WO2025207802A1 - Souches microbiennes recombinantes comprenant des phénotypes à oxydation bêta réduite et procédés associés - Google Patents

Souches microbiennes recombinantes comprenant des phénotypes à oxydation bêta réduite et procédés associés

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WO2025207802A1
WO2025207802A1 PCT/US2025/021599 US2025021599W WO2025207802A1 WO 2025207802 A1 WO2025207802 A1 WO 2025207802A1 US 2025021599 W US2025021599 W US 2025021599W WO 2025207802 A1 WO2025207802 A1 WO 2025207802A1
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pex
protein
seq
yeast cell
gene
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Min QI
Zhixiong Xue
Quinn Qun Zhu
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Nutrition and Biosciences USA 4 Inc
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Nutrition and Biosciences USA 4 Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/40—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Candida
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida
    • C12R2001/73—Candida lipolytica

Definitions

  • This invention is in the field of biotechnology, it pertains to methods useful for manipulating the beta-oxidation in eukaryotic organisms, based on disruption of peroxisome biogenesis factor (PEX) proteins. More specifically, this invention relates to microbes such as yeast, genetically engineered to have reduced or eliminated betaoxidation.
  • PX peroxisome biogenesis factor
  • Ambrettolide, iso-Ambrettolide(s), Hexadecanolide and innumerable related molecules are macrocyclic molecules that have exceptional diffusion properties and fine musk characteristics.
  • These fragrance molecules can be produced from C16 omega- hydroxylated (co-hydroxy) fatty acids.
  • co-hydroxy fatty acid derivatives are made chemically from petroleum-based starting materials or through the bioconversion of paraffin. The required chemical methods for producing these compounds involve the use of hazardous organic reagents, are energy intensive and are environmentally costly.
  • Y. lipolytica is an oleaginous yeast that can produce large amounts of oil inside the cells, wherein Y. lipolytica cells have strong beta-oxidation capability and can use fatty acid or oil as sole carbon source for its growth.
  • the strong beta-oxidation capability of Y. lipolytica converts most fatty acid substrates to carbon dioxide, resulting in limited amounts of oil produced.
  • modified yeasts such as modified Yarrowia spp, for example modified Yarrowia lipolytica, which are modified to have a reduced, or even a blocked, fatty acid p-oxidation pathway compared to the parent non-modified yeasts.
  • modified yeast cells suitable for producing dicarboxylic acids.
  • modified yeast cells able to have an increase of dicarboxylic acids production compared to the parent non-modified yeast cells.
  • modified yeast cells suitable for producing hydroxy fatty acids.
  • modified yeast cells able to have an increase of hydroxy fatty acids production compared to the parent non-modified yeast cells.
  • a modified yeast cell having a reduction of betaoxidation activity compared to beta-oxidation activity of a non-modified parental yeast cell, wherein the modified cell comprises a genetic modification reducing or eliminating the expression, activity and/or function of one or more native peroxisome biogenesis factor (Pex) proteins encoded by one or more endogenous peroxisome biogenesis factor (PEX) genes.
  • Pex peroxisome biogenesis factor
  • PEX endogenous peroxisome biogenesis factor
  • a modified yeast cell having a reduction of betaoxidation activity, the modified yeast having a reduced ability to grow in an oleic acid- enriched medium compared to a non-modified parental yeast cell, wherein the modified yeast cell comprises a genetic modification reducing or eliminating the expression, activity and/or function of one native peroxisome biogenesis factor (Pex) proteins encoded by one endogenous peroxisome biogenesis factor (PEX gene.
  • Pex peroxisome biogenesis factor
  • a modified yeast cell having a reduction of betaoxidation activity in which less acetyl-CoA is produced as compared to beta-oxidation activity of an otherwise identical (isogenic) parental yeast cell, wherein the modified cell comprises a genetic modification reducing or eliminating the expression, activity and/or function of one or more native peroxisome biogenesis factor (Pex) proteins encoded by one or more endogenous peroxisome biogenesis factor (PEX genes.
  • Pex peroxisome biogenesis factor
  • a method of increasing the weight percent of at least one polyunsaturated fatty acid relative to the weight percent of total fatty acids in a yeast cell comprising genetically modifying said yeast cell for reducing or eliminating the expression, activity and/or function of one or more native peroxisome biogenesis factor (Pex) proteins encoded by one or more endogenous peroxisome biogenesis factor (PEX) genes of said yeast cell.
  • Pex peroxisome biogenesis factor
  • PEX endogenous peroxisome biogenesis factor
  • the one or more native PEX proteins may be selected from a group consisting of a PEX3 protein, a PEX5 protein and a PEX 20 protein.
  • the one or more genes encoding the native PEX proteins may comprise a PEX3 gene.
  • the one or more genes encoding the native PEX proteins may comprise a PEX5 gene.
  • the one or more genes encoding the native PEX proteins may comprise a PEX20 gene.
  • the genetic modification which eliminates or reduces the activity and/or function of the one more native PEX proteins may comprise disrupting, partially deleting and/or mutating a subsequence of a PEX gene encoding a PEX protein ATP binding site, a PEX protein transmembrane (TM) domain or a PEX protein zinc-finger domain or protein-protein interaction domain, or combinations thereof.
  • TM PEX protein transmembrane
  • the modified yeast cell is Yarrowia lipolytica.
  • Figure 1 shows a cycle of beta-oxidation.
  • Figures 2A-2C show examples of the genetically modified Y. lipolytica strains.
  • Figure 4 shows a physical and functional map of plasmid pO4L2P3.
  • Figure 5 shows a physical and functional map of the Cla ⁇ large fragment of plasmid pO4L2P3.
  • Figure 7 shows physical and functional map of the C/al large fragment of plasmid pPOX4-LEU2.
  • Figure 8 is an illustration of pex20 deletion by Pop-in and Pop-out approach.
  • Figure 9 is an illustration of pex5 deletion by Pop-in and Pop-out approach.
  • Figure 10 depicts the physical and functional map of plasmid pY-P3.
  • Figure 11 shows physical and functional map of Sph ⁇ and Asci large fragment of plasmid pY-P3.
  • ambrox refers to (3aR,5aS,9aS,9bR)-dodecahydro- 3a,6,6,9a-tetramethylnaphtho [2,1-b]furan), which is known commercially as AMBROX (Firmenich), Ambroxan (Henkel) AMBROFIX® (Givaudan), AMBERLYN® (Quest), CETALOX® Laevo (Firmenich), AMBERMOR® (International Flavors and Fragrances, and AROMOR® and/or norambrenolide Ether ( Pacific).
  • ambrox come from the (-) stereoisomer rather than the (+) enantiomer.
  • the odor of the (- ) stereoisomer is described as musk-like, woody, warm or ambery whereas the (+) enantiomer has a relatively weak odor note.
  • a “functional gene” is a gene capable of being used by cellular components to produce an active gene product, typically a protein.
  • a “nonfunctional gene” cannot be used by cellular components to produce an active gene product (/.e., a functional protein), or has a reduced ability to be used by cellular components to produce an active gene product (/.e., a functional protein).
  • a “functional protein” is a protein that possesses a function (or activity), such as an enzymatic function/activity, a binding function/activity (e.g., DNA binding), an ATP-binding and/or hydrolysis function/activity, a surface-active property, signal transduction function/activity or transporter function/activity, and the like, and which has not been mutagenized, truncated, or otherwise modified to abolish or reduce that function/activity.
  • a functional protein includes and relates to the ability of a particular protein to function such as normal peroxisomal function (e.g., peroxisome membrane function). Such function includes ability of proteins to be involved in peroxisome biogenesis and/or that participates in the process of importing cellular proteins into peroxisomes.
  • nucleic acid molecule refers to polynucleotides of the disclosure which can be DNA, cDNA, genomic DNA, synthetic DNA, or RNA, and can be double-stranded or single-stranded, the sense and/or an antisense strand.
  • proteins are considered to be “related proteins,” or “homologs.” Such proteins can be derived from organisms of different genera and/or species, or different classes of organisms (e.g., bacteria and fungi), or artificially designed. Related proteins also encompass homologs determined by primary sequence analysis, determined by secondary or tertiary structure analysis, or determined by immunological cross-reactivity, or determined by their functions.
  • the degree of homology between sequences can be determined using any suitable method known in the art (see, e.g., Smith and Waterman (1981 ) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol., 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, Wl); and Devereux et al. (1984) Nucleic Acids Res. 12:387-95).
  • PILEUP is a useful program to determine sequence homology levels.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pair-wise alignments. It can also plot a tree showing the clustering relationships used to create the alignment.
  • PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle, (Feng and Doolittle (1987) J. Mol. Evol. 35:351 -60). The method is similar to that described by Higgins and Sharp ((1989) CAB/OS 5:151 -53).
  • Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
  • BLAST algorithm Another example of a useful algorithm is the BLAST algorithm, described by Altschul et al. ((1990) J. Mol. Biol. 215:403-10) and Karlin et al. ((1993) Proc. Natl. Acad. Sci. USA 90:5873-87).
  • One particularly useful BLAST program is the WU-BLAST-2 program (see, e.g., Altschul et al. (1996) Meth. Enzymol. 266:460-80). Parameters “W,” “T,” and “X” determine the sensitivity and speed of the alignment.
  • the BLAST program uses as defaults a wordlength (W) of 11 , the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M'5, N'- 4, and a comparison of both strands.
  • Percent sequence identity is calculated using CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithm
  • Gap extension penalty 0.05
  • Toggle Residue specific penalties ON Toggle hydrophilic penalties: ON
  • polypeptides are substantially identical.
  • first polypeptide is immunologically cross-reactive with the second polypeptide.
  • polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive.
  • a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions (e.g., within a range of medium to high stringency).
  • yeast of the genus Yarrowia e.g., Yarrowia lipolytica
  • yeast of the genus Yarrowia are generally haploid, therefore a single copy of a specified gene (/.e., a single allele) is sufficient to confer a specified phenotype, for example, reduced betaoxidation (reduced Acetyl-CoA production).
  • expressing a polypeptide refers to the cellular process of producing a polypeptide using the translation machinery (e.g., ribosomes) of the cell.
  • translation machinery e.g., ribosomes
  • wild-type and “native” may be used interchangeably and refer to genes, proteins or strains found in nature, or that are not intentionally modified for the advantage of the presently described cells/strains.
  • “Otherwise identical (isogenic) parental yeast cell” and “non-modified parental yeast cell” are used interchangeably and intend to refer to a yeast cell that is genetically identical to a modified yeast cell except for the specific genetic modification or mutation introduced for the intent of the disclosure.
  • modification and “genetic modification” are used interchangeably and include, but are not limited to: (a) the insertion, substitution, or removal of one or more nucleotides in a gene or an ORF thereof, or the insertion, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of the gene or ORF thereof, (b) a gene disruption, (c) a gene deletion, (d) the down-regulation of a gene, (e) specific mutagenesis and/or (f) random mutagenesis of any one or more the genes disclosed herein.
  • genetic modification eliminates or reduces the activity and/or function of the one more native PEX proteins comprises disrupting, partially deleting and/or mutating a subsequence of a PEX gene encoding a PEX protein ATP binding site, a PEX protein transmembrane (TM) domain, and a PEX protein zinc-finger domain, and protein-protein interaction domain, and the like.
  • TM PEX protein transmembrane
  • down-regulated when used to describe the expression of a gene or polynucleotide sequence, the terms “disruption”, “down -regulated”, “down-regulation”, “inhibition”, “inactivation”, “silencing” and the like are used interchangeably herein to refer to instances when the transcription of the polynucleotide sequence is reduced or eliminated, which results in a reduction or elimination of protein expression derived from the polynucleotide sequence (if the gene comprised an ORF).
  • down-regulation can refer to instances where protein translation from transcripts produced by the polynucleotide sequence is reduced or eliminated.
  • down-regulation can refer to instances where a protein expressed by the polynucleotide sequence has reduced activity
  • Exemplary methods of disruption include complete gene knockout such that the whole gene is deleted from the genome or partial deletion of any portion of a gene, including a polypeptide-coding sequence, a promoter, an enhancer, or another regulatory element, or mutagenesis of the same, where mutagenesis encompasses substitutions, insertions, deletions, inversions, and combinations and variations, thereof, any of which mutations substantially prevent the production of a function gene product.
  • a gene can also be disrupted using CRISPR, RNAi, antisense, or any other method that abolishes gene expression.
  • a gene can be disrupted by deletion or genetic manipulation of non-adjacent control elements.
  • the deletion of a gene refers to the deletion of the coding sequence, and optionally adjacent enhancer elements, including but not limited to, for example, promoter and/or terminator sequences, but does not require the deletion of non-adjacent control elements.
  • Deletion of a gene also refers to the deletion a part of the coding sequence, or a part of promoter immediately or not immediately adjacent to the coding sequence, where there is no functional activity of the interested gene existed in the engineered cell.
  • the terms “genetic manipulation,” “genetic alteration”, “genetic engineering”, and similar terms are used interchangeably and refer to the alteration/change of a nucleic acid sequence.
  • the alteration can include but is not limited to a substitution, deletion, insertion or chemical modification of at least one nucleic acid in the nucleic acid sequence.
  • “Reducing the expression, activity and/or function of a protein” intends to any method or process, and the results thereof, that decreases the production (expression) of a protein, diminishes its biological activity, or impairs its functional role within a cell. This decrease is perceptible and measurable by comparison with a non-modified parental cell.
  • Methods for measuring a reduction of expression, activity and/or function of a protein include, but are not limited to, Western Blotting, Quantitative PCR (qPCR), Enzyme activity assay, Enzyme-Linked Immunosorbent Assay (ELISA), Flow Cytometry, or Reporter Assays.
  • “Eliminating the expression, activity and/or function of a protein” intends to any method or process, and the results thereof, that abolishes the production (expression) of a protein, its biological activity, or its functional role within a cell. This elimination is perceptible and measurable by comparison with a non-modified parental cell, and within the limits of the used measuring methods.
  • Methods for measuring an elimination of expression, activity and/or function of a protein include, but are not limited to, Western Blotting, Quantitative PCR (qPCR), Enzyme activity assay, Enzyme-Linked Immunosorbent Assay (ELISA), Flow Cytometry, or Reporter Assays.
  • the terms “down-regulation” of gene expression and “upregulation” of gene expression include any method that results in lower (down-regulated) or higher (up-regulated) expression of a gene.
  • the down-regulation of a gene in yeast can be achieved by RNA-induced gene silencing, genetic modifications of control elements such as the promoter, untranslated regions (UTRs), codon changes, and the like.
  • a modified strain of Yarrowia ⁇ e.g., Y. lipolytica
  • TALENs transcriptional activator like endonucleases
  • ZFNs zinc-finger endonucleases
  • homing mimerase endonuclease and the like.
  • the portion of the gene to be modified ⁇ e.g., a coding region, a non-coding region, a leader sequence, a pro-peptide sequence, a signal sequence, a transcription terminator, a transcriptional activator, or other regulatory elements required for expression of the coding region
  • a modified strain of Yarrowia e.g., Y. lipolytica
  • CRISPR/Cas9 editing is constructed by means of CRISPR/Cas9 editing.
  • a gene encoding a PEX protein can be disrupted, deleted, mutated, or otherwise genetically modified by means of nucleic acid guided endonucleases, that find their target DNA by binding either a guide RNA (e.g., Cas9) or a guide DNA (e.g., NgAgo), which recruits the endonuclease to the target sequence on the DNA, wherein the endonuclease can generate a single or double stranded break in the DNA.
  • a guide RNA e.g., Cas9
  • a guide DNA e.g., NgAgo
  • the gene encoding the nucleic acid guided endonuclease (e.g., a Cas9 from S. pyogenes, or a codon optimized gene encoding the Cas9 nuclease) is operably linked to a promoter active in the Yarrowia (e.g., Y. lipolytics) cell and a terminator active in Yarrowia (e.g., Y. lipolytics) cell, thereby creating a Yarrowia (e.g., Y. lipolytics) Cas9 expression cassette.
  • a promoter active in the Yarrowia e.g., Y. lipolytics
  • a terminator active in Yarrowia e.g., Y. lipolytics
  • Yarrowia e.g., Y. lipolytics
  • one or more target sites unique to the gene of interest are readily identified by a person skilled in the art.
  • variable targeting domain will comprise nucleotides of the target site which are 5' of the (PAM) proto-spacer adjacent motif (TGG), which nucleotides are fused to DNA encoding the Cas9 endonuclease recognition domain for S. pyogenes Cas9 (CER).
  • PAM proto-spacer adjacent motif
  • CER S. pyogenes Cas9
  • the combination of the DNA encoding a VT domain and the DNA encoding the CER domain thereby generate a DNA encoding a gRNA.
  • a filamentous fungal expression cassette for the gRNA is created by operably linking the DNA encoding the gRNA to a promoter active in filamentous fungal cells and a terminator active in filamentous fungal cells.
  • the DNA break induced by the endonuclease is repaired/replaced with an incoming sequence.
  • a nucleotide editing template is provided, such that the DNA repair machinery of the cell can utilize the editing template.
  • about 500bp 5’ of targeted gene can be fused to about 500bp 3' of the targeted gene to generate an editing template, which template is used by the filamentous fungal host’s machinery to repair the DNA break generated by the RGEN (RNA-guided endonuclease).
  • the Cas9 expression cassette, the gRNA expression cassette and the editing template can be co-delivered to filamentous fungal cells using many different methods (e.g., protoplast fusion, electroporation, natural competence, or induced competence).
  • the transformed cells are screened by PCR, by amplifying the target locus with a forward and reverse primer. These primers can amplify the wild-type locus or the modified locus that has been edited by the RGEN. These fragments are then sequenced using a sequencing primer to identify edited colonies.
  • the term “transformed” refers to the introduction of an exogenous or heterologous DNA into a cell.
  • the transforming DNA may or may not be integrated, i.e., covalently linked into the genome of the cell.
  • the phrase “engineered cells,” “modified yeast cells” or similar phrases, refer to cells that include genetic modifications and characteristics described herein. Engineered/modified/recombinant yeast do not include naturally occurring yeast.
  • the common approach to reduce or eliminate beta-oxidation is to down- regulate enzymes involved in the beta-oxidation process (Fig. 1 ), especially the acyl-CoA oxidase that carries out the first reaction.
  • Fig. 1 enzymes involved in the beta-oxidation process
  • acyl-CoA oxidase that carries out the first reaction.
  • acyl-CoA oxidases there are six different acyl-CoA oxidases in Y. lipolytica, each has its own substrate specificity, and some also have regulatory functions (http://dx.doi.org/10.1083/jcb.200305055).
  • the diacylglycerol acyltransferases are the key enzymes for oil biosynthesis in Yarrowia, it includes acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1 ) and acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2).
  • DGAT1 acyl-CoA:diacylglycerol acyltransferase 1
  • DGAT2 acyl-CoA:diacylglycerol acyltransferase 2
  • Either or both DGAT1 and DGAT2 can be downregulated to reduce oil amounts (Zhang et al., Yeast, 29: 25-38).
  • Down-regulation of DGAT1 or DGAT2 or both can be performed using any of the strategies disclosed herein useful for such as for example (e.g., deletion, insertion, other type of mutation).
  • peroxisomes refers to ubiquitous organelles found in all eukaryotic cells. They have a single lipid bilayer membrane that separates their contents from the cytosol and that contains various membrane proteins essential to the functions described below. Peroxisomes selectively import proteins via an “extended shuttle mechanism”. More specifically, there are at least 32 known peroxisomal proteins, also known as peroxins, which participate in the process of importing proteins by means of ATP hydrolysis through the peroxisomal membrane. Some peroxins comprise a specific protein signal, i.e., a peroxisomal targeting signal or “PTS” at either the N-terminus or C-terminus to signal that importation through the peroxisomal membrane should occur.
  • PTS peroxisomal targeting signal
  • peroxisome biogenesis factor protein proteins involved in peroxisome biogenesis and/or that participate in the process of importing cellular proteins by means of ATP hydrolysis through the peroxisomal membrane.
  • the acronym of a gene that encodes any of these proteins is “PEX gene”.
  • the terms “peroxisome biogenesis factor protein”, “peroxin” and “PEX protein” are interchangeable and refer to proteins involved in peroxisome biogenesis and/or that participate in the process of importing cellular proteins by means of ATP hydrolysis through the peroxisomal membrane.
  • a system for nomenclature of PCX genes is described by Distel et al., J. Cell Biol., 135:1 -3 (1996).
  • PEX3 refers to a polynucleotide sequence encoding peroxisome biogenesis factor-3 (Pex3 protein ["Pex3p”]; GenBank Acc. No. KAG5359845, YALI0F22539g).
  • PEX3 protein is a peroxisomal integral membrane protein believed to play a role in peroxisomal membrane formation during peroxisome biogenesis (e.g., Baerends et al., J. Biol. Chem. 271 :8887-8894; Bascom et al., Mai. Biol. Cell 14:939- 957).
  • PEX5 refers to a polynucleotide sequence encoding peroxisome biogenesis factor-5 (Pex5 protein [‘Pex5’], GenBank Acc. No. KAG5370090; YALI0F28457g). PEX5 is a peroxisomal targeting signal receptor; it binds to the C-terminal PTS1 -type tripeptide peroxisomal targeting signal (SKL-type) and plays an essential role in peroxisomal protein import (Szilard and Rachubinski, Biochem. J. 346 (Pt 1 ): 177-184). [0084] PEX 20.
  • PEX2CT refers to a polynucleotide sequence encoding peroxisome biogenesis factor-20 (Pex20 protein [‘Pex20’]; GenBank Acc. No. XP_503644; YALI0_E06831 g).
  • PEX20 is a coreceptor required for the peroxisomal import of proteins, it contains a C-terminal PTS2-type peroxisomal targeting signal, such as 3- oxoacyl-CoA thiolase (Vladimir et al., J. Cell Biol. 142: 403-420; Chang & Rachubinski, Traffic 20: 504-515).
  • the PEX3, PEX5, and PEX20 individually or combining two of them, or all three of them in order to downregulate or delete can be useful strategies to reduce or eliminate beta-oxidation.
  • Down-regulation of PEX3, PEX5 or PEX20, or combining two of them or all three together for downregulation or deletion can be performed using any of the strategies disclosed herein useful for such as for example (e.g., deletion, insertion, other type of mutation).
  • the disruption may occur in a PEX gene that encodes a peroxisome biogenesis factor protein that includes the all following PEXs, and two or three of their different combinations: Pexl p, Pex2p, Pex3p, Pex3Bp, Pex4p, Pex5p, Pex5Bp, Pex5Cp, Pex5/20p, Pex6p, Pex7p, Pex8p, Pex10p, Pex12p, Pex13p, Pex14p, Pex15p, Pex16p, Pex17p, Pex14/17p, Pex18p, Pex19p, Pex20p, Pex21 p, Pex21 Bp, Pex22p, Pex22p-like and PeX26p (US 9,617571 B2).
  • the disruption may be a gene knockout or a deletion in a portion of the gene.
  • Described herein is also a pex- disrupted Y. lipolytica, having a disruption in a native gene encoding Pex3p or Pex5p or Pex20p, or Pex3P and PEX5P, or Pex3P and Pex20P, or Pex3p and pex5p and Pex20p together.
  • US Patent 9,617,571 and US Patent 10,626,424 the disclosures of each of which is incorporated by reference, further describe certain genetic modifications of native genes encoding one or more PEX proteins.
  • Certain embodiments of the disclosure provide methods for increasing the weight percent of at least one polyunsaturated fatty acid relative to the weight percent of total fatty acids in a modified yeast cell having a reduction of beta-oxidation activity in which less acetyl-CoA is produced as compared to beta-oxidation activity of an otherwise identical (isogenic) parental yeast cell.
  • PEX proteins Peroxisome Biogenesis Factor Proteins
  • Disruption of 2 PEX proteins is more effective than disrupting one PEX protein; and disrupting three PEX proteins is the most effective way to reduce betaoxidation.
  • AH291 control strain with genotype as: MATA, dgatl-, dgat2-, and ura3-.
  • Yarrowia strains shown in Table 1 with a disruption of a single pex3 gene (AH246), pex5 gene (AH299) or pex20 gene (AH292) can still grow on plates with palmitate as a sole carbon source and produce a good amount of biomass at 72 hours, suggesting that these strains have some beta-oxidation capabilities, and the time courses of their growth are shown in Figures 2A-2C.
  • Figure 2A shows strain growth for 24 hours.
  • Figure 2B shows strain growth for 48 hours.
  • Figure 2C shows strain growth for 72 hours.
  • CM Complete Media
  • leucin plates 0.13% Amino acid dropout powder minus leucine, 0.17% yeast nitrogen base, 0.5% ammonium sulfate, 2.0% glucose, 2.1% agar.
  • the colonies from transformation were analyzed by PGR.
  • a PGR fragment of 426 bp (SEQ ID NO. 9) was produced with primers of AH088 (GAACGACCATTTCCCGAGCGTTTTC), referred to as SEQ ID. NO. 10 and P216 (GTCGATCAATGAGCATCTGTGTTCGTC), referred to as SEQ ID. No. 11 indicating the DNA fragment with PEX3 was integrated into cells.
  • a PCR fragment of 517 bp (SEQ ID NO. 12) was produced with primers of P189 (CGTTATTGGATACACCGATGCCTTCAAC), referred to as SEQ ID. NO. 13 and P190 (AGCGGGAATATGATGATAGCTCTA-GATGATG), referred to as SEQ ID. NO. 14 indicating the DNA fragment of POX4 was integrated into cells.
  • Figure 5 shows a physical and functional map of the C/al large fragment of plasmid pO4L2P3.
  • Strain AH246 (dgatl-, dgat2-, pex3-, ura3-) was generated from strain AH1314 in order to restore the LEU2 and P0X4 genes back to their wild types (Table 1 , Fig. 2 & 3).
  • Construct pPOX4-Leu2 (Fig. 6; SEQ ID No: 15, Table 3) contains wild type genes of LEU2, and P0X4, the components of the C/al large fragment of plasmid pPOX4- Leu2, used for generation of strain AH246, are further described in Table 3, Fig. 7 and SEQ ID NO: 16
  • Figure 6 depicts the physical and functional map of plasmid pPOX4-LEU2.
  • a PGR fragment of 2,743 bp (SEQ ID NO: 17) was produced with primers of P240 (AATGATAGCCCACGCATAAGCTACAAGAG), referred to as SEQ ID No. 18 and P241 (CCTAACA-GCTGGAGCCACTATATAGTTG), referred to as SEQ ID. No. 19, indicating the DNA fragment (Fig. 7) with wild type POX4 integrated into cells.
  • a colony with PCR positive was designated as strain AH246 (dgatl-, dgat2-, pex3-, ura3-).
  • Figure 7 provides a physical and functional map of the C/al large fragment of plasmid pPOX4-LEU2
  • EXAMPLE 3 Generation of strain AH292 from strain AH291 for deletion of PEX 20 [00130] Strain AH292 (table 1 , Figs. 2 & 3) with pex20 deletion was generated from strain AH291 (dgatl-, dgat2-, ura3-, table 1 ) by deletion of PEX20 gene encoding proteins (SEQ ID NOs: 20 & 21).
  • plasmid pYRH-YPEX20-5-3N (SEQ ID NO: 22) was linearized with restriction enzyme BglW and transformed into strain AH291 Table 1 , Fig. 2 & 3).
  • Figure 8 is an illustration of PEX20 deletion by a Pop-in and Pop-out approach.
  • the transformed cells were growing on CM glucose minus uracil plates (0.13% amino acid dropout mixture minus uracil, with 0.17% yeast nitrogen base, 2.0% glucose, 0.5% ammonium sulfate, and 2.1% agar). The colonies from transformation were analyzed by PCR with primers of YPEX20-5-confirm (GATTCTAGTATATCCAAATACC), referred to as SEQ ID. No. 23 and YPEX20-3-confirmA (AAATATCCGTTGCTATATCCTC), referred to as SEQ ID No. 24.
  • Transformant with a PCR fragment of 1 ,436 bp indicates the BglW digested fragment integrated into right chromosome position (Fig. 8).
  • YPD (Yeast extract, Peptone and Dextrose (glucose)
  • Re-patched colonies were analyzed by PCR with primers of YPEX20-5- confirmA (SEQ ID NO: 26) and YPEX20-3-confirmA (SEQ ID NO: 27).
  • strain AH292 (dgatl-, dgat2-, pex20-, ura3-).
  • Strain AH256 (Table 1 , Fig. 2 & 3) with double deletion of pex3 (SEQ ID NO: 4 and 5) and pex5 (SEQ ID NO: 29 and 30) was generated from strain AH246 (dgatl-, dgat2-, pex3-, ura3-) by deletion of pex5 gene.
  • the pex5 was deleted by Pop-in and pop-out approach as illustrated in Fig. 9. Specifically, the Sa/I DNA fragment containing PEX5-5’ and PEX5-3’ arms of plasmid pYRH-PEX5-5-3N (SEQ ID NO.31 , Fig. 9) was transformed into strain AH246.

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Abstract

L'invention concerne des cellules de levure génétiquement modifiées ayant des perturbations des gènes pex3, pex5 ou pex20 et des combinaisons de celles-ci ayant une beta-oxydation réduite et des procédés pour augmenter les acides gras polyinsaturés.
PCT/US2025/021599 2024-03-27 2025-03-26 Souches microbiennes recombinantes comprenant des phénotypes à oxydation bêta réduite et procédés associés Pending WO2025207802A1 (fr)

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