Metabolic engineered cells for jasmonic acid biosynthesis

Abstract

The present invention relates in some aspects to a host cell comprising a metabolic pathway for producing jasmonic acid, the host cell overexpressing a polypeptide having 12-oxophytodienoic acid reductase (OPR) activity relative to a corresponding parent host cell.

Description

[Technical field]

[0001] The field of the invention relates to cells, enzymes, and methods useful for the production of jasmonic acid molecules. More specifically, the disclosure relates to recombinant host cells that overexpress a polypeptide having 12-oxophytodienoic acid reductase (OPR) activity compared to a corresponding parent host cell. [Background technology]

[0002] The production of chemicals from cell cultures is an important application of biotechnology. Typically, the steps in the development of such bioproduction methods may include: (1) selection of an appropriate host cell, (2) elimination of metabolic pathways that result in undesirable by-products, (3) deregulation of the desired pathway at both the enzyme activity level and the transcription level, and (4) overexpression of appropriate enzymes in the desired pathway.

[0003] Jasmonic acid ("JA") is an organic compound with the chemical formula of C6H6O2. Jasmonic acid and its derivatives, commonly known as "jasmonates," have found numerous applications in agriculture, flavors and fragrances, and biopharmaceutical fields. Traditionally, filamentous fungi have been the only commercially viable source of biosynthetic jasmonates. However, yields tend to be highly correlated with fungal morphology, which poses substantial challenges for the production of jasmonic acid in conventional fermenter equipment.

[0004] Higher plants are thought to synthesize jasmonic acid following the biosynthetic pathway outlined in Figure 4. The enzyme 12-oxophytodienoic acid reductase (OPR, EC 1.3.1.42) catalyzes the reduction of 12-oxophytodienoic acid (12-OPDA) to 3-oxo-2-(cis-2'-pentenyl)-cyclopentane-1-octanoic acid (OPC8), a key step in the jasmonic acid biosynthetic pathway. [ka] OPDA reductase

[0005] OPR belongs to the Old Yellow Enzyme (OYE) family and utilizes NADPH and flavin mononucleotide (FMN) for the reduction of the double bond of OPDA (Schaller and Weiler, 1997). Plant OPR enzymes dedicated to jasmonic acid biosynthesis have been well characterized and the corresponding genes have been cloned (Schaller and Weiler, 1997). In contrast, relatively little is known about the fungal pathway of jasmonic acid biosynthesis, despite the fact that jasmonic acid as a natural product was first isolated from the filamentous fungus Lasiodiplodia theobromae (Aldridge et al., 1971).

[0006] Recent studies have shown that, like higher plants, fungi also use arene oxide and OPDA as key intermediates for JA biosynthesis (Oliw and Hamberg, 2017), indicating that homology searches can be used to identify fungal counterparts of plant genes related to jasmonic acid biosynthesis. However, it has been demonstrated that the cyclopentenone reduction mechanism in L. theobromae differs from that in plants. In plants, the reduced flavin cofactor appears to be located on the α-face of the OPDA side chain, whereas in known fungal enzymes, this occurs on the β-face of the cyclopentenone plane (Tsukada et al., 2010). Such differences in the stereoselectivity of cyclopentenone olefin reduction in plant and fungal OPR enzymes may suggest sequence and structural differences. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Aldridge DC, Galt S, Giles D, and Turner WB.(1971)Metabolites of Lasiodiplodia theobromae.Journal of the Chemical Society C:1623-1627.

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[0008] In a first aspect, the present invention provides a recombinant host cell comprising a metabolic pathway for producing jasmonic acid. The host cell overexpresses a polypeptide having 12-oxophytodienoic acid reductase (OPR) activity compared to a corresponding parent host cell. The polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0009] In a second aspect, the present invention encompasses a biosynthetic method for producing jasmonic acid, comprising culturing a recombinant host cell according to the first aspect above, and recovering jasmonic acid from at least one of the recombinant cell and the culture medium.

[0010] In a third aspect, provided herein is a recombinant host cell comprising a metabolic pathway for producing jasmonic acid, wherein the host cell overexpresses a gene encoding a polypeptide having 12-oxophytodienoic acid reductase (OPR) activity compared to a corresponding parent host cell, and the gene comprises a polynucleotide sequence having at least 90% identity to the polynucleotide sequence set forth in SEQ ID NO:2.

[0011] In a fourth aspect, provided herein is a biosynthetic method for producing jasmonic acid, comprising culturing a recombinant cell of the third aspect above, and recovering jasmonic acid from at least one of the recombinant cell and the culture medium.

[0012] In a fifth aspect, provided herein is a biosynthetic method for producing a polypeptide having 12-oxophytodienoic acid reductase (OPR) activity, the method comprising culturing a recombinant cell of the third aspect above, and recovering the polypeptide having 12-oxophytodienoic acid reductase (OPR) activity from at least one of the recombinant cell and the culture medium.

[0013] While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the drawings and detailed description presented herein are not intended to limit the disclosure to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims.

[0014] Other features and advantages of the present invention will become apparent in the following detailed description of preferred embodiments of the invention, which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0015] [Figure 1] Figure 1A shows the purification of g5119 protein on a Ni-NTA column, and Figure 1B shows the purification of g5119 protein on an SDS-PAGE gel. [Diagram 2] FIG. 1 includes a typical HPLC profile of the g5119 catalyzed reaction. [Diagram 3] FIG. 1 includes LC / MS analysis of g5119 catalyzed reactions. [Figure 4] FIG. 1 shows the jasmonic acid biosynthetic pathway in higher plants. [Diagram 5] FIG. 1 is a schematic diagram of cassette hph-gs5119 cloning in the gateway destination binary vector pPm43GW. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Definition: As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. To the extent that terms such as "include," "having," and the like are used in the specification or claims, such terms are intended to be inclusive in the same manner as the term "comprise," as such term is interpreted when used as a transitional term in a claim. The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

[0017] A "cell system" is any cell that provides for expression of ectopic proteins. It includes bacteria, yeast, filamentous fungi, plant cells and animal cells. It includes both prokaryotic and eukaryotic cells. It also includes in vitro expression of proteins based on cellular components such as ribosomes.

[0018] "Coding sequence" is to be given its ordinary and customary meaning to those of skill in the art and is used without limitation to refer to a DNA sequence that codes for a specific amino acid sequence.

[0019] "Growing a cell line." Growing includes providing the appropriate medium to allow the cells to grow and divide. It also includes providing resources so that the cells or cell components can translate and make recombinant proteins.

[0020] "Protein Expression". Protein production can occur after gene expression. It consists of the step after DNA is transcribed into messenger RNA (mRNA). The mRNA is then translated into a polypeptide chain, which is finally folded into a protein. DNA can be present in a cell by transfection, a process of deliberately introducing nucleic acid into a cell. This term is often used for non-viral methods in eukaryotic cells. It can also refer to other methods and cell types, but other terms are preferred. "Transformation" is more frequently used to describe non-viral DNA transfer in bacteria, non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term, since transformation is also used to refer to the progression to a cancerous state (carcinogenesis) in these cells. Transduction is frequently used to describe virus-mediated DNA transfer. Transformation, transduction and viral infection are included in the definition of transfection in this application.

[0021] According to the present disclosure, "filamentous fungi" or "molds" are eukaryotic microorganisms classified as members of the fungal kingdom. Filamentous fungi typically grow in the form of multicellular filaments called hyphae. In contrast, fungi that can adopt a single-cell growth habit are called "yeasts." Yeasts are single-celled organisms that evolved from a multicellular ancestor. Yeast species useful in the present disclosure include, but are not limited to, those that have the ability to express multicellular properties by forming strings of connected budding cells known as pseudohyphae or pseudohyphae.

[0022] The term "complementary" should be given its ordinary and customary meaning to those of skill in the art and is used without limitation to describe the relationship between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the subject technology includes isolated nucleic acid fragments that are complementary to the complete sequences reported in the accompanying sequence listing, as well as substantially similar nucleic acid sequences thereof.

[0023] The terms "nucleic acid" and "nucleotide" are to be given their ordinary and customary meanings to those of skill in the art and are used without limitation to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0024] As used herein, the term "nucleotide sequence" refers to a heteropolymer of nucleotides or a sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule, including DNA or RNA molecules, including cDNA, DNA fragments or portions, genomic DNA, synthetic, e.g., chemically synthesized DNA, plasmid DNA, mRNA, and antisense RNA, any of which may be single-stranded or double-stranded. The terms "nucleotide sequence," "nucleic acid," "nucleic acid molecule," "oligonucleotide," and "polynucleotide" are also used interchangeably herein to refer to a heteropolymer of nucleotides. The nucleic acid molecules and / or nucleotide sequences provided herein are presented herein in a 5' to 3' orientation from left to right and are represented using the standard code for representing nucleotide characters as set forth in the United States Sequencing Rules, 37 CFR §§ 1.821-1.825 and World Intellectual Property Organization (WIPO) Standard ST.25. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified or degenerate variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.

[0025] As used herein, the term "gene" refers to a nucleic acid molecule that can be used to produce mRNA, antisense RNA, miRNA, anti-microRNA antisense oligodeoxyribonucleotides (AMOs), and the like. A gene may or may not be capable of being used to produce a functional protein or gene product. A gene may include both coding and non-coding regions, such as introns, regulatory elements, promoters, enhancers, termination sequences, and / or 5' and 3' untranslated regions. A gene may be "isolated," meaning a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and / or various chemicals used in the chemical synthesis of the nucleic acid.

[0026] A "codon-optimized" version of a gene refers to an exogenous gene that is introduced into a cell and the codons of the gene are optimized for a particular cell. Typically, not all tRNAs are expressed equally or at the same level across species. Codon optimization of a gene sequence thereby involves changing codons to match the most common tRNAs, i.e., changing codons recognized by less common tRNAs to synonymous codons recognized by relatively more common tRNAs in a given fungal cell. In this way, mRNA from a codon-optimized gene is translated more efficiently. The codons and synonymous codons preferably code for the same amino acid.

[0027] As used herein, "genetic modification" or "genetically modified" includes such genetic modification to the genome of a fungal cell, such as a yeast cell, and / or the introduction of an exogenous nucleotide sequence, such as in the form of one or more plasmids, into a fungal cell, such as a yeast cell.

[0028] The term "isolated" should be given its ordinary and customary meaning to one of skill in the art, and when used in the context of an isolated nucleic acid or isolated polypeptide, is used without limitation to refer to a nucleic acid or polypeptide that exists apart from its native environment by the hand of man and thus is not a product of nature. An isolated nucleic acid or polypeptide can exist in a purified form or can exist in a non-native environment, such as, for example, a transgenic host cell.

[0029] As used herein, the terms "incubate" and "incubating" refer to the process of mixing two or more chemical or biological entities (such as chemical compounds and enzymes) and allowing them to interact under conditions favorable for producing a composition comprising a jasmonate compound.

[0030] The term "degenerate variant" refers to a nucleic acid sequence having a residue sequence that differs from a reference nucleic acid sequence by one or more degenerate codon substitutions. Degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. A nucleic acid sequence and all of its degenerate variants express the same amino acid or polypeptide.

[0031] The terms "polypeptide", "protein" and "peptide" should be given their ordinary and customary meaning to those of skill in the art. The three terms may be used interchangeably and are used without limitation to refer to a polymer of amino acids or amino acid analogs, regardless of their size or function. Although "protein" is often used in reference to relatively large polypeptides and "peptide" is often used in reference to small polypeptides, the use of these terms in the art overlaps and varies. As used herein, the term "polypeptide" refers to peptides, polypeptides, and proteins, unless otherwise indicated. The terms "protein", "polypeptide" and "peptide" are used interchangeably herein when referring to polynucleotide products. Thus, exemplary polypeptides include polynucleotide products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the above.

[0032] The terms "polypeptide fragment" and "fragment", when used in reference to a reference polypeptide, should be given their ordinary and customary meaning to one of ordinary skill in the art, and are used, without limitation, to refer to a polypeptide that has deleted amino acid residues compared to the reference polypeptide itself, but where the remaining amino acid sequence is typically identical to the corresponding positions in the reference polypeptide. Such deletions may occur at the amino or carboxy termini of the reference polypeptide, or both.

[0033] The term "functional fragment" of a polypeptide or protein refers to a peptide fragment that is a portion of a full-length polypeptide or protein and has substantially the same biological activity or performs substantially the same function (e.g., performs the same enzymatic reaction) as the full-length polypeptide or protein.

[0034] The terms "variant polypeptide," "modified amino acid sequence," or "modified polypeptide," used interchangeably, refer to an amino acid sequence that differs from a reference polypeptide by one or more amino acids, e.g., the substitution, deletion, and / or addition of one or more amino acids. In one aspect, the variant is a "functional variant" that retains some or all of the capability of the reference polypeptide.

[0035] The term "functional variant" further includes conservatively substituted variants. The term "conservatively substituted variant" refers to a peptide having an amino acid sequence that differs from a reference peptide by one or more conservative amino acid substitutions and maintains some or all of the activity of the reference peptide. A "conservative amino acid substitution" is the replacement of an amino acid residue with a functionally similar residue. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue, such as isoleucine, valine, leucine, or methionine, for another; the substitution of one charged or polar (hydrophilic) residue, such as between arginine and lysine, between glutamine and asparagine, between threonine and serine, for another; the substitution of one basic residue, such as lysine or arginine, for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid, for another; or the substitution of one aromatic residue, such as phenylalanine, tyrosine, or tryptophan, for another. Such substitutions are expected to have little or no effect on the apparent molecular weight or isoelectric point of a protein or polypeptide. The phrase "conservatively substituted variants" also includes peptides in which residues are replaced with chemically derivatized residues, provided that the resulting peptide retains some or all of the activity of the reference peptide described herein.

[0036] The term "variant" in reference to the polypeptides of the subject technology further includes functionally active polypeptides having an amino acid sequence that is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identical to the amino acid sequence of a reference polypeptide.

[0037] The term "homologous," in all its grammatical forms and spelling variations, refers to the relationship between polynucleotides or polypeptides that have a "common evolutionary origin," including polynucleotides or polypeptides from superfamilies and homologous polynucleotides or proteins from different species (Reeck et al., Cell 50:667, 1987). Such polynucleotides or polypeptides have sequence homology reflected by their sequence similarity, whether in terms of percent identity or the presence of particular amino acids or motifs at conserved positions. For example, two homologous polypeptides may have amino acid sequences that are at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identical.

[0038] "Suitable regulatory sequences" should be given its ordinary and customary meaning to those of skill in the art and are used without limitation to refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence and that influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0039] "Promoter" should be given its ordinary and customary meaning to those skilled in the art, and is used without limitation to refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Typically, the coding sequence is located 3' to the promoter sequence. A promoter may be derived entirely from a native gene, or may be composed of different elements derived from different promoters found in nature, or may even include synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions. In many cases, a promoter that causes a gene to be expressed in most cell types is commonly referred to as a "constitutive promoter". It is further recognized that in many cases, the exact boundaries of a regulatory sequence are not fully defined, so that DNA fragments of different lengths may have identical promoter activity.

[0040] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence if it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation.

[0041] As used herein, the term "expression" should be given its ordinary and customary meaning to those of skill in the art and is used without limitation to refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment of the subject technology.

[0042] The terms "overexpress", "overexpresses" or "overexpression" as used herein refer to a higher level of activity of a gene, e.g., transcription of the gene, translation of a higher level of mRNA into protein, and / or production of a higher level of a gene product, e.g., a polypeptide, than is present in a cell in its native or control, e.g., state not transformed with the particular heterologous or recombinant polypeptide being overexpressed. A typical example of an overexpressed gene is a gene that is under the transcriptional control of another promoter compared to the gene's native promoter. Additionally or alternatively, other changes in the gene's control elements, such as enhancers, can be used to overexpress a particular gene. Furthermore, modifications that affect, i.e., increase, the translation of mRNA transcribed from the gene may alternatively or additionally be used to achieve an overexpressed gene as used herein. These terms may also refer to an increase in the copy number of a gene in a cell and / or an increase in the amount of mRNA and / or gene product. Overexpression can result in 25%, 50%, 100%, 200%, 500%, 1000%, 2000% or higher levels in a cell compared to control levels.

[0043] "Transformation" should be given its ordinary and customary meaning to those of reasonable skill in the art and is used without limitation to refer to the transfer of a polynucleotide into a target cell. The transferred polynucleotide can be integrated into the genome or chromosomal DNA of the target cell, resulting in genetically stable inheritance, or can replicate independently of the host chromosome. The host organism containing the transformed nucleic acid fragment is referred to as "transgenic" or "transformed."

[0044] The terms "transformed," "transgenic," and "recombinant," when used herein in reference to a host cell, shall be given their ordinary and customary meaning to one of skill in the art, and are used without limitation to refer to a cell of a host organism, such as a plant or microbial cell, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule may be stably integrated into the genome of the host cell, or the nucleic acid molecule may exist as an extrachromosomal molecule. Such extrachromosomal molecules are capable of autonomous replication. A transformed cell, tissue, or subject is understood to encompass not only the end product of the transformation process, but also its transgenic progeny.

[0045] The terms "recombinant," "heterologous," and "exogenous," when used herein in reference to polynucleotides, should be given their ordinary and customary meaning to those of skill in the art, and are used without limitation to refer to polynucleotides (e.g., DNA sequences or genes) that are derived from a source foreign to a particular host cell, or that, if derived from the same source, have been modified from their original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to a particular host cell, but that has been modified, for example, by the use of site-directed mutagenesis or other recombinant techniques. The term also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the term refers to a DNA segment that is foreign or heterologous to the cell, or that is homologous to the cell, but in a location or form within the host cell in which the element is not normally found.

[0046] Similarly, the terms "recombinant," "heterologous," and "exogenous," as used herein in reference to a polypeptide or amino acid sequence, refer to a polypeptide or amino acid sequence that is derived from a source foreign to a particular host cell or, if derived from the same source, has been modified from its original form. Thus, recombinant DNA segments can be expressed in a host cell to produce a recombinant polypeptide.

[0047] An "endogenous," "native," or "wild-type" nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence. Thus, for example, a "wild-type mRNA" is an mRNA that occurs naturally in or is endogenous to an organism. A "homologous" nucleic acid sequence is a nucleotide sequence that is naturally associated with a host cell into which it is introduced.

[0048] The terms "plasmid", "vector" and "cassette" are to be given their ordinary and customary meanings to those skilled in the art and are used without limitation to refer to extrachromosomal elements that often carry genes that are not part of the central metabolism of the cell and are usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome-integrating sequences, phages or nucleotide sequences, linear or circular, single-stranded or double-stranded DNA or RNA from any source, in which several nucleotide sequences are linked or recombined into a unique construct that allows the introduction of a promoter fragment and a DNA sequence for a selected gene product together with appropriate 3' untranslated sequences into a cell. A "transformation cassette" refers to a specific vector that contains a foreign gene and has elements in addition to the foreign gene that facilitate the transformation of a specific host cell. An "expression cassette" refers to a specific vector that contains a foreign gene and has elements in addition to the foreign gene that allow the expression of that gene to be enhanced in a foreign host.

[0049] As used herein, the term "increased copy number" means that at least one extra copy of at least a polypeptide coding sequence of a given gene is present in a recombinant cell compared to the copy number of the same gene in a parent cell, e.g., a wild-type cell, from which the recombinant cell is derived. For example, a recombinant cell may contain 1, 2, 3, 4, 5, 10 or more extra copies of at least a polypeptide coding sequence of a given gene compared to the copy number of that same gene in a parent cell. The extra copies of a given gene may be integrated into the genome of the recombinant cell or may be present on one or more autonomously replicating vectors or plasmids present in the recombinant cell.

[0050] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide or peptide sequences are invariant over the entire window of alignment of components, such as nucleotides or amino acids.The "percent identity" for the aligned segments of test sequence and reference sequence is the number of identical components shared by two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence.

[0051] As used herein, the term "percent sequence identity" or "percent identity" refers to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (the appropriate nucleotide insertion, deletion or gap is less than 20 percent of the reference sequence in total over the comparison window). Optimal alignment of sequences to align a comparison window is well known to those skilled in the art and may be performed by tools such as Smith and Waterman local homology algorithm, Needleman and Wunsch homology alignment algorithm, Pearson and Lipman similarity search method, preferably by computer implementations of these algorithms such as GAP, BESTFIT, FASTA and TFASTA available as part of GCG® Wisconsin Package® (Accelrys Inc., Burlington, MA). The "percent identity" for an aligned segment of a test sequence and a reference sequence is the number of identical elements shared by the two aligned sequences divided by the total number of elements in the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. The percent sequence identity is expressed as the percent identity multiplied by 100. The comparison of one or more polynucleotide sequences can be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. For the purposes of this disclosure, "percent identity" can also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0052] Percent sequence identity is preferably determined using the "Best Fit" or "Gap" programs of the Sequence Analysis Software Package™ (Version 10; Genetics Computer Group, Inc., Madison, WI). "Gap" utilizes the algorithm of Needleman and Wunsch (Needleman and Wunsch, Journal of Molecular Biology 48:443-453, 1970) to find the alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. "BestFit" uses the local homology algorithm of Smith and Waterman (Smith and Waterman, Advances in Applied Mathematics, 2:482-489, 1981; Smith et al., Nucleic Acids Research 11:2205-2220, 1983) to optimally align the best segments of similarity between two sequences and insert gaps to maximize the number of matches. The percent identity is most preferably determined using the "Best Fit" program.

[0053] Useful methods for determining sequence identity are also disclosed in the Basic Local Alignment Search Tool (BLAST) program, publicly available from the National Center for Biotechnology Information (NCBI), National Library of Medicine, National Institutes of Health, Bethesda, Md. 20894. See BLAST Manual, Altschul et al., NCBI, NLM, NIH, Altschul et al., J. Mol. Biol. 215:403-410 (1990). BLAST programs version 2.0 and above allow for the introduction of gaps (deletions and insertions) into the alignment. For peptide sequences, BLASTX can be used to determine sequence identity; for polynucleotide sequences, BLASTN can be used to determine sequence identity.

[0054] As used herein, the term "substantial percent sequence identity" refers to percent sequence identity of at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% identity, at least about 90% sequence identity, or even greater sequence identity, such as about 98% or about 99% sequence identity. Thus, one embodiment of the present invention is a polynucleotide molecule having at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% identity, at least about 90% sequence identity, or even greater sequence identity, such as about 98% or about 99% sequence identity, with the polynucleotide sequences described herein. Polynucleotide molecules encoding proteins having the activity of the gs5119 sequences of the present disclosure can direct the production of various jasmonate compounds and have substantial percent sequence identity with the polynucleotide sequences provided herein and are encompassed within the scope of the present disclosure.

[0055] As used herein, the term "identity" refers to the percentage of amino acids that are the same between a pair of sequences after alignment of the sequences (which can be done using only sequence or structural information or some other information, but is usually based on sequence information alone), and the term "similarity" refers to a score assigned based on alignment using some similarity matrix. The similarity index can be any one of the following: BLOSUM62, PAM250, or GONNET, or any matrix used by those skilled in the art for protein sequence alignment.

[0056] Identity is the degree of correspondence between two subsequences (no gaps between sequences). An identity of 25% or more implies functional similarity, while 18-25% implies structural or functional similarity. Note that two completely unrelated or random sequences (more than 100 residues) may have higher than 20% identity. Similarity is the degree of similarity between two sequences when they are compared. This depends on their identity.

[0057] As is apparent from this specification, certain aspects of the disclosure are not limited by the specific details of the examples set forth herein, and it is therefore contemplated that other modifications and applications, or their equivalents, will occur to those skilled in the art. Accordingly, it is intended that the claims cover all such modifications and applications that do not depart from the spirit and scope of the disclosure.

[0058] Furthermore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described above.

[0059] Metabolically engineered cells for jasmonic acid biosynthesis: In one aspect, the present disclosure provides a host cell having an effective metabolic pathway comprising at least one enzyme activity. The pathway produces 12-oxophytodienoic acid (12-OPDA) which is then reduced to 3-oxo-2-(cis-2'-pentenyl)-cyclopentane-1-octanoic acid (OPC8). Such cells can be naturally occurring, but in certain embodiments are recombinant cells produced by genetic engineering.

[0060] In representative embodiments, OPC8 is produced in a reaction in which endogenous 12-OPDA is a substrate and the reaction is catalyzed by a polypeptide having OPR activity. The polypeptide may be a "gs5119-like protein" (or "gs5119 protein" or "gs5119"), including an amino acid sequence as set forth in SEQ ID NO:1, or a variant thereof having OPR activity and comprising an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO:1. In aspects of these embodiments, the variant polypeptide has the same or substantially the same OPR activity as gs5119 as described herein, e.g., an enzyme comprising the amino acid sequence of SEQ ID NO:1.

[0061] The polypeptide having OPR activity is overexpressed compared to the corresponding parent host cell from which the recombinant host cell is derived. In some embodiments, overexpression can be achieved by genetically modifying the host cell to contain one or more ectopic copies of a native gene encoding a polypeptide having OPR activity. In a further non-exclusive embodiment, overexpressing the polypeptide having OPR activity is carried out by replacing the native promoter of the gene expressing the polypeptide having OPR activity with a promoter having a higher expression level than the promoter native to the host cell. Exemplary higher level promoters include ToxA and ToxB from Pyrenophora tritici-repentis, although any suitable promoter for high level recombinant expression in fungal host cells can serve this role. Alternatively, or in combination with the aforementioned approach, overexpression of the polypeptide having OPR activity can be achieved by recombinantly introducing into the host cell at least one copy of an exogenous polynucleotide sequence that is not native to the host cell and that encodes a polypeptide having OPR activity.

[0062] A nucleic acid encoding a polypeptide having OPR activity may include a polynucleotide sequence encoding a gs5119-like polypeptide. A polynucleotide sequence, also known as a "gs5119-encoding polynucleotide" or "gs5119 polynucleotide", may be, for example, the sequence set forth in SEQ ID NO:2 or a variant thereof, such as the codon-optimized sequence set forth in SEQ ID NO:3. Thus, in some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO:2. Also provided is a nucleic acid comprising a nucleotide sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO:3. In some embodiments, the nucleotide sequence encodes a polypeptide that comprises the enzyme gs5119, an enzyme comprising the amino acid sequence of SEQ ID NO:1.

[0063] In certain embodiments of this aspect, the host cell can be a bacteria. Exemplary bacterial genera include: Escherichia, Salmonella, Bacillus, Acinetobacter, Corynebacterium, Methylosinus, Methylomonas, Rhodococcus, Pseudomonas, Rhodobacter, Synechocystis, and the like. ), Aspergillus, Arthrobotlys, Brevibacteria, Mycobacterium, Arthrobacter, Citrobacter, Klebsiella, Pantoea, Salmonella, Corynebacterium, and Clostridium.

[0064] In further embodiments, the host cell is a eukaryotic cell selected from the group consisting of a yeast cell, a filamentous fungal cell, a plant cell, and an animal cell.

[0065] One aspect of the embodiment relates to genetically modified filamentous fungal cells capable of producing jasmonates. Exemplary fungal cells include those of the genera Lasiodiplodia, Rhizopus, Fusidium, Gibberella, Trichoderma, Hypocrea, Aspergillus, Fusarium, Penicillium, Neurospora, Chaetomium, and the like, including their anamorphs and teleomorphs, as well as recognized synonymous genera. These include species of the genera Acremonium, Glomerella, Myceliophthora, Sporotrichum, Thielavia, Chrysosporium, Corynascus, Ctenomyces, Verticillium, Cordyceps, Nectria, and Magnaporthe.

[0066] The phrase "a fungal species belonging to the genus Lasiodiplodia" may mean that the fungus is classified in the genus Lasiodiplodia according to classifications known to those skilled in the art of mycology. Specifically, those classified into the group of the genus Lasiodiplodia according to the taxonomy used by the NCBI (National Center for Biotechnology Information) database (www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi) can be used. Examples of fungi belonging to the genus Lasiodiplodia include, but are not limited to, Lasiodiplodia abnormis, Lasiodiplodia citri, Lasiodiplodia citricola, Lasiodiplodia crassispora, Lasiodiplodia fiorii, Lasiodiplodia frezaliana, Lasiodiplodia gyranensis, and the like. gilanensis, Lasiodiplodia gonubiensis, Lasiodiplodia hormozganensis, Lasiodiplodia iraniensis, Lasiodiplodia margaritacea, Lasiodiplodia missouriana, Lasiodiplodia paraphysaria, Lasiodiplodia parva, Lasiodiplodia plurivora, Lasiodiplodia pseudotheobromae, Lasiodiplodia ricini ricini, Lasiodiplodia rubropurpurea, Lasiodiplodia theobromaetheobromae, Lasiodiplodia thomasiana, Lasiodiplodia undulata, Lasiodiplodia venezuelensis, and Lasiodiplodia viticola. Fungal species belonging to other genera include, but are not limited to, T. reesei, H. jecorina, A. niger, A. fumigatus, A. orzyae, A. nidulans, F. oxysporum, N. crassa, C. thermophilum, A. thermophilum, G. Examples of suitable microbial species include G. graminicola, M. thermophila, S. thermophile, T. terrestris, T. heterothallica, C. thermophile, V. dahlia, C. militaris, N. heamatococca, or M. orzyae.

[0067] In another aspect of the embodiment, the host cell is selected from the group consisting of soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice, broccoli, cauliflower, cabbage, parsnip, melon, carrot, celery, parsley, tomato, potato, strawberry, peanut, grape, grass seed crops, sugar beet, sugar cane, beans, peas, rye, flax, broadleaf trees, coniferous trees, forage grasses, Arabidopsis thaliana, rice (Oryza sativa), barley (Hordeum yulgare), switchgrass (Panicum vigratum), Brachypodium spp., Brassica spp., and Crambe abyssinica. abyssinica.

[0068] In some embodiments, the host cell is genetically modified to overexpress a polypeptide having OPR activity compared to an unmodified parent cell, e.g., a wild-type host cell, and the polypeptide comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to the amino acid sequence shown in SEQ ID NO:1. The protein having an OPR-like protein may be homologous or heterologous to the host cell. For purposes herein, in certain embodiments, a homologous polypeptide having OPR activity is encoded by a polynucleotide sequence that naturally occurs in, is isolated from, or is derived from the same or a taxonomically equivalent taxonomic species as the host cell. Furthermore, as will be recognized by those skilled in the art, a homologous protein may contain one or more insertions, deletions, and substitutions and still be considered to be "derived from" the same species as the wild-type host cell. Such one or more insertions, deletions, and substitutions may result in an increase or decrease in expression or activity of the homologous polypeptide. Similarly, a polynucleotide encoding a homologous polypeptide having OPR activity may contain one or more insertions, deletions and substitutions, including substitutions that optimize codon usage without altering the sequence of the encoded protein.

[0069] A heterologous polypeptide having OPR activity is encoded by a polynucleotide sequence that naturally occurs in, is isolated from, or is derived from a taxonomic species different from the host cell. Moreover, as will be appreciated by those skilled in the art, a heterologous polypeptide having OPR activity can contain one or more insertions, deletions, and substitutions and still be considered to be "derived" from a taxonomic species different from the host cell. Such one or more insertions, deletions, and substitutions can result in an increase or decrease in the expression or activity of the heterologous polypeptide having OPR activity. Similarly, a polynucleotide encoding a heterologous polypeptide having OPR activity can contain one or more insertions, deletions, and substitutions, including substitutions that optimize codon usage without changing the sequence of the encoded protein.

[0070] As used herein with respect to a polynucleotide sequence, "derived from" refers to the isolation of a target polynucleotide sequence using one or more molecular biology techniques known to those of skill in the art, including, but not limited to, back-translation of a polypeptide or amino acid sequence, cloning, subcloning, amplification by PCR, in vitro synthesis, and the like. Additionally, as will be appreciated by those of skill in the art, a polynucleotide sequence derived from a target polynucleotide sequence may be modified by one or more insertions, deletions, and substitutions and still be considered "derived from" the target nucleotide sequence. Such one or more insertions, deletions, and substitutions may result in an increase or decrease in expression or activity of a protein of interest encoded by the polynucleotide sequence, and may be located within the promoter sequence, 5' or 3' untranslated region, or within the coding region of the protein of interest.

[0071] As used herein with respect to a polynucleotide sequence, "isolated" or "isolated" means that the nucleic acid sequence has been altered from its natural state by separating it from some or all of the naturally-occurring nucleic acid sequence with which it is essentially associated.

[0072] In some embodiments, a host cell may be genetically modified by transformation of the host cell with a gs5119 gene construct. As used herein, a "gs5119 gene construct" refers to an isolated polynucleotide that contains elements necessary to increase expression of a gs5119-like protein. These elements may include, but are not limited to, a polynucleotide sequence that encodes a gs5119-like protein (coding sequence), a promoter that includes a polynucleotide sequence operably linked to the coding sequence and directs the transcription and translation of the coding sequence.

[0073] The recombinant host cells of the invention may further comprise one or more genetic constructs directing the production and secretion of one or more homologous or heterologous polypeptides having OPR activity, such as gs5119 or gs5119-like proteins. Such constructs include, but are not limited to, a coding sequence for the polypeptide, a promoter operably linked to the coding sequence and comprising a polynucleotide sequence directing transcription of the coding region, and a polynucleotide element comprising a sequence encoding a secretory signal peptide operably linked to the coding sequence, as well as a targeting polynucleotide sequence directing homologous recombination of the construct into the genome of the host cell. The terms "secretory signal peptide", "secretory signal" and "signal peptide" refer to any sequence of nucleotides and / or amino acids that may be involved in the secretion of the mature or precursor form of a secreted protein. The signal sequence may be endogenous or exogenous to the host cell. The signal sequence may be one normally associated with a gene encoding the protein of interest or another secreted protein. The signal sequence may also be a "hybrid signal sequence" that comprises partial sequences from two or more genes encoding secreted proteins.

[0074] As will be appreciated by those skilled in the art, the coding sequence, promoter and / or secretion signal may be derived from a parent host cell, a different organism, and / or may be synthesized in vitro. For example, the promoter and secretion signal may be derived from one or more genes encoding proteins that are highly expressed and secreted when the parent host cell is grown in a fermentation process as defined below, e.g., genes that are typically highly expressed in filamentous fungi. These polynucleotide elements may also be altered or modified by the replacement, substitution, addition or elimination of one or more nucleic acids compared to the naturally occurring polynucleotide. However, it should be understood that the practice of the present invention is not limited by the selection of the promoter in the gs5119 gene construct or by the selection of the promoter and secretion signal in the gene construct expressing a gs5119-like enzyme.

[0075] The above genetic constructs may contain a selection marker for the identification of transformed host cells. The selection marker may be present on the genetic construct or it may be a separate isolated polynucleotide co-transformed with the genetic construct. The choice of selection marker is well known to those skilled in the art and includes genes (synthetic or natural) that confer the ability to utilize a metabolite that is not normally metabolized by microorganisms (e.g., the A. nidulans amdS gene, which encodes acetamidase and confers the ability to grow on acetamide as the sole nitrogen source), or genes that confer antibiotic resistance (e.g., the Escherichia coli hph gene, which encodes hygromycin-beta-phosphotransferase and confers resistance to hygromycin). Alternatively, if the host cell expresses little or no selected marker activity, the corresponding gene can be used as a marker. Examples of such markers include trp, pyr4, pyrG, argB, leu, etc. The corresponding host strain must therefore lack a functional gene corresponding to the selected marker, ie lack expression of trp, pyr, arg, leu, etc.

[0076] The gene construct may contain a transcription terminator functional in the host cell, as known to those skilled in the art. The transcription terminator may be located immediately downstream of the coding sequence. The practice of the present invention is not limited by the selection of a transcription terminator sufficient to direct the termination of transcription in the host cell.

[0077] Gene constructs may contain additional polynucleotide sequences between the various sequence elements described herein. These sequences, which may be natural or synthetic, may add one or more amino acids to the protein encoded by the construct. The implementation of the present invention is not restricted by the presence of additional polynucleotide sequences between the various sequence elements of the gene constructs present in the host cell.

[0078] As disclosed above, some embodiments relate to recombinant filamentous fungal host cells. Methods for introducing genetic constructs into fungal cells are well known to those skilled in the art and include, but are not limited to, Agrobacterium tumefaciens-mediated transformation, calcium chloride treatment of fungal protoplasts to weaken cell membranes, addition of polyethylene glycol to allow cell membrane fusion, depolarization of cell membranes by electroporation, or firing of the construct through cell walls and membranes by microprojectile bombardment using a particle gun. The practice of the present invention is not limited by the method for introducing genetic constructs into fungal cells.

[0079] The recombinant fungal cells presented herein are understood to encompass both fective and imperfect forms, as well as other taxonomic equivalents, such as anamorphs and teleomorphs, regardless of the species name by which they are known. Further examples of taxonomic equivalents can be found, for example, in Cannon, Mycopathologica 111:75-83, 1990; Moustafa et al., Persononia 14:173-175, 1990; Stalpers, Stud. Mycol. 24, 1984; Upadhyay et al., Mycopathologia 87:71-80, 1984; Guarro et al., Mycotaxon 23:419-427, 1985; Awao et al., Mycotaxon 16:436-440, 1983; von Klopotek, Arch. Microbiol. 98:365-369, 1974; and Long et al., 1994, ATCC Names of Industrial Fungi, ATCC, Rockville Md. Those skilled in the art will readily recognize the identity of the appropriate equivalents. Thus, unless otherwise indicated, use of a particular genus and / or species designation in this disclosure will also be understood to refer to genera and species related by anamorphic or teleomorphic relationships, genera and species recognized as synonyms, and those that have been or may in the future be reclassified into one of the claimed genera or species.

[0080] In plant cells, the expression vector of the subject technology can include a coding region operably linked to a promoter capable of directing expression of the recombinant polypeptide of the subject technology in a desired tissue at a desired developmental stage. Conveniently, the polynucleotide to be expressed can include a promoter sequence and a translation leader sequence derived from the same polynucleotide. A 3' non-coding sequence that codes for a transcription termination signal should also be present. The expression vector can also include one or more introns to facilitate polynucleotide expression.

[0081] For plant host cells, any combination of promoters and terminators capable of inducing the expression of coding regions can be used in the vector sequences of the subject technology. Some suitable examples of promoters and terminators include those from nopaline synthase (nos), octopine synthase (ocs) and cauliflower mosaic virus (CaMV) genes. One type of efficient plant promoter that can be used is a high-level plant promoter. Such a promoter must be operably linked to the expression vector of the subject technology to promote the expression of the vector. High-level plant promoters that can be used in the subject technology include, for example, the promoter of the small subunit (ss) of ribulose-l,5-bisphosphate carboxylase from soybean (Berry-Lowe et al., J. Molecular and App. Gen., 1:483 498 (1982), which is incorporated herein in its entirety to the extent that it is consistent with this specification), and the promoter of chlorophyll alb-binding protein. These two promoters are known to be light-inducible in plant cells (see, e.g., Genetic Engineering of Plants, an Agricultural Perspective, A. Cashmore, Plenum, NY (1983), pages 29-38; Coruzzi, G. et al., The Journal of Biological Chemistry, 258:1399 (1983) and Dunsmuir, P. et al., Journal of Molecular and Applied Genetics, 2:285 (1983), each of which is incorporated by reference herein to the extent consistent herewith).

[0082] synthetic biology Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described, for example, in Sambrook, J., Fritsch, E F and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1989 (hereinafter "Maniatis"); and Silhavy, T J, Bennan, M L and Enquist, L W Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1984; and Ausubel, F M et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, 1987 (each of which is hereby incorporated by reference in its entirety).

[0083] Production Expression of proteins in recombinant host cells is often carried out using vectors that contain constitutive or inducible promoters directing the expression of one or more recombinant proteins. Fusion vectors add several amino acids to the protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (1) increase the expression of the recombinant protein; (2) increase the solubility of the recombinant protein; and (3) aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction between the fusion moiety and the recombinant protein to allow separation of the recombinant protein from the fusion moiety after purification of the fusion protein. Such vectors are within the scope of the present disclosure.

[0084] In one embodiment, an expression vector contains genetic elements for expression of a recombinant polypeptide in a bacterial cell or other microorganism. Elements for transcription and translation in a microbial cell can include a promoter, a coding region for the protein complex, and a transcription terminator.

[0085] Those skilled in the art will be aware of the molecular biology techniques available for the preparation of expression vectors. As noted above, the polynucleotides used for incorporation into expression vectors of the subject technology can be prepared by routine techniques such as polymerase chain reaction (PCR).

[0086] Several molecular biology techniques have been developed to operably link DNA to vector via complementary cohesive ends.In one embodiment, complementary homopolymer tracts can be added to the nucleic acid molecule that is inserted into vector DNA.The vector and nucleic acid molecule are then linked by hydrogen bonds between complementary homopolymer tails to form recombinant DNA molecules.

[0087] In an alternative embodiment, a synthetic linker containing one or more restriction sites provided is used to operably link the polynucleotides of the subject technology to an expression vector. In one embodiment, the polynucleotides are generated by restriction endonuclease digestion. In one example, the nucleic acid molecules are treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding 3'-single-stranded ends with their 3'-5'-exonuclease activity and fill in recessed 3'-ends with their polymerization activity, thereby generating blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that can catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are polynucleotides carrying polymer linker sequences at their ends. These polynucleotides are then cleaved with the appropriate restriction enzyme and ligated into expression vectors that have been cleaved with an enzyme that produces ends compatible with the ends of the polynucleotides.

[0088] Alternatively, a vector with ligation-independent cloning (LIC) site can be utilized. The required PCR amplified polynucleotide can then be cloned into the LIC vector without restriction digestion or ligation (Aslanidis and de Jong, Nucl. Acid. Res. 18 6069-74, (1990); Haun et al., Biotechniques 13, 515-18 (1992), which is incorporated herein by reference to the extent consistent with the present specification).

[0089] In one embodiment, it is suitable to use PCR to isolate and / or modify the polynucleotide of interest for insertion into a selected plasmid. Suitable primers for use in PCR preparation of the sequence can be designed to isolate the required coding region of the nucleic acid molecule, add restriction endonuclease or LIC sites, and place the coding region in the desired reading frame.

[0090] In one embodiment, the polynucleotide for incorporation into the expression vector of the subject technology is prepared using PCR with suitable oligonucleotide primers. The coding region is amplified, but the primers themselves are incorporated into the amplified sequence product. In one embodiment, the amplification primers contain restriction endonuclease recognition sites that allow the amplified sequence product to be cloned into a suitable vector.

[0091] The expression vector can be introduced into a host cell, for example, bacteria or other microorganisms such as yeast or fungi, by conventional transformation or transfection techniques. Transformation of suitable cells with the expression vector of the subject technology is accomplished by methods known in the art and typically depends on both the type of vector and the cell. Suitable techniques include calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, chemoporation or electroporation. Microbial host cell expression systems and expression vectors containing regulatory sequences directing high-level expression of foreign proteins are well known to those skilled in the art. Any of these can be used to construct vectors for the expression of the recombinant polypeptides of the subject technology in microbial host cells. These vectors can then be introduced into suitable microorganisms by transformation to allow high-level expression of the recombinant polypeptides of the subject technology.

[0092] Vectors or cassettes useful for transformation of suitable microbial host cells are well known in the art. Typically, the vector or cassette contains sequences directing the transcription and translation of the associated polynucleotide, a selection marker, and sequences allowing autonomous replication or chromosomal integration. A suitable vector contains a region 5' of the polynucleotide that has transcription initiation control, and a region 3' of the DNA fragment that controls transcription termination. It is preferred that both control regions are derived from genes homologous to the transformed host cell, but it is understood that such control regions need not be derived from genes native to the particular species selected as the host.

[0093] Initiation control regions or promoters useful for driving the expression of recombinant polypeptides in desired microbial host cells are numerous and well known to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the subject technology, including, but not limited to, CYCI, HIS3, GALI, GALIO, ADHI, PGK, PH05, GAPDH, ADCI, TRPI, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces), AOXI (useful for expression in Pichia), lac, trp, JPL, IPR, T7, tac and trc (useful for expression in Escherichia coli), and ToxA and ToxB (useful for expression in filamentous fungi).

[0094] Successfully transformed cells, i.e., cells containing expression vectors, can be identified by techniques well known in the art. For example, cells transfected with the expression vector of the subject technology can be cultured to produce the polypeptides described herein. Cells can be tested for the presence of expression vector DNA by techniques well known in the art. Transformed host cells can contain a single copy of the expression vector described above, or alternatively, multiple copies of the expression vector.

[0095] Jasmonate biosynthesis In a further aspect, the invention comprises culturing a recombinant host cell comprising a metabolic pathway to produce jasmonic acid, wherein the host cell overexpresses a polypeptide having 12-oxophytodienoic acid reductase (OPR) activity relative to a corresponding parent host cell. The recombinant host cell may be cultured in submerged liquid fed-batch culture or continuous culture.

[0096] As used herein, the terms "cultivating" and "culturing" refer to growing a cell culture, e.g., a population of microbial cells, under suitable conditions in a liquid or solid medium. Cultivation may be carried out using conventional fermentation equipment suitable for such purposes (e.g., shake flasks, fermentors, and bioreactors).

[0097] As defined herein, a "submerged liquid culture" is a cell culture in which the cells are suspended or significantly suspended in a liquid medium containing the nutrients necessary to maintain cell viability. The culture is generally agitated at a rate sufficient to ensure distribution of the cells throughout the medium. The agitation rate is also typically selected to prevent the formation of nutrient concentration gradients.

[0098] In a "batch process" or "batch fermentation", all of the necessary culture and medium components, except oxygen for an aerobic process, are placed into a reactor at the start of the operation and the fermentation is allowed to proceed to completion, at which time the product is removed from the reactor. In a "fed-batch process" or "fed-batch fermentation", a culture is continuously or sequentially fed with one or more medium components without removing the culture medium.

[0099] In a "continuous process" or "continuous fermentation", fresh medium is supplied and culture liquid is continuously removed at volumetrically equal or substantially equal rates to maintain the culture at a steady growth rate. With respect to a continuous process, "steady state" refers to a state in which the concentrations of reactants do not change appreciably, and "quasi-steady state" refers to a state in which, after initiation of the reaction, the concentrations of reactants fluctuate within a range consistent with normal operation of a continuous hydrolysis process. A continuous fermentation process may also be referred to as a CSTR (continuous stirred tank reactor) fermentation. One example of a continuous fermentation process is a chemostat, where the growth rate of a microorganism is controlled by the supply of one limiting nutrient in the medium.

[0100] In the fermentation process of the present invention, the recombinant host cells may be initially cultured in batch fermentation, typically with a non-inducing carbon source. Typically, upon completion of the batch fermentation, e.g., as determined by the exhaustion of essentially all of the available carbon source, the concentration of the carbon source in the culture filtrate is 1 g / L or less, the recombinant host cells are cultured in fed-batch, continuous or combined fed-batch and continuous submerged liquid culture.

[0101] Fed-batch and continuous processes are typically carried out in one or more bioreactors. Typical bioreactors used in cell culture fermentation processes include, but are not limited to, those with mechanically agitated vessels or other agitation means (such as air injection). The bioreactor may be temperature and pH controlled. Typically, a means is provided for washing the reactor in place. A means may also be provided for disinfecting or sterilizing the bioreactor prior to introduction of the target organism to minimize or prevent competition for the carbon source from other organisms. Bioreactors can be constructed from many materials, but are most often glass or stainless steel. Generally, provisions are made for sampling (in a manner that prevents or minimizes the introduction of undesirable competing organisms). Means are often provided for obtaining other measurements (e.g., ports and probes for measuring dissolved oxygen concentration or the concentration of other solutes such as ammonium ions). The practice of the present invention is not limited by the choice of bioreactor.

[0102] In the fermentation process of the present invention, a fed-batch, continuous or combined fed-batch and continuous submerged liquid culture is provided with a feed liquid containing a carbon source. In some embodiments, the carbon source consists of one or more carbohydrates. As used herein, the term "carbon source" refers to a carbon-containing substance that provides the majority of the carbon required for the growth of the parent or recombinant cell culture and the production of jasmonates therefrom. For purposes herein, the carbon source may be one or more carbohydrates, non-carbohydrate substances such as sugar alcohols, organic acids, or alcohols, or combinations thereof. However, for purposes herein, an organic nitrogen source that may be provided to the cell culture may or may not be considered a carbon source.

[0103] In the fermentation process of the present invention, the feed solution may contain one or more additional components, such as nitrogen sources, vitamins, minerals and salts, necessary for the growth of fungal cells, as known to those skilled in the art. The nitrogen sources may be inorganic and / or organic in nature, including, but not limited to, one or more amino acids, peptides and proteins in pure or raw form (e.g., corn steep liquor), any number of protein hydrolysates (peptone, tryptone, casamino acids), yeast extract, ammonia, ammonium hydroxide, ammonium salts, urea, nitrates and combinations thereof. The implementation of the fermentation process of the present invention is not limited by the additional components of the feed solution.

[0104] The feed solution is provided to the fermentation process at a rate, feed rate or "carbon addition rate" or "CAR" (measured as g carbon per liter per hour). In an exemplary fermentation process according to the invention, the feed solution may be provided to the fed-batch culture at a carbon addition rate of about 0.2 to about 4 g carbon / L culture / h, or any rate therebetween, such as 0.2, 0.3, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.5, 3.0, 3.5, and 4.0 g carbon / L culture / h, or any rate therebetween. Alternatively, the feed solution may be provided to the fed-batch culture at a carbon addition rate of about 0.001 to 0.1 h. -1or any dilution rate therebetween, e.g., about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 h -1 , or any dilution rate therebetween.

[0105] The fermentation process of the present invention may be carried out at a temperature between about 20°C and about 55°C, or any temperature therebetween, such as between about 30°C and about 45°C, or any temperature therebetween, or at 20, 22, 25, 28, 30, 32, 35, 38, 40, 42, 45, 48, 50°C, 55°C, or any temperature therebetween.

[0106] The fermentation process of the present invention may be carried out at a pH of about 2.5 to 8.5, or any pH therebetween, such as about pH 3.5 to pH 7.0, or any pH therebetween, such as about pH 2.5, 3.0, 3.2, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.2, 5.4, 5.5, 5.7, 5.8, 6.0, 6.2, 6.5, 6.8, 7.0, 7.2, 7.5, 7.8, 8.0, 8.5, or any pH therebetween. The pH may be controlled by the addition of a base, such as ammonium or sodium hydroxide, or by the addition of an acid, such as phosphoric acid.

[0107] The fermentation process of the present invention may be carried out for about 1 to 90 days, or any period therebetween, for example, 3 to 30 days, or any amount therebetween, 3 to 8 days, or any amount therebetween, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 60, 70, 80, or 90 days, or any amount therebetween.

[0108] The fermentation process of the present invention may comprise at least 0.5 liters, e.g., from about 0.5 to about 1,000,000 liters, or any amount therebetween, e.g., from 5 to about 400,000 liters, or any amount therebetween, from 20 to about 200,000 liters, or any amount therebetween, or from 2,000 to about 200,000 liters, or any amount therebetween, or from about 0.5, 1, 10, 50, 100, 200, 400, 600, 800, 1000, 2000, 4000, 6000, 8000, 10 In some embodiments, the method may be carried out in a culture having a volume of 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 150,000, 200,000, 300,000, 400,000, 500,000, 750,000 or 1,000,000 liters, or any amount therebetween.

[0109] The fermentation process of the present invention may be carried out aerobically, in the presence of oxygen, or anaerobically, in the absence of oxygen. For example, the process may be carried out aerobically such that air or oxygen gas is provided to the submerged liquid culture at a superficial gas velocity of about 0.001 to about 100 cm / s, or any rate therebetween, such as a rate of about 0.01 to about 20 cm / s, or any rate therebetween. An alternative parameter for measuring aeration rate known to those skilled in the art is vessel volumes per minute (vvm). In the fermentation process of the present invention, air or oxygen gas is provided to the submerged liquid culture at a rate of about 0.5 to about 5 vvm, or any rate therebetween. Antifoam agents (either silicone or non-silicone based) can be added to control excessive foaming during the process, if necessary, as known to those skilled in the art.

[0110] As used herein, the term "specific productivity", alternatively expressed as "qp", refers to the rate at which one or more jasmonates are produced from a given mass of recombinant host cells. Typically, the specific productivity of a fermentation process is expressed as mg protein per g of host cells per hour (mg protein / g cells / h) and is calculated by measuring the concentration in mg / L of jasmonate in the culture filtrate (culture medium from which the recombinant cells have been removed), divided by the concentration of cultured cells in the culture medium (in g dry weight units per L), and divided by the total time in hours since the feed solution was first provided to the culture. The fermentation process of the present invention may also be characterized by a "maximum productivity" (or "max qp"), which is the maximum value of qp calculated during the course of the fermentation process, or an "average productivity" (or "average qp"), which is the average of all values ​​of qp calculated during the course of the fermentation process.

[0111] In some embodiments, a fermentation process according to the invention in which one or more jasmonates are produced from a culture of cells overexpressing a polypeptide having 12-oxophytodienoic acid reductase (OPR) activity compared to a corresponding parent host cell exhibits at least a 50% increase in maximum specific productivity (qp) compared to that exhibited by a comparable process utilizing the parent host cell from which the recombinant host cell is derived. For example, such a fermentation process may exhibit at least a 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more increase in maximum specific productivity (qp) compared to that exhibited by a comparable process utilizing the parent host cell from which the recombinant host cell is derived.

[0112] As used herein, the term "equivalent fermentation process" or "equivalent process" refers to a fermentation process in which a parent fungal cell is cultured under conditions of the same or nearly the same medium composition, time, cell density, temperature and pH as those used to culture the isolated fungal cell derived from that parent fungal cell.

[0113] After cultivation, the product, such as jasmonic acid, can be extracted directly from the liquid medium, or solid matter, such as cells, can be removed from the medium by centrifugation or membrane filtration, and the product can be collected and purified by ion exchange, concentration, distillation, and crystallization. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Accordingly, the descriptions and examples should not be construed as limiting the scope of the invention which is delineated by the appended claims.

[0114] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred materials and methods are described below.

[0115] The present disclosure will be more fully understood by considering the following non-limiting examples. It should be understood that these examples, while showing preferred embodiments of the subject technology, are given by way of illustration only. From the above discussion and these examples, those skilled in the art can ascertain the essential features of the subject technology, and can make various changes and modifications thereof to adapt the subject technology to various applications and conditions without departing from the spirit and scope thereof.

[0116] example The subject technology is further defined in the following examples. It should be understood that these examples, although showing preferred embodiments of the subject technology, are given as examples only. From the above discussion and these examples, those skilled in the art can ascertain the essential features of the subject technology, and can make various changes and modifications thereof to adapt the subject technology to various applications and conditions without departing from the spirit and scope thereof.

[0117] Example 1 Genomic and transcriptomic analyses were performed on Lasiodiplodia iraniensis, a species of jasmonic acid-producing fungus (Zheng et al., 2019), to identify several candidate sequences homologous to plant OPR genes (Schaller and Weiler, 1997). The candidate genes were then expressed in E. coli bacteria with N-terminal His tags, and the product proteins were purified with a Ni-NTA purification system. An HPLC-based OPR activity assay was used to screen the candidate proteins, and the results showed that g5119 (SEQ ID NO:2) encodes a 12-oxophytodienoic acid reductase enzyme (SEQ ID NO:1) in Lasiodiplodia iraniensis.

[0118] Codon-optimized g5119 (SEQ ID NO: 3) was synthesized by Gene Universal Inc. (Newark, Delaware) and cloned into the pET-28a vector (Novagen, Wisconsin) between the NdeI and XhoI sites. The resulting construct was transformed into BL21(DE3) competent Escherichia coli (E. coli) cells for expression. In a typical experiment, overnight cultures were used to inoculate liquid Luria-Bertani (LB) medium (2%) containing 100 mg / L carbenicillin. Cultures were initially grown to an OD of 0.6 at 37°C. 600 The cells were grown at 37 °C for 1 h and cooled to 16 °C. Protein expression was then induced by adding 1 mM IPTG. After incubation at 16 °C for 18 h, the cells were harvested by centrifugation and stored at -80 °C until use.

[0119] Total soluble protein was extracted from frozen cells using B-PER™ Bacterial Cell Lysis Reagent (Thermo Fisher Scientific, Massachusetts) according to the manufacturer's instructions and further purified by Ni-NTA column (Qiagen, Maryland) as shown in Figure 1 A. Purified g5119 protein was visualized on an SDS-PAGE gel as shown in Figure 1 B.

[0120] OPR activity assays were performed in 100 mM K-Pi buffer, pH 7.0, containing 50 mg / L OPDA (Cayman Chemical, Michigan), 1 mM NADPH, and purified g5119 protein. The reaction was stopped by adding HCl to pH 2, followed by ethyl acetate extraction. The ethyl acetate phase was used for HPLC analysis.

[0121] HPLC analysis was performed on a Thermo Scientific Vanquish UHPLC system using an Acclaim™ 120, C18 column (3 μm 120A, 3 × 150 mm). The mobile phase was A, 0.1% TFA (trifluoroacetic acid) and B, acetonitrile with a gradient: 0–5 min, 5% B; 5–9 min, 5–80% B; 9–13 min, 80% B; 13–14 min, 80–5% B; 14–17 min, 5% B. The detector wavelengths for OPDA and OPC8 were set at 200 nm. Figure 2 contains a typical HPLC profile of the g5119-catalyzed reaction. Figure 3 contains the LC / MS analysis of the g5119-catalyzed reaction. In #3-5199, the reaction was catalyzed by g5199 in the absence of NADPH. In #6-5199, the reaction was catalyzed by g5199 in the presence of 1 mM NADPH, where no OPDA was detected after the reaction, indicating complete conversion.

[0122] Example 2 Agrobacterium tumefaciens-mediated transformation (ATMT) has long been used to transfer genes into a wide variety of fungi, including plant pathogenic or symbiotic fungi (Vieira and Camilo, 2011). A. tumefaciens has the natural ability to transfer a segment of its Ti plasmid, known as "T-DNA", into plant or fungal cells, where it randomly integrates into nuclear chromosomes. A binary vector system can be used by ATMT. In this system, the T-DNA and the pathogenicity region are separated on two different plasmids, allowing easier genetic manipulation of smaller binary vectors containing T-DNA (Hoekema et al., 1983), in mushrooms, industrial fungi and biological control fungi (Ando et al., 2009; Sharma and Kuhad, 2010; Vieira and Camilo, 2011). A. tumefaciens has the natural ability to transfer a segment of its Ti plasmid, known as "T-DNA", into plant or fungal cells, where it randomly integrates into nuclear chromosomes. A binary vector system can be used by ATMT, in which the T-DNA and the virulence region are separated on two different plasmids, allowing easier genetic manipulation of smaller binary vectors containing the T-DNA (Hoekema et al., 1983).

[0123] As reported in the literature, ATMT was successfully performed in Lasiodiplodia theobromae (Muniz et al., 2014). Similar ATMT-based transformation protocols can be used to achieve expression and / or overexpression of gs5119-like proteins in species of the genus Lasiodiplodia, such as Lasiodiplodia iraniensis.

[0124] Microbial sources and growth conditions Wild-type L. iraniensis is stored at 5 °C on potato dextrose agar (PDA) (Muniz et al., 2012). Escherichia coli strain DH5α is used as a host for propagation of plasmid DNA. A. tumefaciens strain AGL1 harboring the binary vector is maintained on Luria-Bertani (LB) medium supplemented with 250 μg / mL spectinomycin.

[0125] Plasmids The backbone of pPm43GW (VIB, Gent, Belgium) is used to construct a binary vector for transforming L. iraniensis. It contains a cassette in which the E. coli hygromycin B (Hyg B) phosphotransferase (hph) resistance gene is under the control of the Aspergillus nidulans trpC promoter and a PtGFP cassette, which contains the promoter toxA-5'-UTR from Pyrenophora tritici-repentis, a gene encoding a polynucleotide sequence encoding a gs5119-like protein ("gs5119"), and a nos terminator (Tnos). The resulting plasmid is designated pPm43GW-gs5119-HPH. As shown in Figure 5 , gs5119 is under the control of PtoxA-5′-UTR, with LB and RB representing the left and right borders.

[0126] This vector is transformed into A. tumefaciens strain AGL1 using electroporation method Bacteria are spread onto LB plates supplemented with 250 μg / mL spectinomycin and incubated at room temperature for 2 days.

[0127] Sensitivity to the hygromycin B test Prior to transformation, cultures obtained from germinated pycnidiospores (Muniz et al., 2012) are subjected to a minimal inhibitory concentration of hygromycin B. The cultures are inoculated onto PDA containing different concentrations of hygromycin B (0, 50, 100, 150, 200, 250 and 300 μg / mL) and then incubated at 28 °C for 14 days. For each treatment, five Petri dishes were used. The hygromycin B concentration that completely inhibits mycelial growth on all five plates is used for the transformation experiment. This test is repeated twice.

[0128] Fungal transformation Transformation is performed as previously described with minor modifications (de Groot et al., 1998; dos Reis et al., 2004; Staats et al., 2007). An isolated colony of A. tumefaciens strain AGL1 is grown overnight at 27°C, 150 rpm in 20 mL LB-mannitol (10 g bactopeptone, 5 g yeast extract, 2.5 g NaCl, 10 g mannitol) supplemented with 250 μg / mL spectinomycin and 75 μg / mL carbenicillin. Subsequently, A. tumefaciens cells are centrifuged and resuspended in 20 mL minimal medium (MM) (11.4 mM K2HPO4, 10.6 mM KH2PO4, 2.4 mM MgSO4-7H2O, 5.4 mM NaCl, 68 μM CaCl2-2H2O, 6.6 μM FeSO4, 1.74 mM ZnSO4-7H2O, 2 mM CuSO4-5H2O, 8 mM H3BO3, 2.96 mM MnSO4-H2O, 2 mM Na2MoO4-2H2O, 6.3 mM NH4NO3, 11 mM glucose) containing 75 μg / mL carbenicillin and 250 μg / mL spectinomycin. After overnight incubation at 27 °C and 150 rpm, cultures were cultured in 20 mL of induction medium (IM) (same as MM but amended with 40 mM (2-[N-morpholino]ethanesulfonic acid), 54 mM glycerol and 200 µL acetosyringone (AS), containing 75 µg / mL carbenicillin and 250 µg / mL spectinomycin) to an OD of 0.15. 660 Dilute to OD660 Cultures are returned to the same growth conditions until a β-spore ratio of 0.25 is reached. L. iraniensis monospore strains are grown on PDA at 28 °C for 14 days. Pycnidiospores are obtained according to the method of Muniz et al. (2012), collected by flooding a Petri dish with sterile water, and counted under a microscope equipped with a Neubauer chamber. The final pycnidiospore concentration is approximately 10 in saline. 7 Adjust the concentration to spores / mL. Co-cultivation of A. tumefaciens with pycnidiospore solution is performed by adding 100 µL of bacterial culture to 100 µL of fungal suspension. The mixture is placed on a square cellulose membrane (120 x 120 x 17 mm) on co-cultivation medium (IM + 1.5% agar) in the presence and absence of 200 µL AS and incubated at 22 °C for 2 days. After 2 days, the sample is transferred to a selective medium (SM) amended with 150 µg / mL hygromycin and 200 µM / mL cefotaxime to inhibit the growth of A. tumefaciens. The colonies that appear after incubation at 28 °C (putative transformants) are transferred to PDA containing 150 µg / mL hygromycin and incubated at 28 °C.

[0129] Transgene stability and viability of Lasiodiplodia transformants All transgenic isolates from ATMT of Lasiodiplodia are subcultured on PDA amended with 150 μg hygromycin. The plates are incubated at 28 °C for 8 days. Every 2 days, the colony growth rate is checked. After 8 days, pycnidiospore production is tested. This procedure is repeated three times in succession. The fungal colonies are then analyzed for GFP expression by fluorescence microscopy. HygB minimum inhibitory concentration of L. iraniensis determined by culturing monospore cultures on PDA containing different HygB concentrations. The minimum inhibitory concentration is then used for the initial selection of transformants. [Industrial Applicability]

[0130] The present disclosure has applicability in the nutraceutical and pharmacological industries.The present disclosure relates generally to methods for the biosynthetic production of jasmonic acid, for example, via modified microbial strains. [ka] TIFF2025512101000003.tif127165 SEQ ID NO:1 Amino acid sequence of wild-type 12-oxophytodienoic acid reductase enzyme gs5119 in Lasiodiplodia iraniensis: SEQ ID NO:2 DNA sequence of wild-type 12-oxophytodienoic acid reductase enzyme gs5119 in Lasiodiplodia iraniensis: SEQ ID NO:3 DNA sequence of wild-type 12-oxophytodienoic acid reductase enzyme gs5119 in Lasiodiplodia iraniensis, codon-optimized for Escherichia coli:

Claims

1. Recombinant host cells comprising a metabolic pathway for producing jasmonic acid, wherein the host cells overexpress a polypeptide having 12-oxophytodienoic acid reductase (OPR) activity compared to the corresponding parental host cell, and the polypeptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:

1.

2. The recombinant host cell according to claim 1, wherein the overexpression of the polypeptide having OPR activity is provided by replacing the native promoter of the gene expressing the polypeptide having OPR activity with a promoter having a higher expression level than the native promoter.

3. The recombinant host cell according to claim 2, wherein the native promoter is replaced with the ToxA promoter of Pyrenophora tritici-repentis or the ToxB promoter of Pyrenophora tritici-repentis.

4. The recombinant host cell according to claim 1, wherein the overexpression of the polypeptide having OPR activity is provided by recombinantly introducing at least one copy of an exogenous polynucleotide sequence encoding the polypeptide having OPR activity into the host cell.

5. Recombinant host cells comprising a metabolic pathway for producing jasmonic acid, wherein the host cells overexpress a gene encoding a polypeptide having 12-oxophytodienoic acid reductase (OPR) activity compared to the corresponding parental host cell, and the gene comprises a polynucleotide sequence having at least 90% identity with the polynucleotide sequence shown in SEQ ID NO: 2, or a polynucleotide sequence having at least 90% identity with the polynucleotide sequence shown in SEQ ID NO:

3.

6. The recombinant host cell according to claim 5, wherein the overexpression of the gene encoding a polypeptide having 12-oxophytodienoic acid reductase (OPR) activity is provided by replacing the native promoter of the gene expressing the polypeptide having OPR activity with a promoter having a higher expression level than the native promoter.

7. The recombinant host cell according to claim 6, wherein the native promoter is replaced with the ToxA promoter of Pyrenophora tritici-repentis or the ToxB promoter of Pyrenophora tritici-repentis.

8. The recombinant host cell according to claim 5, wherein the overexpression of the gene encoding a polypeptide having 12-oxophytodienoic acid reductase (OPR) activity is provided by recombinantly introducing at least one copy of the exogenous sequence encoding the polypeptide having OPR activity into the host cell.

9. The recombinant host cell according to any one of claims 1 to 8, wherein the host cell is selected from the group consisting of bacteria, yeast, filamentous fungi, cyanobacteria, algae, and plant cells.

10. The aforementioned host cells are from the genera Lasiodiplodia, Rhizopus, Fusidium, Gibberella, Trichoderma, Hypocrea, Aspergillus, Fusarium, Penicillium, Neurospora, Chaetomium, Acremonium, and Glomerella. Recombinant host cells according to any one of claims 1 to 8, wherein the host cell is a filamentous fungus belonging to a genus selected from the group consisting of Myceliophora, Sporotrichum, Thielavia, Chrysosporium, Corynascus, Ctenomyces, Verticillium, Cordyceps, Nectria, and Magnaporte.

11. Recombinant host cell according to any one of claims 1 to 8, wherein the host cell is a Lasiodiplodia iraniensis cell.

12. The aforementioned host cells include genera such as Escherichia, Salmonella, Bacillus, Acinetobacter, Corynebacterium, Methyrosinus, Methyromonas, Rhodococcus, Pseudomonas, Rhodobacter, Synechocystis, and Aspergillus. Recombinant host cells according to any one of claims 1 to 8, selected from the group consisting of s), the genera Arthrobotlys, Brevibacteria, Mycobacterium, Arthrobacter, Citrobacter, Klebsiella, Pantoea, Salmonella, Corynebacterium, and Clostridium.

13. The recombinant host cell according to any one of claims 1 to 8, wherein the host cell is an Escherichia cell.

14. The recombinant host cell according to any one of claims 1 to 8, wherein the host cell is an Escherichia coli cell.

15. The aforementioned host cells include soybeans, rapeseed, sunflowers, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice, broccoli, cauliflower, cabbage, parsnips, melons, carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts, grapes, grass seed crops, sugar beets, sugarcane, beans, peas, rye, flax, broad-leaved trees, coniferous trees, forage grasses, Arabidopsis thaliana, rice (Oryza sativa), barley (Hordeum yulgare), switchgrass (Panicum vigratum), Brachypodium spp., and Brassica. Recombinant host cells according to any one of claims 1 to 8, which are cells isolated from a plant selected from the group consisting of ), and Crambe abyssinica.

16. A biosynthetic method for producing jasmonic acid, Culture recombinant host cells according to claim 1 or 5, and To recover the jasmonic acid from at least one of the recombinant cells and culture medium. A biosynthesis method including the above.

17. A biosynthetic method for producing polypeptides having 12-oxophytodienoic acid reductase (OPR) activity, Culture the recombinant cells described in claim 5, and To recover the polypeptide having 12-oxophytodienoic acid reductase (OPR) activity from at least one of the recombinant cells and culture medium. A biosynthesis method including the above.

18. A vector containing a polynucleotide sequence having at least 90% identity with the polynucleotide sequence shown in Sequence ID No.

2.

19. The vector according to claim 18, further comprising a promoter operably ligated to a polynucleotide sequence having at least 90% identity with the polynucleotide sequence shown in SEQ ID NO: 2, wherein the promoter has a higher expression level than the native promoter of the gene expressing the polynucleotide sequence shown in SEQ ID NO: 2.

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