amino acid sequences coding for allene oxide synthases from guayule ((
1995) J. Biol.
Chem. 270( 15):8487-8494), flaxseed (( 1993) Proc Natl Acad Sci USA 90(
18):8519-8523) and
Arabidopsis.
The results demonstrate that the T15 nucleic acid sequence is approximately
85%
similar to the bell pepper HPO lyase DNA sequence and about 88% similar in the
amino acid
sequence. Furthermore, the T15 sequence is also at least about 55% similar to
other HPO
2 0 lyase nucleic acid sequences and at least about 57% similar in the amino
acid sequence. In
addition, the T15 amino acid sequence is only about 41% similar to the allene
oxide synthase
sequences. The C17 sequence also follows a similar pattern of similarity to
the HPO lyase
sequences. Thus, the T15 and C17 sequences encode proteins highly similar to
HPO lyase.
However, the results of the sequence comparisons (Figure 8) demonstrate that
the C15
2 5 nucleic acid sequence is between 50% and 54% similar to the other HPO
lyase nucleic acid
sequences and about 58% similar to the allene oxide synthase DNA sequences.
Furthermore,
the deduced amino acid sequence of C15 is between about 38% and 42% similar to
the HPO
lyase amino acid sequences and about 51 % similar to the AOS amino acid
sequences. Thus,
the C 15 sequence encodes a protein which is divergent from both the known HPO
lyase
3 0 sequences, and is more similar to allene oxide synthase sequences.
The nucleotide sequence of each PCR product was determined by automated
sequencing. The sequences obtained are used to search Genbank. Search results
identify the
CA 02301856 2000-02-24
WO 00/00627 PCT/US99/14777
sequences from T15 and C17 as being similar to HPO lyase sequences, while the
sequence of
C15 is similar to allene oxide synthase sequences.
In order to obtain full length coding sequence for T 15, C 15 and C 17, RACE
PCR
reactions are employed using the Marathon cDNA Amplification kit (Clontech)
according to
the manufacturers protocol, and the oligonucleoddes shown in Table 3.
Table 3
1 KMC 10-1: 5'-CGGTGGAGATCCTCGCCACCGGTGCCGACCC-3'
2KMC 10-2: 5'-CTTCCTTCACGGTTGTCCTCACTTCCTCCGCCAG-3'
3KMC17-1:5'-TCCAGCAGCGCTGCCCCTTTCTCTCCCCGG-3'
4KMC17-2: 5'-CACTGTTTGTTCTTCTCGCTCGGTGTCCCCG-3'
SKMC 10-3: 5'-GGGTCGGCACCGGTGGCGAGGATCTCCACCG-3'
6KMC 10-4: 5'-CTGGCGGAGGAAGTGAGGACAACCGTGAAGGAAG-3'
7KMC17-3: 5'-CCGGGGAGAGAAAGGGGCAGCGCTGCTGG-3'
8KMC17-4:5'-CGGGGACACCGAGCGAGAAGAACAAACAGTG-3'
9KM'f 15-1: 5'-GACTTGGTACTGGTGGACTAAGCCTAAGTGTTTC-3'
l OKMT 15-2: 5'-GGCTGATAACCACAAAGAAGCTCCCCTTTC-3'
11KMT15-3: 5'-GAAACACTTAGGCTTAGTCCACCAGTACCAAGTC-3'
12KMT 15-4: 5'-GAAAGGGGAGCTTCTTTGTGGTTATCAGCC-3'
PCR products from the amplification reactions with DNA obtained from tomato
and
cucumber are cloned into pCR2.1 TOPO. The sequences of the 5' and 3'-RACE
products
from tomato (pCGN8303 (5' RACE) and pCGN8304 (3' RACE)), cucumber, C15
(pCGN8302 (5'RACE) and pCGN8306 (3' RACE)) and C17 (pCGN8301 (5' RACE) and
2 5 pCGN8307 (3' RACE)) are sequenced and aligned with the respective
sequences obtained
from pCGN8305, pCGN8309, and pCGN8308 to obtain preliminary full length
sequences
corresponding to a tomato HPO lyase-like sequence (Figure 4), a cucumber HPO
lyase-like
sequence (Figure 6) and a cucumber allene oxide synthase-like sequence (Figure
5).
3 0 Example 9 Preparation of Expression Constructs
A set of constructs are prepared for transformation into either plant or
bacterial hosts
to further characterize the novel sequence from cucumber. To create a full
length coding
sequence for the cucumber (C15) allene oxide synthase-like sequence, the
sequences from the
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CA 02301856 2000-02-24
WO 00/00627 PCT/US99/14777
5' RACE (pCGN8302) and 3' RACE (pCGN8306) were PCR amplified and combined at a
unique restriction endonuclease site.
The 5' C15 sequence is amplified using primers (4KMC15ES1 5'-
CGGGATCCATGGCTTCTTCCTCCCCTGAACTTC-3' and 5KMC15EAS2 5'-
TGCCGACCCATTTCAGTATAGTGGG-3' ) in PCR amplification reactions described
above. The primer 4KMC 15EAS 1 amplifies from the 5' region and contains the
start codon
(ATG), and a BamHI site. The 3' C 15 sequence is amplified using the AP 1
primer provided
in the Marathon Kit (BRL-Lifetechnologies, Gaithersburg, MD) and the primer
6KMC15ES3
(5'- TTCACACCATTCCCCTGCCTTTCTTCCC-3'). The sequence of the C15 full length
clone is shown in Figure 6.
A. Bacterial Expression Construct
The 5' .RACE PCR amplification product is digested with BamHI and XbaI (unique
site endogenous to the C 15 sequence) and cloned into the expression vector
pQE30
(Invitrogen) with the amplification product of the 3' RACE PCR reaction
digested with XbaI
and SmaI. This construct provides a full length encoding sequence of the C 15
cDNA in the
E. coli expression vector to create the vector pCGN8333. The full length
sequence is also
cloned into the plasmid pUC 119 to create the vector pCGN8334.
B. Plant Expression Construct
A binary vector for plant transformation, pCGN5138, was constructed from
2 0 pCGN 1558 (McBride and Summerfelt, ( 1990) Plant Molecular Biology, 14:269-
276). The
polylinker of pCGN 1558 was replaced as an HindIIIIEcoRI fragment with a
polylinker
containing unique restriction endonuclease sites, HindIII, SseIIPstI, NotI,
BamHI, Swal, XbaI,
PacI, Ascl, and Asp718.
The full length coding sequence of C15 is cloned to be expressed from the
plant
constitutive promoter 35S for.expression in plants. The expression cassette is
cloned into the
binary vector pCGN5138 to create the vector pCGN8337.
Example 10 Expression of cucumber C15 in E. coli
The expression vector pCGN8333 was transformed into E coli (strain M15,
Qiagen,
3 0 Hilden, Germany) using a calcium chloride procedure described in Maniatis,
et al. (( 1989)
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor
Laboratory,
Cold Spring Harbor, New York). Transformed colonies were screened by Western
immunoblot analysis for expression of the HPO Lyase protein using antibodies
raised to the
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WO 00/00627 PC'T/U899/14777
bell pepper HPO lyase as described in Shibata, et al. (1995) Plant Cell
Physiol. 97:1059-
1072.
Hydroperoxide lyase activity was determined by spectrophotometric and gas
chromotography (GC) methods described by Matsui, et al. ( 1991 ),
Phytochemistry, 30:2109
2113, using both linolenic acid 13-hydroperoxide and linolenic acid 9-
hydroperoxide as
substrates.
The results of the gas chromotography assay (Figure 9) demonstrate that the
protein
encoded by the cucumber C 15 sequence has greater activity toward linolenic
acid 9-
hydroperoxide (Figure 9B) substrates than linolenic acid 13-hydroperoxide
substrates (Figure
9A). The results of the spectrophotometric assays further demonstrate the
preference of the
protein encoded by cucumber HPO lyase nucleic acid sequence for 9-
Hydroperoxide
substrates. The results of the spectrophotometric assay are presented in
Figure 10.
Thus, the cucumber C15 sequence represents the first known cloning of a
nucleic acid
sequence encoding a 9-hydroperoxide lyase.
All publications and patent applications mentioned in this specification are
indicative
of the level of skill of those skilled in the art to which this invention
pertains. All
publications and patent applications are herein incorporated by reference to
the same extent as
if each individual publication or patent application was specifically and
individually indicated
2 0 to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it will be
obvious that
certain changes and modifications may be practiced within the scope of the
appended claim.
38
