WO2000050575A2 - Sequences d'acides nucleiques de 9-hydroperoxyde lyase d'acides gras - Google Patents

Sequences d'acides nucleiques de 9-hydroperoxyde lyase d'acides gras Download PDF

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WO2000050575A2
WO2000050575A2 PCT/US2000/005311 US0005311W WO0050575A2 WO 2000050575 A2 WO2000050575 A2 WO 2000050575A2 US 0005311 W US0005311 W US 0005311W WO 0050575 A2 WO0050575 A2 WO 0050575A2
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sequence
plant
lyase
sequences
hpo lyase
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Kenji Matsui
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Calgene Llc
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • 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 .
  • Compounds, such as jasmonic acid are produced from hydroperoxides, such as 13-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. Alternatively, 13-hydroperoxylinolenic can be catabolyzed by hydroperoxide lyase eventually forming volatile aldehydes and traumatic acid.
  • hydroperoxides such as 13-hydroperoxylinolenic acid
  • AOS allene oxide synthase
  • ACS allene oxide cyclase
  • Jasmonic acid is involved in stress and disease resistance signaling responses via the octadecanoid pathway.
  • Fatty acid hydroperoxide lyase catalyzes the cleavage of carbon-carbon bonds in polyunsaturated fatty acid hydroperoxides to produce short-chain aldehydes and (D-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 aromas/flavors produced are a function of the specific products produced by the lysis of specific fatty acid hydroperoxides.
  • 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.
  • 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.
  • melon melon aroma and/or a melon flavor
  • Such characteristics are important to industries concerned with fragrances and flavorings .
  • short-chain aldehydes are also thought to be involved in disease resistance. For example, Croft, et al ((1993) Plant Physiol .
  • hydroperoxide also referred to as HPO lyase or HPOL
  • HPO lyase HPOL
  • HPOL hydroperoxide
  • the present invention provides nucleic acid sequences encoding for proteins which catalyze the formation of short- chain aldehydes and oxo-acids from 9-hydroperoxide fatty acids. Such proteins are referred to herein as 9- hydroperoxide lyases or 9-HPO lyase.
  • nucleic acid sequences encoding plant 9-HPO lyase may now be characterized with respect to enzyme activity.
  • this invention encompasses 9-HPO lyase nucleic acid sequences and the corresponding amino acid sequences, and the use of these nucleic acid sequences in the preparation of oligonucleotides containing 9-HPO lyase encoding sequences for analysis and recovery of plant 9-HPO lyase gene sequences.
  • the 9-HPO lyase encoding sequence may encode a complete or partial sequence depending upon the intended use. All or a portion of the genomic sequence, or cDNA sequence, is intended.
  • constructs which provide for transcription or transcription and translation (expression) of the 9-HPO lyase sequence in a host cell.
  • constructs which are capable of transcription or transcription and translation in plant host cells are preferred.
  • a reduction in 9-HPO lyase may be desired.
  • recombinant constructs may be designed having the plant 9-HPO lyase sequences in a reverse orientation for expression of an anti-sense sequence or use of co-suppression, also known as " transwitch" , constructs may be useful .
  • Such constructs may contain a variety of regulatory regions including transcriptional initiation regions obtained from genes preferentially expressed in plant seed tissue.
  • this invention relates to a method for producing a 9-HPO lyase in a host cell or progeny thereof via the expression of a construct in the cell.
  • Cells containing the 9-HPO lyase as a result of the production of the 9-HPO lyase encoding sequence are also contemplated herein.
  • modified plants, tissues and aldehyde compositions obtained by expression of the 9-HPO lyase sequence and protein of this invention.
  • 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 8A) of the bell pepper HPOL (CaHPOL) , tomato fruit HPOL (LeHPOL) , cucumber hypocotyl HPOL (CsC17HPOL, pseudogene) , Arabidopsis inflorecence HPOL (AtHPOL) , banana leaf HPOL (MsHPOL) , cucumber hypocotyl 9-HPOL (Csl5HPOL) , 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 using linoleic acid 13-hydroperoxide ( Figure 9A) and linoleic acid 9- hydroperoxide (figure 9B) substrates.
  • GC gas chromatography
  • Figure 10 provides the results of the spectrophotometric assay of the cucumber 9-HPO lyase expressed from E. coli using linoleic acid 13 -hydroperoxide and linoleic acid 9- hydroperoxide substrates.
  • nucleotide sequences are provided which are capable of coding sequences of amino acids, such as, a protein, polypeptide or peptide, which demonstrate the ability to form short-chain aldehydes and oxo-acids from 9-hydroperoxide fatty acids under plant enzyme reactive conditions.
  • 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.
  • nucleic acid sequences are provided which encode for 9-hydroperoxide lyase (hereinafter referred to as 9-HPO lyase) .
  • 9-HPO lyase 9-hydroperoxide lyase
  • Such nucleic acid sequences are demonstrated herein to encode enzymes which form (3Z , 6Z) -nonadienal and 9-oxo-nonanoic acid from Linolenic acid -hydroperoxide or 9-hydroperoxy- (10E, 12Z, 15Z)- octadecadienoic acid.
  • nucleic acid sequence from cucumber is identified from cDNA libraries made from total RNA isolated from 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.
  • expression constructs are provided which direct the expression of nucleic acid sequences encoding 9-HPO lyase in bacterial and plant tissues.
  • the 9-HPO lyase sequence of the present invention may be used in expression constructs to produce transgenic plants with improved organoleptic properties, in particular improved fruity (or "melon") note flavor and aroma characteristics.
  • 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 ( 3 Z) -hexenal and (2-3) -hexenal (Croft, et al . (1993) Plant Physiol . 101:13-24).
  • Aldehydes, such as (2 E) -hexenal have also been shown to be effective anti-bacterial agents, further contributing to enhanced disease resistance (Croft, et al . (1993), supra) .
  • these compounds may be involved in a general wounding response in plants .
  • 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 .
  • assays are performed on protein extracts of cultured bacterial cells expressing the protein.
  • cucumber 9-HPO lyase expression constructs are prepared to direct the expression of the 9-HPO lyase sequence in E coli .
  • the expressed 9-HPO lyase enzymes demonstrate the ability to produce (3Z, 6Z) -nonoadienal and 9-oxo-nonanoic acid from linolenic acid 9-hydroperoxide or 9-hydroperoxy- (10E, 12Z, 15Z) -octadecadienoic acid by gc analysis.
  • nucleic acid probes DNA and RNA
  • nucleic acid probes are labeled to allow detection, preferably with radioactivity although enzymes or other methods may also be used.
  • antibody preparations either monoclonal or polyclonal may be utilized. Polyclonal antibodies, although less specific, typically are more useful in gene isolation.
  • detection the antibody is labeled using radioactivity or any one of a variety of second antibody/enzyme conjugate systems that are commercially available. Examples of some of the available antibody detection systems are described by Oberfilder ( Focus (1989) BRL Life Technol . , Inc ., 11 : 1-5) .
  • a genomic or other appropriate library prepared from the candidate plant source of interest may be probed with conserved sequences from one or more plant HPO lyase (s) to identify homologously related sequences. Positive clones may be analyzed by restriction enzyme digestion and/or sequencing. When a genomic library is used, one or more sequences may be identified providing both the coding region, as well as the transcriptional regulatory elements of the HPO lyase gene from such plant source. Probes can also be considerably shorter than the entire sequence. Oligonucleotides may be used, for example, but should be at least about 10, preferably at least about 15, more preferably at least 20 nucleotides in length.
  • nucleic acid fragments When longer nucleic acid fragments are employed (>100 bp) as probes, especially when using complete or large cDNA sequences, one can still screen with moderately high stringencies (for example using 50% formamide at 37°C with minimal washing) in order to obtain signal from the target sample with 20-50% deviation, i.e., homologous sequences.
  • moderately high stringencies for example using 50% formamide at 37°C with minimal washing
  • homologous sequences are found when there is an identity of sequence and 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.
  • Glu/Asp Val/Ile, Ser/Thr, Arg/Lys and Gln/Asn may also be considered in determining sequence homology.
  • a lengthy nucleic acid sequence may show as little as 50-60% sequence identity, and more preferably at least about 70% sequence identity, between the target sequence and the given plant HPO lyase of interest excluding any deletions which may be present, and still be considered related.
  • Amino acid sequences are considered homologous by as little as 20% sequence identity between the two complete mature proteins . ( See generally, Doolittle, R.F., OF URFS and ORFS (University Science Books, CA, 1986.)
  • sequences provided herein be used to identify homologous 9-hydroperoxide lyases, but the resulting sequences obtained therefrom may also provide a further method to obtain plant 9-hydroperoxide lyase sequences from other plant sources.
  • PCR may be a useful technique to obtain related 9-HPO lyases from sequence data provided herein.
  • additional sequences may be added to the 5' end of the primer to include restriction endonuclease cleavage sites to allow for convenient cloning of the PCR fragment into cloning vectors. Addition of such 5' "tails" is well known in the art, and do not interfere with amplification as mismatched nucleotides at the 5' end of primers are not required for amplification .
  • amino acid sequence alignments between sequences encoding 13- hydroperoxy lyase from bell pepper (Matsui, et al . (1996) FEBS Letters 394:21-24), banana (European Patent Application, Publication Number EP 0 801 133 A2 , the entirety of which is incorporated herein by reference) and Arabidopsis identify highly conserved peptide sequences.
  • Synthetic oligonucleotide primers are constructed based on these peptide sequences and used in PCR reactions to amplify cDNAs from several plant tissue sources which are highly similar to known HPO lyase sequences.
  • Complimentary DNA sequences are identified from tomato hypocotyl and fruit tissues and cucumber hypocotyl tissue which are highly homologous to the bell pepper 13 -HPO lyase.
  • a second cDNA sequence is obtained from the cucumber hypocotyl tissue which is divergent from the 13 -HPO lyase sequences.
  • the sequence shows a slightly higher similarity to allene oxide synthases .
  • the nucleotide sequence obtained from any method may be molecularly cloned into an appropriate vector for propagation of the DNA by methods known in the art.
  • Many cloning vectors are available commercially, and may be employed used with the nucleotide sequences of the present invention.
  • the DNA may be inserted into a pBluescript vector (Stratgene, La Jolla, CA) .
  • useful cloning vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids which are in turn derived from the well known cloning vector pBR322 (ATCC 37017) .
  • cloning vectors include the pGEM vectors (Promega Biotec, Madison, WI) .
  • linear cloning vectors with single thymine ("T") overhangs are available for convenient cloning of PCR fragments amplified utilizing Taq DNA polymerase which usually adds an adenine ("A") to the end of the amplified fragment.
  • vectors include pCR2.1 vector (Invitrogen, La Jolla, CA) .
  • Cloned DNA sequences may be expressed in hosts to confirm HPO lyase activity. Techniques and expression vectors for expression in such hosts are well known in the art and are available from a wide variety of commercial sources. Examples of such vectors include pQE30 (Qiagen, Hilden, Germany) .
  • sequences of the DNA coding regions can be optimized by gene resynthesis, based on codon usage, for maximum expression in particular hosts.
  • expression of the cucumber HPO lyase sequence in E coli demonstrates that the sequence encodes a 9-hydroperoxide lyase. This is the first reported sequence cloned, to the inventor's knowledge, encoding a hydroperoxide lyase with activity on linolenic acid 9-hydroperoxide substrates.
  • the transcription, or transcription and translation (expression) , of the 9-HPO lyase in a host cell is desired to produce a ready source of the enzyme and/or modify the composition of fatty acids and/or volatile compound found therein.
  • Other useful applications may be found when the host cell is a plant host cell, in vi tro and in vivo .
  • Nucleic acids (genomic DNA, plasmid DNA, cDNA, synthetic DNA, mRNA, etc.) encoding HPO lyase or amino acid sequences of the purified enzymes, which permit design of nucleic acid probes facilitating the isolation of DNA coding sequences therefor, are known in the art and are available for use in the methods of the present invention. It is generally recognized to an artisan skilled in the field to which the present invention pertains that the nucleic acid sequences provided herein and the amino acid sequences derived therefrom may be used to isolate other potential HPO lyase genes from GenBank or other database using DNA and peptide search techniques generally known in the art.
  • DNA coding sequences useful in the present invention can be derived from algae, fungi, bacteria, mammalian sources, plants, etc. Homology searches in existing databases using signature sequences corresponding to conserved nucleotide and amino acid sequences of 9-HPO lyase can be employed to isolate equivalent, related genes from other sources such as plants and microorganisms . Searches in EST databases can also be employed. Furthermore, the use of DNA sequences encoding enzymes functionally enzymatically equivalent to those disclosed herein, wherein such DNA sequences are degenerate equivalents of the nucleic acid sequences disclosed herein in accordance with the degeneracy of the genetic code, is also encompassed by the present invention. Demonstration of the functionality of coding sequences identified by any of these methods can be carried out by expression of such sequences in various expression systems employing various organisms.
  • constructs for the expression of the 9-HPO lyase sequences in plants are described. Also, the use of such 9-HPO lyase expression constructs for increasing aldehyde and oxo-acid production in plant tissues is contemplated in the present invention.
  • the 9-HPO lyase expression constructs may be employed to produce a host plant having modified organoleptic properties .
  • organoleptic properties is meant as relating to qualities, such as taste, color, odor, and feel, of a substance, such as a food, that stimulate the sense organs.
  • constructs may be prepared to direct the expression of the 9-HPO lyase sequence in a host cell to provide for the production of aldehydes and oxo-acids for use in flavorings and fragrances .
  • nucleic acid sequences which encode 9-hydroperoxide lyase may be used in various constructs, for example, as probes to obtain further sequences. Alternatively, these sequences may be used in conjunction with appropriate regulatory sequences to increase levels of the respective 9- HPO lyase of interest in a host cell for recovery or study of the enzyme in vi tro or in vivo or to decrease levels of the respective 9-HPO lyase of interest for some applications when the host cell is a plant entity, including plant cells, plant parts (including but not limited to seeds, cuttings or tissues) and plants.
  • the constructs may contain the nucleic acid sequence which encodes the entire 9- HPO lyase protein, or a portion thereof.
  • the entire 9-HPO lyase sequence is not required.
  • nucleic acid sequence encoding a plant or other 9-HPO lyase of this invention may include genomic , cDNA or mRNA sequence.
  • encoding is meant that the sequence corresponds to a particular amino acid sequence either in a sense or anti-sense orientation.
  • extrachromosomal is meant that the sequence is outside of the plant genome of which it is naturally associated.
  • “recombinant” is meant that the sequence contains a genetically engineered modification through manipulation via mutagenesis, restriction enzymes, and the like.
  • a cDNA sequence may or may not contain pre-processing sequences, such as transit peptide sequences or targeting sequences to facilitate delivery of the 9-HPO lyase protein to a given organelle or membrane location.
  • pre-processing sequences such as transit peptide sequences or targeting sequences to facilitate delivery of the 9-HPO lyase protein to a given organelle or membrane location.
  • the use of any such precursor 9-HPO lyase DNA sequences is preferred for uses in plant cell expression.
  • a genomic 9-HPO lyase sequence may contain the transcription and translation initiation regions, introns, and/or transcript termination regions of the plant 9- HPO lyase, which sequences may be used in a variety of DNA constructs, with or without the 9-HPO lyase structural gene.
  • nucleic acid sequences corresponding to the 9-HPO lyase of this invention may also provide signal sequences useful to direct protein delivery into a particular organellar or membrane location, 5' upstream non-coding regulatory regions (promoters) having useful tissue and timing profiles, 3' downstream non-coding regulatory regions useful as transcriptional and translational regulatory regions, and may lend insight into other features of the gene.
  • the desired plant or other 9-HPO lyase nucleic acid sequence may be manipulated in a variety of ways .
  • the flanking regions may be subjected to resection, mutagenesis, etc.
  • transitions, transversions , deletions, and insertions may be performed on the naturally occurring sequence.
  • all or part of the sequence may be synthesized.
  • one or more codons may be modified to provide for a modified amino acid sequence, or one or more codon mutations may be introduced to provide for a convenient restriction site or other purpose involved with construction or expression.
  • the structural gene may be further modified by employing synthetic adapters, linkers to introduce one or more convenient restriction sites, or the like.
  • nucleic acid or amino acid sequences encoding a plant or other 9-HPO lyase of this invention may be combined with other non-native, or “heterologous” , sequences in a variety of ways.
  • heterologous sequences is meant any sequence which is not naturally found joined to the native (or wild-type) 9- HPO lyase, including, for example, combinations of nucleic acid sequences from the same plant which are not naturally found joined together.
  • the DNA sequence encoding a plant or other 9-HPO lyase of this invention may be employed in conjunction with all or part of the gene sequences normally associated with the 9-HPO lyase.
  • a DNA sequence encoding 9-HPO lyase is combined in a DNA construct having, in the 5' to 3 ' direction of transcription, a transcription initiation control region capable of promoting transcription and translation in a host cell, the DNA sequence encoding plant 9-HPO lyase and a transcription and translation termination region.
  • 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 a 9-HPO lyase foreign to the wild- type cell present therein, for example, by having a recombinant nucleic acid construct encoding a 9-HPO lyase therein not native to the host species.
  • 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.
  • an HPO lyase isolated from cucumber hypocotyl is expressed in E coli, strain Ml5.
  • 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 .
  • the HPO lyase isolated from the cucumber hypocotyl encodes a 9-HPO lyase.
  • 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).
  • methods for the expression of 13-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 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 plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the fungal host.
  • 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.
  • the vector may be one which, when introduced into the fungal cell, is integrated into the genome and replicated together with the chromosome (s) into which it has been integrated.
  • 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.
  • 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 location (s) in the chromosome (s) .
  • 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 lOOObp, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination.
  • 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.
  • the vector may further comprise an origin of replication 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 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 phosphotransferase) , niaD (nitrate reductase) , pyrG (orotidine-5 ' -phosphate decarboxylase) and sC (sulfate adenyltransferase) and trpC (anthranilate synthase) .
  • amdS acetamidase
  • argB ornithine carbamoyltransferase
  • bar phosphinothricin acetyltransferase
  • hygB hygromycin phosphotransferase
  • niaD nitrate reducta
  • 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 may be obtained from genes encoding polypeptides either homologous or heterologous to the host cell.
  • 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 encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awa ori glucoamylase (glaA) , Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase and
  • a useful promoter is the Sac char o yces 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.
  • 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 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 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 . 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 and Aspergillus 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 .
  • 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.
  • 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.
  • 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.
  • the 5' end of the coding 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.
  • the foreign signal peptide coding region may be obtained from a glucoamylase 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 signal, Aspergillus niger neutral amylase signal, the Rhizomucor miehei aspartic proteinase signal, the Humicola lanuginosus cellulase signal, or the Rhizomucor miehei lipase signal.
  • 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) .
  • 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)).
  • 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 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 ( Endomyce tales) , Basidiosporogenous yeast and yeast belonging to the Fungi Imperfect! (Blastomycetes) .
  • the Ascosporogenous yeasts are divided into the families Spermophthoraceae and 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, Sporidiobolus , Filobasidium and Filobasidiella .
  • Yeast belonging to the Fungi Imperfect! 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) .
  • yeast and manipulation of yeast genetics are well known in the art (see, for example, Biochemistry and Genetics of Yeast, Bacil 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.
  • an activator e.g., a trans-acting factor
  • 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 .
  • the nucleic acid sequence encoding an activator may be obtained from the genes encoding Saccharomyces cerevisiae heme activator protein 1 (hapl), Saccharomyces cerevisiae galactose metabolizing protein 4 (gal4) and Aspergillus nidulans ammonia regulation protein (areA) .
  • hapl Saccharomyces cerevisiae heme activator protein 1
  • gal4 Saccharomyces cerevisiae galactose metabolizing protein 4
  • areA Aspergillus nidulans ammonia regulation protein
  • a chaperone is a protein which assists another protein in folding properly (Hartl et al . , TIBS 19 : 20-25 (1994); Bergeron et al . , TIBS 19:124-128 (1994); Demolder et al . , J. Biotechnology 32 : 119- 189 (1994); Craig, Science 250:1902-1903(1993); Gething and Sambrook, Nature 355:33-45 (1992); Puig and Gilbert, J Biol . Chem .
  • the nucleic acid sequence encoding a chaperone may be obtained from the genes encoding Aspergillus oryzae protein disulphide isomerase, Saccharomyces cerevisiae calnexin, Saccharomyces cerevisiae BiP/GRP78 and Saccharomyces cerevisiae Hsp70. For further examples, see Gething and Sambrook, Nature 355:33-45 (1992); Hartl et al .
  • a processing protease is a protease that cleaves a propeptide to generate a mature biochemically active polypeptide (Enderlin and Ogrydziak, Yeast 10 : 61 -19 (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 encoding Aspergillus 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 .
  • 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 cultivated in a nutrient medium suitable for production of the protein or fragment thereof using methods known in the art.
  • 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 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.
  • Suitable media are available from commercial 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, if polyclonal 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.
  • 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 like.
  • plant host cells are employed in transformation experiments with constructs containing DNA sequences coding 9-HPO lyase enzymes expressed from constitutive or tissue enhanced promoters.
  • the regulatory regions will vary, including regions from viral, plasmid or chromosomal genes, or the like.
  • constitutive or regulatable promoters may be employed.
  • transcriptional initiation regions which have been described are regions from bacterial and yeast hosts, such as E. coli , B . subtilis, Sacchromyces cerevisiae, including genes such as beta-galactosidase, T7 polymerase, tryptophan E and the like.
  • the constructs will involve regulatory regions functional in plants which provide for modified production of 9-HPO lyase, and, possibly, modification of the fatty acid composition.
  • the open reading frame coding for the plant HPO lyase or functional fragment thereof will be joined at its 5' end to a transcription initiation regulatory region.
  • the use of all or part of the complete plant HPO lyase gene is desired; namely all or part of the 5' upstream non- coding regions (promoter) together with the structural gene sequence and 3' downstream non-coding regions may be employed.
  • a different promoter such as a promoter native to the plant host of interest or a modified promoter, i.e., having transcription initiation regions derived from one gene source and translation initiation regions derived from a different gene source
  • numerous transcription initiation regions are available which provide for a wide variety of constitutive or regulatable, e.g., inducible, transcription of the structural gene functions.
  • the transcription/translation initiation regions corresponding to such structural genes are found immediately 5' upstream to the respective start codons.
  • transcriptional initiation regions used for plants are such regions associated with the T-DNA structural genes such as for nopaline and mannopine synthases, the 19S and 35S promoters from CaMV, and the 5 ' upstream regions from other plant genes such as napin, ACP, SSU, PG, zein, phaseolin E, and the like.
  • Enhanced promoters, such as double 35S, are also available for expression of HPO lyase sequences .
  • 5' upstream non-coding regions are obtained from other genes regulated during seed maturation, those preferentially expressed in plant embryo tissue, such as ACP and napin-derived transcription initiation control regions, are desired.
  • seed-specific promoters may be obtained and used in accordance with the teachings of issued U.S. Patent Numbers 5,608,152 and 5,530,194, which references are hereby incorporated by reference.
  • Transcription initiation regions which are preferentially expressed in seed tissue, i.e., which are undetectable in other plant parts, are considered desirable for expression in order to minimize any disruptive or adverse effects of the gene product.
  • Regulatory transcript termination regions may be provided in DNA constructs of this invention as well.
  • Transcript termination regions may be provided by the DNA sequence encoding the plant 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. Where the transcript termination region is from a different gene source, it will contain at least about 0.25 kb, preferably about 1-3 kb of sequence 3 ' to the structural gene from which the termination region is derived.
  • Plant expression or transcription constructs having a 9- 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 tissues for edible uses. Most especially preferred are seed, fruit, vegetable and leaf crops. Plants of interest include, but are not limited to, Brassica species, soybean, 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 dicotyledenous and monocotyledenous species alike and will be readily applicable to new and/or improved transformation and regulation techniques .
  • coexpression of a plant or other 9-HPO lyase in a plant tissue with a second gene involved in lipid peroxidation may also find use in the present invention.
  • coexpression of a 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 "melon" flavor in an edible plant tissue .
  • 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.
  • techniques of microinjection, DNA particle bombardment, and electroporation have been developed which allow for the transformation of various monocot and dicot plant species.
  • 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.
  • a cytotoxic agent e.g. antibiotic, heavy metal, toxin, etc.
  • complementation providing prototrophy to an auxotrophic host, viral immunity or the like.
  • one or more markers may be employed, where different conditions for selection are used for the different hosts.
  • 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 vir genes are present in the transformed Agrobacterium host.
  • the armed plasmid can give a mixture of normal plant cells and gall.
  • the expression or transcription construct bordered by the T-DNA border region (s) will be inserted into a broad host range vector capable of replication in E. coli and 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) 77:7347-7351) and EPA 0 120 515, which are incorporated herein by reference.
  • a vector containing separate replication sequences one of which stabilizes the vector in E. coli , and the other in Agrobacterium.
  • McBride and Summerfelt Plant Mol . Biol . (1990) 14:269-276
  • the pRiHRI Jaanin, 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.
  • 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 G418, 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.
  • 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 .
  • Host cells expressing the 9-HPO lyases of the present invention provide a novel source of volatile aldehydes contributing to "melon" notes for use in various applications.
  • the host cells may also contain constructs providing for a increased production of enzymes involved in lipid peroxidation, for example lipoxygenase .
  • 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 an altered fatty acid composition.
  • plant host cells containing a construct providing for the expression of the 9-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 and in PCT Publication WO 95/26413 the entireties of which are incorporated herein by reference.
  • 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.
  • 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.
  • 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.
  • NADH nicotine adenine dinucleotide
  • a nucleic acid fragment encoding hydroperoxide lyase from Bell pepper has been previously cloned and sequenced (Matsui, et al . (1996) supra) .
  • the nucleotide sequence was used to search Genbank for HPO lyase related sequences .
  • Genbank accession identified from Genbank (Accession Number Z97339, (http: //www.ncbi .nlm.nih.gov/web/Genbank/Index.html) ) containing a genomic sequence from Arabidopsis was reported to encode an allene oxide synthase.
  • 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 10ml REC buffer (50mM Tris-HCI, pH 9 , 0.8M NaCl, 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.
  • RNA is then precipitated by addition of 1 volume RecP (50mM Tris-HCL pH9 , lOmM EDTA and 0.5% (w/v) CTAB) and collected by brief centrifugation as before.
  • the R ⁇ A pellet is redissolved in 0.4 ml of 1M ⁇ aCl .
  • the R ⁇ A 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 R ⁇ A. After washing with ethanol, this final R ⁇ A precipitate is dissolved in water and stored frozen.
  • total R ⁇ As may be obtained using TRIzol reagent (BRL Life Technologies, Gaithersburg, MD) following the manufacturers protocol.
  • cD ⁇ A Complementary D ⁇ As
  • a pair of primers were designed to amplify the 5' and 3' regions from the Arabidopsis HPO lyase-like cD ⁇ A from the libraries described in example 2 above. These two primers,
  • HPOL28 for 3' RACE, 5 ' -CGGTTCCTCTGCGCCTCTCTCGCCGGCG-3 '
  • HPOL21 for 5' RACE, 5 ' -GCGGAACCGGAGGACTAAAACGCAGC-3 '
  • Adapter specific primers API 5'- CCATCCTAATACGACTCACTATAGGGC-3 '
  • the primers API and HPOL 21 were used, and for the amplification of the 3 ' region the API 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 bp was obtained from the 3 ' RACE reaction with RNA obtained from the silique tissue described above.
  • a second round of PCR reactions using the same conditions described above was performed with the gel purified 1100 bp fragment.
  • a reaction was performed with the primers HPOL13 (5'- CTTGGCGTAGTTCCTCAGCCTCTTG-3 ' ) and AP2 (5'- ACTCACTATAGGGCTCGAGCGGC-3 ' ) to amplify an approximately 1000 bp fragment as a confirmation of the HPO lyase-like sequence.
  • the reamplified 1000 bp fragment was gel purified and cloned into the pCR2.1 TOPO vector (Invitrogen, Carlsbad, CA) to create the plasmid pCGN8094.
  • the 5' RACE reaction produced many non-specific fragments.
  • a 1000 bp 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.
  • 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 Ba-nHI 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.
  • the PCR reaction mix contained 0.2 mM each of dATP, dCTP, dGTP and dTTP, 1.0% glycerol, 0.2 mM Tris-HCI (pH 8.3), 4.6 mM KC1, 1.5 mM EDTA, 15 ⁇ M dithiothreitol , 7.3 ⁇ gm/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 72°C for 7 minutes in a Perkin-Elmer 9800 thermocycler .
  • the resulting PCR product was digested with Ba-r-HI and Hindi11 and ligated into the vector pQE30 to create the vector pCGN8099.
  • the 3' terminus of the Arabidopsis HPO lyase was cloned into the Hindlll site of pCGN8099 from pCGN8094 to create the E. coli expression vector pCGN8100
  • a binary vector for plant transformation was constructed from pCGNl558 (McBride and Summerfelt, (1990) Plant Molecular Biology, 14:269-276).
  • the polylinker of pCGN1558 was replaced as an Hindlll/.EcoRI fragment with a polylinker containing unique restriction endonuclease sites, Hindlll , Ssel /Pstl , Not! , BamHl , Swal , Xbal , Pad , Ascl , 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 bp nucleotides from pCGN8091 were cloned as an EcoRI fragment into the plasmid pBluescript II SK (Stratagene, La Jolla, CA) to create the vector pCGN8093.
  • the 3' RACE product from pCGN8090 was cloned as a Hindlll fragment into pCGN8093 to create a full length HPO lyase coding sequence in the plasmid PCGN8094.
  • the Xp.nI site of pCGN8094 was removed by digesting with Kpnl and filling in the site with Klenow fragment, and the HPO lyase coding sequence was cloned from this plasmid as a Smal fragment into the Stul 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 . , Proc . Natl .
  • 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, 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. Hydroperoxide lyase activity was determined by gas chromotography (GC) methods described by Matsui, et al .
  • GC gas chromotography
  • HPO lyase-like sequences are obtained from tomato (Lycopersicon esculentum Mill. Cv UC82B) green immature fruit (ca 20 mm) and cucumber ⁇ Cucumis sativus L. cv Suyo) hypocotyl (of 3 day old seedlings) tissues.
  • Total RNA was isolated using TRIzol reagent (Gibco-BRL Life Technologies, Gaithersburg, MD) following the manufacturers protocol.
  • cDNA Complementary DNAs
  • HPO lyase 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.ac.jp/) and seven conserved peptide sequences were identified (see Figure 7 for positions, Table 2 for a listing).
  • 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 (Clonetech, Palo Alto,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.
  • the letters "N” represents an A, C, G, or a T
  • the letter “S” represents a C or a G in that position
  • the letter “Y” represents a C or a T
  • the letter “R” represents an A or a G in that position.
  • the 475 bp PCR product from tomato and cucumber were cloned into the plasmid pCR2.1TOPO (invitrogen) to yield the plasmids T15 (pCGN8305) and C15 (pCGN8309) respectively.
  • 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 bp product was cloned into pCR2.1 TOPO (Invitrogen), to create the plasmid C17 (PCGN8308) .
  • 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 .
  • 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
  • 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.
  • the T15 and C17 sequences encode proteins highly similar to HPO lyase.
  • the results of the sequence comparisons demonstrate that the C15 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.
  • 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.
  • the C15 sequence encodes a protein which is divergent from both the known HPO lyase sequences, and is more similar to allene oxide synthase sequences.
  • the T15, C15 and C17 sequences are used to search Genbank. Search results further confirm the sequences from T15 and C17 as being similar to HPO lyase sequences, while the sequence of C15 is more similar to allene oxide synthase sequences .
  • RACE PCR reactions are employed using the Marathon cDNA Amplification kit (Clontech) according to the manufacturers protocol, and the oligonucleotides shown in Table 3.
  • 2KMC10-2 5 ' -CTTCCTTCACGGTTGTCCTCACTTCCTCCGCCAG-3 '
  • 3KMC17-1 5 ' -TCCAGCAGCGCTGCCCCTTTCTCTCCCCGG-3 '
  • 4KMC17-2 5 ' -CACTGTTTGTTCTTCTCGCTCGGTGTCCCCG-3 '
  • 5KMC10-3 5 ' -GGGTCGGCACCGGTGGCGAGGATCTCCACCG-3 '
  • 6KMC10-4 5 ' -CTGGCGGAGGAAGTGAGGACAACCGTGAAGGAAG-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 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) .
  • a set of constructs are prepared for transformation into either plant or bacterial hosts to further characterize the novel sequence from cucumber.
  • the sequences from the 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 4KMC15EAS1 amplifies from the 5' region and contains the start codon (ATG) , and a BamEl site.
  • the 3 ' C15 sequence is amplified using the API 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.
  • the 5' RACE PCR amplification product is digested with BamHI and Xbal (unique site endogenous to the C15 sequence) and cloned into the expression vector pQE30 (Invitrogen) with the amplification product of the 3 ' RACE PCR reaction digested with Xbal and Smal .
  • This construct provides a full length encoding sequence of the C15 cDNA in the E. coli expression vector to create the vector pCGN8333.
  • the full length sequence is also cloned into the plasmid pUC119 to create the vector pCGN8334.
  • pCGN5138 A binary vector for plant transformation, pCGN5138, was constructed from pCGN1558 (McBride and Summerfelt, (1990) Plant Molecular Biology, 14:269-276).
  • the polylinker of pCGN1558 was replaced as an Hindlll / EcoRl fragment with a polylinker containing unique restriction endonuclease sites, Hindlll, Ssel /Pstl , No tl , BamHl , Swal , Xbal , Pad , 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.
  • the expression vector pCGN8333 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, 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.
  • Hydroperoxide lyase activity was determined by spectrophotometric and gas chromotography (GC) methods described by Matsui, et al . (1991), Phytochemistry, 30:2109-
  • the results of the gas chromotography assay demonstrate that the protein encoded by the cucumber C15 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.
  • the cucumber C15 sequence represents the first known cloning of a nucleic acid sequence encoding a 9- hydroperoxide lyase.

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Abstract

L'invention concerne la 9-hydroperoxyde lyase ou des enzymes de HPO lyase; des produits de recombinaison d'ADN utiles pour l'expression d'une HPO lyase de plante dans une cellule; des produits de recombinaison d'ADN utiles pour l'expression antisens d'une 9-HPO lyase dans une cellule de plante. Ces produits de recombinaison contiennent une séquence d'ADN codant pour la 9-HPO lyase sous le contrôle d'éléments régulateurs capables de diriger de façon préférentielle l'expression de cette 9-HPO lyase dans le tissu de la plante, quand ce produit de recombinaison est exprimé dans une plante transgénique. L'invention concerne également des procédés de mise en application d'une séquence d'ADN codant pour une 9-HPO lyase afin de modifier les aldéhydes volatils dans le tissu de la plante, ainsi que des procédés servant à augmenter la résistance d'une plante à des maladies. Les séquences de 9-HPO lyase illustrées ici sont obtenues à partir d'Arabidopsis.
PCT/US2000/005311 1999-02-26 2000-02-25 Sequences d'acides nucleiques de 9-hydroperoxyde lyase d'acides gras WO2000050575A2 (fr)

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WO2002050291A1 (fr) * 2000-12-18 2002-06-27 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada Region regulatrice de la peroxyde lyase
WO2002099112A2 (fr) * 2001-06-07 2002-12-12 Rhobio Sur-expression d'une lipoxygenase dans les plantes et diminution de la sensibilite des plantes aux maladies et aux agressions par des organismes pathogenes
FR2825578A1 (fr) * 2001-06-07 2002-12-13 Rhobio Sur-expression d'une lipoxygenase dans les plantes et diminution de la sensibilite des plantes aux maladies et aux agressions par des organismes pathogenes
JP2003528613A (ja) * 2000-03-29 2003-09-30 フイルメニツヒ ソシエテ アノニム マスクメロンＣｕｃｕｍｉｓＭｅｌｏヒドロペルオキシドリアーゼ及びその使用
US7317012B2 (en) 2005-06-17 2008-01-08 Bristol-Myers Squibb Company Bicyclic heterocycles as cannabinoind-1 receptor modulators

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003528613A (ja) * 2000-03-29 2003-09-30 フイルメニツヒ ソシエテ アノニム マスクメロンＣｕｃｕｍｉｓＭｅｌｏヒドロペルオキシドリアーゼ及びその使用
JP4951773B2 (ja) * 2000-03-29 2012-06-13 フイルメニツヒ ソシエテ アノニム マスクメロンＣｕｃｕｍｉｓＭｅｌｏヒドロペルオキシドリアーゼ及びその使用
WO2002050291A1 (fr) * 2000-12-18 2002-06-27 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada Region regulatrice de la peroxyde lyase
US7223855B2 (en) 2000-12-18 2007-05-29 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food Hydroperoxide Lyase regulator region
WO2002099112A2 (fr) * 2001-06-07 2002-12-12 Rhobio Sur-expression d'une lipoxygenase dans les plantes et diminution de la sensibilite des plantes aux maladies et aux agressions par des organismes pathogenes
FR2825578A1 (fr) * 2001-06-07 2002-12-13 Rhobio Sur-expression d'une lipoxygenase dans les plantes et diminution de la sensibilite des plantes aux maladies et aux agressions par des organismes pathogenes
FR2825580A1 (fr) * 2001-06-07 2002-12-13 Rhobio Sur-expression d'une lipoxygenase dans les plantes et diminution de la sensibilite des plantes aux maladies et aux agressions par des organismes pathogenes
WO2002099112A3 (fr) * 2001-06-07 2003-12-04 Rhobio Sur-expression d'une lipoxygenase dans les plantes et diminution de la sensibilite des plantes aux maladies et aux agressions par des organismes pathogenes
US7317012B2 (en) 2005-06-17 2008-01-08 Bristol-Myers Squibb Company Bicyclic heterocycles as cannabinoind-1 receptor modulators

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