WO2023204053A1 - Modifications de gènes et d'hôtes pour la production d'acide jasmonique - Google Patents

Modifications de gènes et d'hôtes pour la production d'acide jasmonique Download PDF

Info

Publication number
WO2023204053A1
WO2023204053A1 PCT/JP2023/014383 JP2023014383W WO2023204053A1 WO 2023204053 A1 WO2023204053 A1 WO 2023204053A1 JP 2023014383 W JP2023014383 W JP 2023014383W WO 2023204053 A1 WO2023204053 A1 WO 2023204053A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
cell
gene
sequences
polypeptide
Prior art date
Application number
PCT/JP2023/014383
Other languages
English (en)
Inventor
David Nunn
Original Assignee
Sumitomo Chemical Company, Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sumitomo Chemical Company, Limited filed Critical Sumitomo Chemical Company, Limited
Publication of WO2023204053A1 publication Critical patent/WO2023204053A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/02—Thioester hydrolases (3.1.2)
    • C12Y301/02001—Acetyl-CoA hydrolase (3.1.2.1)

Definitions

  • the field of the invention relates to cells, enzymes, and processes useful in the production of jasmonic acid molecules.
  • Jasmonic acid (“JA” ) is an organic compound with a chemical formula of C 6 H 6 O 2 . It and its derivatives are usually known as “jasmonates” and find a great number of applications in the agricultural, flavors and fragrances, and biopharmaceutical arts. Traditionally, filamentous fungi have been the only commercially viable source of biosynthetic jasmonates. However, the yields tend to be highly correlated with fungal morphology which raises a substantial challenge for the production of jasmonic acid in conventional fermenter apparatuses.
  • NPL4 Farmer EE, Ryan CA. (1990) Interplant communication: airborne methyl jasmonate induces synthesis of proteinase inhibitors in plant leaves. Proc Natl Acad Sci U S A. 87:7713-7716.
  • NPL5 Farmer EE, Johnson RR, Ryan CA. (1992) Regulation of expression of proteinase inhibitor genes by methyl jasmonate and jasmonic Acid. Plant Physiol. 98:995-1002.
  • NPL6 Farmer EE, Ryan CA. (1992) Octadecanoid Precursors of Jasmonic Acid Activate the Synthesis of Wound-Inducible Proteinase Inhibitors. Plant Cell. 4: 129-134.
  • an acetyl-CoA hydrolase identified from Yarrowia lipolytica pO1f (e.g., an acetyl-CoA hydrolase comprising an amino acid sequence that is at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1) and its uses in the production of jasmonic acid (JA).
  • the gene encoding the Yarrowia lipolytica pO1f acetyl-CoA hydrolase may be cloned into a recombinant host cell for its expression in the host cell.
  • Such host cell may be used in the production (e.g., biosynthetic production) of jasmonic acid.
  • Each cycle of b-oxidation in the conversion of OPC-8 to JA results in a molecule of acetyl-CoA, which then enter the TCA cycle and after further oxidation results in the production of 2 molecules of NADH and one of NADPH.
  • the expression of the acetyl-CoA hydrolase will result in the hydrolysis of acetyl-CoA into acetate and CoA, which can then be recycled in b-oxidation. This will have the effect of preventing the accumulation of excess reducing equivalents resulting in more complete oxidation and higher levels of jasmonic acid produced.
  • FIG. 1 Structure of jasmonic acid and methyl jasmonate.
  • FIG. 2. A diagram of jasmonic acid production by ⁇ -oxidation of the intermediate OPC-8 and the impact of expression of acetyl-CoA hydrolase (ACH) in reducing the accumulation of reduced NADH.
  • FIG. 3. HPLC-UV and MS detection of OPC-8, jasmonic acid (JA) and the b-oxidation intermediates OPC-6 and OPC-4.
  • Cellular system is any cells that provide for the 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 the in vitro expression of proteins based on cellular components, such as ribosomes.
  • Coding sequence is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to refer to a DNA sequence that encodes for a specific amino acid sequence.
  • Growing includes providing an appropriate medium that would allow cells to multiply and divide. It also includes providing resources so that cells or cellular components can translate and make recombinant proteins.
  • Protein Production can occur after gene expression. It consists of the stages after DNA has been transcribed to messenger RNA (mRNA). The mRNA is then translated into polypeptide chains, which are ultimately folded into proteins. DNA may be present in the cells through transfection - a process of deliberately introducing nucleic acids into cells.
  • the term is often used for non-viral methods in eukaryotic cells. It may also refer to other methods and cell types, although other terms are preferred: "transformation” is more often used to describe non-viral DNA transfer in bacteria, non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated DNA transfer. Transformation, transduction, and viral infection are included under the definition of transfection for this application.
  • a “filamentous fungus” or “mold” is a eukaryotic microorganism classified as a member of the fungus kingdom. Filamentous fungi typically grow in the form of multicellular filaments called hyphae. In contrast, fungi that can adopt a single-celled growth habit are called “yeasts”. Yeasts are unicellular organisms which evolved from multicellular ancestors. Yeast species useful for the current disclosure include but are not limited to those that have the ability to develop multicellular characteristics by forming strings of connected budding cells known as pseudohyphae or false hyphae.
  • nucleic acid sequences are complementary to the complete sequences as reported in the accompanying Sequence Listing as well as those substantially similar nucleic acid sequences
  • nucleic acid and “nucleotide” are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally-occurring nucleotides.
  • nucleotide sequence refers to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5′ to 3′ end of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, a DNA fragment or portion, genomic DNA, synthetic, e.g. chemically synthesized, DNA, plasmid DNA, mRNA, and anti-sense RNA, any of which can be single stranded or double stranded.
  • nucleic acid sequence “nucleic acid,” “nucleic acid molecule,” “oligonucleotide” and “polynucleotide” are also used interchangeably herein to refer to a heteropolymer of nucleotides.
  • Nucleic acid molecules and/or nucleotide sequences provided herein are presented herein in the 5′ to 3′ direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR ⁇ 1.821-1.825 and the 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.
  • WIPO World Intellectual Property Organization
  • the term “gene” refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, miRNA, anti-microRNA antisense oligodeoxyribonucleotide (AMO), and the like. Genes may or may not be capable of being used to produce a functional protein or gene product. Genes can include both coding and non-coding regions, e.g. introns, regulatory elements, promoters, enhancers, termination sequences and/or 5′ and 3′ untranslated regions.
  • a gene may be “isolated” by which is meant 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 chemically synthesizing the nucleic acid.
  • a “codon optimized” version of a gene refers to an exogenous gene introduced into a cell and where the codons of the gene have been optimized with regard to the 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 prevalent tRNAs, i.e. to change a codon recognized by a low prevalent tRNA with a synonymous codon recognized by a tRNA that is comparatively more prevalent in the given fungal cell. This way the mRNA from the codon optimized gene will be more efficiently translated.
  • the codon and the synonymous codon preferably encode the same amino acid.
  • Genetical modification or “genetically modified” as used herein involves such genetic modifications to the genome of the fungal cell, such as yeast, and/or introduction of exogenous nucleotide sequences, such as in the form of one or more plasmids, into the fungal cell, such as yeast.
  • isolated is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and when used in the context of an isolated nucleic acid or an isolated polypeptide, is used without limitation to refer to a nucleic acid or polypeptide that, by the hand of man, exists apart from its native environment and is therefore 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, in a transgenic host cell.
  • incubating and “incubation” as used herein means a process of mixing two or more chemical or biological entities (such as a chemical compound and an enzyme) and allowing them to interact under conditions favorable for producing a composition that includes a jasmonate compound.
  • 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 sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxy inosine residues.
  • a nucleic acid sequence and all of its degenerate variants will express the same amino acid or polypeptide.
  • polypeptide refers to peptides, polypeptides, and proteins, unless otherwise noted.
  • polypeptide protein
  • polypeptide peptide
  • exemplary polypeptides include polynucleotide products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
  • polypeptide fragment and “fragment,” when used in reference to a reference polypeptide, are to be given their ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions can occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both.
  • polypeptide or protein refers to a peptide fragment that is a portion of the full-length polypeptide or protein, and has substantially the same biological activity, or carries out substantially the same function as the full-length polypeptide or protein (e.g., carrying out the same enzymatic reaction).
  • variant polypeptide refers to an amino acid sequence that is different from the reference polypeptide by one or more amino acids, e.g., by one or more amino acid substitutions, deletions, and/or additions.
  • a variant is a "functional variant” which retains some or all of the ability of the reference polypeptide.
  • 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 a substitution of an amino acid residue with a functionally similar residue.
  • 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 for another such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; 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.
  • one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another
  • substitution of one charged or polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between threonine and serine
  • substitution of one basic residue such as
  • substitutions are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide.
  • the phrase "conservatively substituted variant” also includes peptides wherein a residue is replaced with a chemically-derivatized residue, provided that the resulting peptide maintains some or all of the activity of the reference peptide as described herein.
  • variant in connection with the polypeptides of the subject technology, further includes a functionally active polypeptide having an amino acid sequence 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.
  • homologous in all its grammatical forms and spelling variations refers to the relationship between polynucleotides or polypeptides that possess a "common evolutionary origin,” including polynucleotides or polypeptides from super families and homologous polynucleotides or proteins from different species (Reeck et al., Cell 50:667, 1987). Such polynucleotides or polypeptides have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or the presence of specific amino acids or motifs at conserved positions.
  • 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 900 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.
  • Suitable regulatory sequences is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is 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 which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
  • Promoter is to be given its ordinary and customary meaning to a person of ordinary skill 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.
  • a coding sequence is located 3' to a promoter sequence.
  • Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise 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 stages of development, or in response to different environmental conditions. Promoters, which cause a gene to be expressed in most cell types at most times, are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
  • operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it can affect the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • expression is to be given its ordinary and customary meaning to a person of ordinary 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 the nucleic acid fragment of the subject technology.
  • overexpress refers to higher levels of activity of a gene, e.g. transcription of the gene; higher levels of translation of mRNA into protein; and/or higher levels of production of a gene product, e.g. polypeptide, than would be in the cell in its native or control, e.g. not transformed with the particular heterologous or recombinant polypeptides being overexpressed, state.
  • a typical example of an overexpressed gene is a gene under transcription control of another promoter as compared to the native promoter of the gene.
  • other changes in the control elements of a gene such as enhancers, could be used to overexpress the particular gene.
  • modifications that affect, i.e. increase, the translation of the mRNA transcribed from the gene could, alternatively or in addition, be used to achieve an overexpressed gene as used herein.
  • These terms can also refer to an increase in the number of copies of a gene and/or an increase in the amount of mRNA and/or gene product in the cell. Overexpression can result in levels that are 25%, 50%, 100%, 200%, 500%, 1000%, 2000% or higher in the cell, as compared to control levels.
  • Transformation is to be given its ordinary and customary meaning to a person of reasonable skill in the craft, and is used without limitation to refer to the transfer of a polynucleotide into a target cell.
  • the transferred polynucleotide can be incorporated into the genome or chromosomal DNA of a target cell, resulting in genetically stable inheritance, or it can replicate independent of the host chromosomal.
  • Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic” or “transformed”.
  • transformed when used herein in connection with host cells, are to be given their respective ordinary and customary meanings to a person of ordinary 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 can be stably integrated into the genome of the host cell, or the nucleic acid molecule can be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating.
  • Transformed cells, tissues, or subjects are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
  • heterologous when used herein in connection with polynucleotides, are to be given their ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to a polynucleotide (e.g., a DNA sequence or a gene) that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of site-directed mutagenesis or other recombinant techniques.
  • the terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence.
  • the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position or form within the host cell in which the element is not ordinarily found.
  • recombinant when used herein in connection with a polypeptide or amino acid sequence, means a polypeptide or amino acid sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
  • recombinant DNA segments can be expressed in a host cell to produce a recombinant polypeptide.
  • 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.
  • a “wild type mRNA” is an mRNA that is naturally occurring in or endogenous to the organism.
  • a “homologous” nucleic acid sequence is a nucleotide sequence naturally associated with a host cell into which it is introduced.
  • Plasmid DNA
  • vector vector
  • cassette are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules.
  • Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.
  • Transformation cassette refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell.
  • “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
  • the term “increased copy number” means at least one extra copy of at least the polypeptide coding sequence of a given gene is present in a recombinant cell as compared to the number of copies of the same gene in a parental cell, e.g., a wild-type cell, from which the recombinant cell is derived.
  • the recombinant cell may contain 1, 2, 3, 4, 5, 10, or more extra copies of at least the polypeptide coding sequence of a given gene relative to the number of copies of that same gene in the parental 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.
  • sequence identity refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids.
  • An "identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence.
  • percent sequence identity refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test ("subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison).
  • Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and preferably by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG (registered trademark) Wisconsin Package (registered trademark) (Accelrys Inc., Burlington, MA).
  • identity fraction for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the 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 part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100.
  • the comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence.
  • percent identity may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.
  • the percent of sequence identity is preferably determined using the "Best Fit” or "Gap” program of the Sequence Analysis Software Package (trademark) (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” performs an optimal alignment of the best segment of similarity between two sequences and inserts gaps to maximize the number of matches using 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). The percent identity is most preferably determined using the "Best Fit” program.
  • BLAST Basic Local Alignment Search Tool
  • the term "substantial percent sequence identity” refers to a 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.
  • one embodiment of the invention is a polynucleotide molecule that has 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 a polynucleotide sequence described herein.
  • Polynucleotide molecules encoding proteins that have the activity of the gs5119 sequence of the current disclosure are capable of directing the production of a variety of jasmonic compounds and have a substantial percent sequence identity to the polynucleotide sequences provided herein and are encompassed within the scope of this disclosure.
  • the term “identity” is the fraction of amino acids that are the same between a pair of sequences after an alignment of the sequences (which can be done using only sequence information or structural information or some other information, but usually it is based on sequence information alone), and the term “similarity” refers to the score assigned based on an alignment using some similarity matrix.
  • the similarity index can be any one of the following BLOSUM62, PAM250, or GONNET, or any matrix used by one skilled in the art for the sequence alignment of proteins.
  • Identity is the degree of correspondence between two sub-sequences (no gaps between the sequences). An identity of 25% or higher implies similarity of function, while 18- 25% implies similarity of structure or function. Keep in mind that two completely unrelated or random sequences (that are greater than 100 residues) can have higher than 20% identity. Similarity is the degree of resemblance between two sequences when they are compared. This is dependent on their identity.
  • Jasmonic acid was first identified as a natural product in 1971 from a filamentous fungus, Lasiodiplodia theobromae (Aldridge et al., 1971). In 1990s, JA and its methyl ester, methyl jasmonate (MeJA), collectively called as jasmonates (FIG.
  • Methyl jasmonate (FIG. 1) which gives an odor pronounced of the floral heart of jasmine has a floral taste with jasmine notes and is used for its floral notes for peach, apricot, grape and other flavors. Flavor and Extract Manufacturers Association (FEMA) has approved its use in food with the number of 3410. In addition, methyl jasmonate has been discovered to induce cytochrome C release in the mitochondria of cancer cells, leading to cell death, but not to harm normal cells (Rotem et al., 2005). Thus, it has a great potential to be developed into a novel class of anticancer drugs.
  • FEMA Flavor and Extract Manufacturers Association
  • jasmonates Due to the importance of jasmonates in agriculture, flavor and fragrance industry, and potentially medicine, large quantities are needed for their various applications. Although jasmonates can be synthesized by organic chemistry, consumer demand for 'natural' flavors has generated a strong need for bio-based processes. More importantly, chemically synthesized jasmonates are a mixture of biologically active and inactive isomers whereas bio-based jasmonates are dominated by biologically active isomers. Unfortunately, like other plant hormones, jasmonate and methyl jasmonate exist only in trace amounts in higher plants e. g. less than 10 ⁇ g in one kg of induced fresh tomato leaves (Chen et al., 2006).
  • the oxidation of the OPC-8 (12-oxophytodieonic acid) intermediate of jasmonic acid synthesis is carried out by the b-oxidation pathway, where two carbons are removed during each cycle, with the removal of six total carbons resulting in jasmonic acid.
  • This oxidation results in the production of excess reducing equivalents in the form of NADH and FADH2, which can then lead to the premature cessation of beta-oxidation and the production of toxic reactive oxygen species. This significantly limits the utility of microbial hosts to produce high levels of jasmonic acid.
  • This invention describes the expression of a heterologous enzyme activity from a gene encoding an acetyl-CoA hydrolase (also called acylase).
  • acetyl-CoA hydrolase also called acylase.
  • Each cycle of b-oxidation in the conversion of OPC-8 to JA results in a molecule of acetyl-CoA, which then enter the TCA cycle and after further oxidation results in the production of 2 molecules of NADH and one of NADPH.
  • the expression of the acetyl-CoA hydrolase will result in the hydrolysis into acetate and CoA, which can then be recycled in b-oxidation. This will have the effect of preventing the accumulation of excess reducing equivalents resulting in more complete oxidation and higher levels of jasmonic acid produced.
  • ACH acetyl-CoA hydrolase
  • OPC-8 1-10g/l
  • HPLC analysis will be used to quantitate the levels of OPC-8, OPC-6, OPC-4, and jasmonic acid produced over time as an indication of the effect of the expression of the water-forming NADH oxidases on increasing the rate and extent of conversion of OPC-8 to jasmonic acid.
  • FIG. 3 shows an example of the HPLC analysis demonstrating the final JA product, substrate JA and intermediates OPC-6 and OPC-4.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne de l'acétyl-CoA hydrolase (ACH) de Yarrowia lipolytica pO1f et ses utilisations dans la production d'acide jasmonique.
PCT/JP2023/014383 2022-04-18 2023-04-07 Modifications de gènes et d'hôtes pour la production d'acide jasmonique WO2023204053A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263331921P 2022-04-18 2022-04-18
US63/331,921 2022-04-18

Publications (1)

Publication Number Publication Date
WO2023204053A1 true WO2023204053A1 (fr) 2023-10-26

Family

ID=88419896

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2023/014383 WO2023204053A1 (fr) 2022-04-18 2023-04-07 Modifications de gènes et d'hôtes pour la production d'acide jasmonique

Country Status (1)

Country Link
WO (1) WO2023204053A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190078098A1 (en) * 2016-03-14 2019-03-14 Board Of Regents, The University Of Texas System Compositions and methods for type iii polyketide production in oleaginous yeast species

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190078098A1 (en) * 2016-03-14 2019-03-14 Board Of Regents, The University Of Texas System Compositions and methods for type iii polyketide production in oleaginous yeast species

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE UniProtKB/TrEMBL 18 January 2017 (2017-01-18), ANONYMOUS : "RecName: Full=Acetyl-CoA deacylase", XP093101215, retrieved from UniProt Database accession no. A0A1D8NKP2 *

Similar Documents

Publication Publication Date Title
CN104854245B (zh) 通过代谢工程生产麦角硫因
CN105683378A (zh) 用于精细化学品的改进生产的重组微生物
US10351880B2 (en) Drimenol synthases I
JP7510187B2 (ja) イソオイゲノールからのバニリンの生合成
KR20220004090A (ko) 헴-함유 단백질의 생산을 위한 균주 및 방법
US20220112525A1 (en) Biosynthesis of vanillin from isoeugenol
EP0967281B1 (fr) 3,4-dihydroxy-2-butanone 4-phosphate synthase
JP7222916B2 (ja) ドリメノール及びそれらの混合物の製造のためのセスキテルペンシンターゼ
EP3140410B1 (fr) Driménol synthases et méthode de production de driménol
US10550409B2 (en) Drimenol synthases III
WO2023204053A1 (fr) Modifications de gènes et d'hôtes pour la production d'acide jasmonique
WO2023204055A1 (fr) Gènes et modifications d'hôtes pour la production d'acide jasmonique
WO2023204106A1 (fr) Souche de levure pour la production d'acide jasmonique
WO2023204107A1 (fr) Souche de levure pour la production d'acide jasmonique
WO2023199843A1 (fr) Thioestérases de jasmonoyl-coenzyme a (ja-coa) et leurs utilisations
US20240060098A1 (en) Amycolatopsis strains for vanillin production with suppressed vanillic acid formation
WO2022133274A1 (fr) Biosynthèse de vanilline à partir d'isoeugénol
WO2023199842A1 (fr) Cellules métaboliquement modifiées pour la biosynthèse de l'acide jasmonique
US20240060097A1 (en) Bioconversion of ferulic acid to vanillin
JP2018201508A (ja) グルタチオンストレス耐性酵母
US20220243230A1 (en) Bioconversion of 4-coumaric acid to resveratrol
Hong et al. Cloning and optimizing the expression of the DHDPS gene in the Medicago truncatula
WO2023064845A1 (fr) Ingénierie rationnelle reposant sur la conception d'enzymes tomo par mutagenèse dirigé vers un site pour la biosynthèse du résorcinol
CN113337485A (zh) 一种抗噬菌体和抗病毒系统
Kim et al. The effect of the cspA 5′-untranslated region on recombinant protein production at low temperature

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23791702

Country of ref document: EP

Kind code of ref document: A1