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WO2024238964A1 - Levure modifiée et utilisation pour la production de triacylglycérol - Google Patents

Levure modifiée et utilisation pour la production de triacylglycérol Download PDF

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Publication number
WO2024238964A1
WO2024238964A1 PCT/US2024/030037 US2024030037W WO2024238964A1 WO 2024238964 A1 WO2024238964 A1 WO 2024238964A1 US 2024030037 W US2024030037 W US 2024030037W WO 2024238964 A1 WO2024238964 A1 WO 2024238964A1
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yeast
engineered
yarrowia
indicated
engineered yeast
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Yulin LU
Aravind Somanchi
Xiunan YI
Zachary HOUSTON
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Yali Biosciences Inc
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Yali Biosciences Inc
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    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/14—Yeasts or derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • 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
    • 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
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
    • 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
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01051—1-Acylglycerol-3-phosphate O-acyltransferase (2.3.1.51)
    • 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)
    • C12Y301/02014—Oleoyl-[acyl-carrier-protein] hydrolase (3.1.2.14), i.e. ACP-thioesterase

Definitions

  • Infant formula is a manufactured food designed to substitute for human breast milk sourced mainly from plants. While vegetable-based fats can be blended to mimic the fatty acyl composition of human milk fat, plant-based fats have a different arrangement of acyl groups on the glycerol backbone of the triglyceride molecules. In human milk fat, more than 70% of the C16:0 is present at the stereospecific numbering (sn) 2 position, with unsaturated fatty acyl groups (mainly C 18:1) occupying the outer sn-1 and sn-3 positions. By contrast, vegetable fats contain almost no saturated long chain fats at the sn-2 position, and saturated long-chain fatty acyl groups such as C16:0 occupy the sn-1/3 positions. Clinical trials suggest that the stereoisomeric structure of the fats is important for digestion of the infant formula. Thus, there remains a need for alternative methods of efficiently producing fats that mimic the structure of human milk fat. BRIEF SUMMARY OF THE INVENTION
  • an engineered Yarrowia yeast comprising a heterologous gene encoding a lysophosphatidic acid acyltransferase specific for C16:0-Coenzyme A (16:0 LPAAT).
  • Figures 1-5 are schematic representations of plasmids that can be used to transform Yarrowia yeast.
  • Figure 6 provides the sequence features of cassette 5’ GSYl::TEFin-HsAGPATl- XPRt-GAPDHp-NAT-GAPDHt::5’ GSY1 (SEQ ID NO: 29) for targeted integration of HsAGPATl at the GSY1 locus, wherein the Y. lipolytica TEF promoter is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of HsAGPATl; the initiator ATG and terminator TGA for HsAGPATl are indicated by uppercase, bold text while the coding region is indicated by uppercase text; the Y.
  • lipolytica XPR terminator (3’-UTR) is indicated by lowercase italics
  • the Y. lipolytica GAPDH promoter is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase), which is indicated by lowercase, bold italic, with the initiator ATG and terminator TAG show n in lowercase, bold, underlined italics
  • NAT ourseothricin N-acetyl transferase
  • the Y. lipolytica GAPDH terminator (3’-UTR) is indicated by uppercase, bold, underlined italics, follo ed by the Y005 GSY1 genomic region (3’) indicated by lowercase text.
  • Figure 7 provides the sequence features of cassette TEFin-HsAGPATl-XPRt- GAPDHp-NAT-GAPDHt (SEQ ID NO: 30) for random integration of HsAGPATl, wherein the Y. lipolytica TEF promoter is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of HsAGPATl; the initiator ATG and terminator TGA for HsAGPATl are indicated by uppercase, bold text while the coding region is indicated by uppercase text; the Y. lipolytica XPR terminator (3’-UTR) is indicated by lowercase italics; the Y.
  • lipolytica GAPDH promoter is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase), which is indicated by low ercase, bold italic, with the initiator ATG and terminator TAG shown in lowercase, bold, underlined italics; the Y. lipolytica GAPDH terminator (3’-UTR) is indicated by uppercase, bold, underlined italics.
  • Figure 8 provides the sequence features of cassette TEFin-NAT-XPRt-GAPDHp- CrLPAAT2-GAPDHt (SEQ ID NO: 31 for random integration of CrLPAAT2, wherein the Y.
  • lipolytica TEF promoter is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of selection marker NAT; a linker between TEFin and NAT is indicated by lowercase text; the initiator ATG and terminator TAG for NAT are indicated by uppercase, bold text while the coding region is indicated by uppercase text; the Y. lipolytica XPR terminator (3’-UTR) is indicated by lowercase italics; and the Y.
  • lipolytica GAPDH promoter is indicated by uppercase, bold italics, driving the expression of CrLPAAT2, which is indicated by lowercase, bold italics, with the initiator ATG and terminator TAA shown in lowercase, bold, underlined italics.
  • the Y. lipolytica GAPDH terminator (3 : -UTR) is indicated by uppercase, bold, underlined italics.
  • Figure 9 provides the sequence features of cassette 5 ’ GSY 1 : : 1 dTEFin- CrLPAAT2-XPRt-GAPDHp-NAT-GAPDHt::5’ GSY1 (SEQ ID NO: 32) for targeted integration of CrLPAAT2 at the Gsy 1 locus, wherein proceeding in the 5 ? to 3’ direction: lowercase sequences represent genomic DNA from Y005 that permit targeted integration at GSY1 locus (5’) via homologous recombination to achieve GSY1 KO; the hybrid Y.
  • lipolytica IdTEF promoter is driving the expression of CrLPAAT2; the upstream activating sequence Id is indicated by bold, underlined text while the TEF promoter is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics: the initiator ATG and terminator TAA for CrLPAAT2 are indicated by uppercase, bold text while the coding region is indicated by uppercase text; the Y. lipolytica XPR terminator (3'-UTR) is indicated by lowercase italics; the Y.
  • lipolytica GAPDH promoter is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase), which is indicated by lowercase, bold italic, with the initiator ATG and terminator TAG shown in lowercase, bold, underlined italics;
  • NAT ourseothricin N-acetyl transferase
  • the Y. lipolytica GAPDH terminator (3’-UTR) is indicated by uppercase, bold, underlined italics, followed by the Y005 GSY1 genomic region (3' J indicated by lowercase text.
  • Figure 10 provides the sequence elements of cassette 5’ GSYl ::7dTEFin- CrLPAAT2-XPRt-GAPDHp-NAT-GAPDHt::5’ GSY1 (SEQ ID NO: 33) for targeted introduction of CrLPAAT2 at Gsyl, wherein, proceeding in the 5’ to 3 ' direction: lowercase sequences represent genomic DNA from Y 005 that permit targeted integration at GSY 1 locus (5’) via homologous recombination to achieve GSY1 KO; the hybrid Y.
  • lipolytica 7dTEF promoter is driving the expression of CrLPAAT2; the upstream activating sequence 7d is indicated by bold, underlined text while the TEF promoter is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics; the initiator ATG and terminator TAA for CrLPAAT2 are indicated by uppercase, bold text while the coding region is indicated by uppercase text; the Y. lipolytica XPR terminator (3’-UTR) is indicated by lowercase italics; the Y.
  • lipolytica GAPDH promoter is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase), which is indicated by lowercase, bold italic, with the initiator ATG and terminator TAG shown in lowercase, bold, underlined italics;
  • NAT ourseothricin N-acetyl transferase
  • the Y. lipolytica GAPDH terminator (3’-UTR) is indicated by uppercase, bold, underlined italics, followed by the Y005 GSY1 genomic region (3’) indicated by lowercase text.
  • Figure 11 provides the sequence elements of cassette 12dTEFin-CrLPAAT2- XPRt-GAPDHp-NAT-GAPDHt (SEQ ID NO: 34) designed for random integration of CrLPAAT2 with the hybrid 12dTEF driving expression of CrLPAAT2.
  • the upstream activating sequence 12d is indicated by bold, underlined text while the TEF promoter is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics
  • the initiator ATG and terminator TAA for CrLPAAT2 are indicated by uppercase, bold text while the coding region is indicated by uppercase text
  • the Y the sequence elements of cassette 12dTEFin-CrLPAAT2- XPRt-GAPDHp-NAT-GAPDHt
  • lipolytica XPR terminator (3’-UTR) is indicated by lowercase italics
  • the Y. lipolytica GAPDH promoter is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase), which is indicated by lowercase, bold italic, with the initiator ATG and terminator TAG shown in low ercase, bold, underlined italics
  • the Y. lipolytica GAPDH terminator (3’-UTR) is indicated by uppercase, bold, underlined italics.
  • Figure 12 provides the sequence of cassette A08::TEFl :ScSUC2:xpr:YlGAPDH:AthFATAl :gapdh::3'A08 (SEQ ID NO: 35) for targeted integration of AthFATAl at the A08 locus, wherein, proceeding in the 5' to 3' direction, the bold, uppercase sequence represents the 5’ flank genomic sequence from strain Y005 that pennit targeted integration at the A08 locus via homologous recombination; tire yeast TEF 1 promoter is indicated by lowercase, underlined text, the yeast sucrose invertase gene (conferring the ability of Y005 to metabolize sucrose) is indicated by italicized uppercase text; the yeast xpr tenninator is indicated by lowercase text; the Y lipolytica GAPDH promoter, indicated by lowercase underlined text drives tire expression of the AthFATAl indicated by uppercase italicized text; and the Y.
  • FIG. 13 provides the sequence of cassette A08::TEFl :ScSUC2:xpr:YlGAPDH:ChFATBl :gapdh::3'A08 (SEQ ID NO: 36) for targeted integration of ChFATAl at the A08 locus, wherein, proceeding in tire 5' to 3' direction: bold, uppercase sequence represent the 5’ flank, genomic sequence from strain Y005 that permit targeted integration at the A08 locus via homologous recombination; the yeast TEF1 promoter is indicated by lowercase, underlined text, the yeast sucrose invertase gene (conferring the ability of Y005 to metabolize sucrose) is indicated by italicized uppercase text; the yeast xpr tenninator is indicated by lowercase text; the Y lipolytica GAPDH promoter, indicated by
  • Figure 14 provides the sequence of cassette 5’A08::TEFl :ScSUC2:xpr:YlGAPDH:JcFATBl :gapdh::A08 3’ (SEQ ID NO: 37) for targeted integration of JcFATBl at the A08 locus, wherein, proceeding in the 5' to 3' direction: bold, uppercase sequence represent the 5‘ flank, genomic sequence from strain Y005 that permit targeted integration at the A08 locus via homologous recombination; the yeast TEF1 promoter is indicated by lowercase, underlined text, the yeast sucrose invertase gene (conferring the ability of Y005 to metabolize sucrose) is indicated by italicized uppercase text; the yeast xpr terminator is indicated by lowercase text; the Y lipolytica GAPDH promoter, indicated by lowercase underlined text drives the expression of the JcFATBl indicated by uppercase italicized text; the Y. lipolytica gapd
  • an engineered Yarrowia yeast comprising a heterologous gene encoding a lysophosphatidic acid acyltransferase specific for C16:0-Coenzyme A (C16:0 LPAAT).
  • the Yarrowia is engineered without disruption of gsyl.
  • Gsyl encodes a glycogen synthase and is a frequently used insertion site for engineering of yeast (e.g., SEQ ID NO: 25 provides a ⁇ 5 kb region of the genome containing gsyl locus from wild-type Yarrowia lipolytica).
  • SEQ ID NO: 25 provides a ⁇ 5 kb region of the genome containing gsyl locus from wild-type Yarrowia lipolytica.
  • the engineered Yarrowia yeast provided herein can comprise an intact gsy 1 gene.
  • the Yarrowia yeast can be engineered to have a heterologous nucleic acid encoding a 16:0 LPAAT at a locus other than gsy 1.
  • the engineered Yarrowia yeast comprises a heterologous gene encoding a 16:0 LPAAT at the A08 locus (e.g., within or overlapping the A08 gene, or within a region defined by YALI0_A08107g) of the genome (i.e., the heterologous gene encoding a 16:0 LPAAT is inserted at the A08 locus of the yeast genome).
  • the heterologous gene encoding a 16:0 LPAAT is inserted at the snfl locus of the yeast genome (e.g.. within or overlapping with snfl. such as within SEQ ID NO: 26, which provides a ⁇ 5 kb region of the genome containing snfl locus from wild-type Yarrowia lipolytica).
  • the heterologous gene encoding a 16:0 LPAAT is inserted at the Mfel locus of the yeast genome (e.g., within or overlapping with snfl.
  • the heterologous gene encoding a 16:0 LPAAT is inserted at the Migl locus of the yeast genome (e.g., within or overlapping with snfl, such as within SEQ ID NO: 28, which provides a ⁇ 4 kb region of the genome containing Migl locus from wild-type Yarrowia lipolytica).
  • the Yarrowia can be engineered to include a heterologous nucleic acid encoding a 16:0 LPAAT (or any other gene as discussed herein) through random integration.
  • the C16:0 LPAAT can be any that preferentially catalyzes the esterification of C16:0 acyl-CoA to the sn-2 position on the glycerol backbone as compared to the esterification of other long chain fatty acyl-CoAs (e.g., C18:0) or other long chain unsaturated fatty acyl-CoAs (e.g., Cl 6: 1 , Cl 8: 1 , or C18:2).
  • the C16:0 LPAAT can be from any suitable species.
  • the gene encodes the Cl 6:0 LPAAT with an endoplasmic reticulum targeting sequence (e.g., KDEL) at the C-terminus.
  • the heterologous nucleic acid encodes a C 16:0 LPAAT from Chlamydomonas reinhardtii (e.g., CrLPAAT2; GenBank Acc. No. A8J0J0), Synechocystis Sp. (SsLPAATl; e.g., GenBank Acc. No. WP_010873224); Microcoleus vaginatus (e.g., MvLPAATl; GenBank Acc. No. WP_006633731); Trichodesmium erythraeum (TeLPAATl; GenBank Acc. No.
  • the engineered Yarrowia comprises a nucleic acid encoding a heterologous LPAAT comprising the amino acid sequence of one of SEQ ID NOs: 1-8.
  • the engineered Yarrowia comprises a nucleic acid encoding a heterologous LPAAT, wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NOs: 9-16.
  • the Yarrowia is engineered to express one or more heterologous thioesterases, such as an acyl-ACP thioesterase (FAT), for instance, a FATA (e.g., FATA1 or FATA2), or FATB (e.g., FATB1 or FATB2).
  • FATA acyl-ACP thioesterase
  • FATB e.g., FATB1 or FATB2
  • the Yarrowia can comprise a heterologous nucleic acid encoding the thioesterase.
  • the thioesterase is AthFATAl Arabidopsis thaliana; e.g., GenBank Acc. No. CAA85387), ChFATBl (Cuphea hookeriana; e.g., GenBank Acc. No.
  • the engineered Yarrowia comprises a nucleic acid encoding a heterologous thioesterase comprising the amino acid sequence of one of SEQ ID NOs: 17-20. In some embodiments, the engineered Yarrowia comprises a nucleic acid encoding a heterologous thioesterase, wherein the nucleic acid comprises one of SEQ ID NOs: 21-24.
  • the Yarrowia also can be engineered to express a heterologous acyltransferase.
  • the Yarrowia comprises a heterologous nucleic acid encoding glycerol-3- phosphate acyltransferase (GPAT), which regulates esterification at the sn-1.
  • GPATs include those from Arabidopsis thaliana (e.g., SEQ ID NO: 38); Ziziphus jujube (e.g., SEQ ID NO: 39), and Haematococcus lacustris (e.g., SEQ ID NO: 40).
  • the Yarrowia comprises a heterologous nucleic acid encoding di acylglycerol acyltransferase (DGAT), which catalyzes the conversion of DAG to TAG.
  • DGATs include those from Brassica napus (e.g., SEQ ID NO: 42), Auxenochlorella prototheocoides (e.g., SEQ ID NO: 43); Camellia sinensis (e.g., SEQ ID NO: 44), and Raphanus sativus (e.g., SEQ ID NO: 45).
  • the heterologous nucleic acid encodes a PDAT, which regulates the transfer of acyl CoA between phosphatidyl choline (PC) and TAG.
  • PDATs include those from Arabidopsis thaliana (e.g., SEQ ID NO: 46); Panicum miliaceum (e.g., SEQ ID NO: 47), and Linnemannia elongate (e.g., SEQ ID NO: 48).
  • the heterologous acyltransferase desirably has higher specificity to 18: 1 fatty acids as compared to other long chain fatty acids.
  • heterologous acyltranferases with higher specificity to 18:1 fatty acids include GPAT9 rom Arabidopsis (GPAT-OE; e.g., SEQ ID NO: 38), DGAT from Brassica napus (e.g., SEQ ID NO: 42), and PDAT from Arabidopsis (PDAT-OE; e.g., SEQ ID NO: 46).
  • the Yarrowia comprises heterologous nucleic acids encoding one or more (or all) of a GPAT, DGAT, and PDAT.
  • the Yarrowia is engineered to express a sucrose invertase enzyme to allow the Yarrowia to grow on sucrose.
  • the Yarrowia can comprise a heterologous nucleic acid encoding a sucrose invertase enzy me, such as ScSUC2 (Saccharomyces cerevisiae').
  • the Yarrowia is engineered to reduce non-homologous end-joining repair (NHEJ) mechanisms.
  • NHEJ non-homologous end-joining repair
  • the Yarrowia can be modified by disrupting or deleting native KU70 and/or KU80.
  • the Yarrowia can be engineered to improve homologous recombination.
  • the Yarrowia can include a heterologous nucleic acid encoding RAD51 or RAD52 (e.g., RAD51 or RAD52 from Saccharomyces cerevisiae (ScRAD51, ScRAD52) or ixom Arabidopsis (AIRAD51 or AtRAD52).
  • the engineered Yarrowia has intact KU70 and/or KU80 genes.
  • the Yarrowia is modified to reduce or eliminate conversion of diacylglycerol to phosphatidylcholine, for instance, by reducing the activity of choline phosphotransferase (CPT1) and/or ethanolamine phosphotransferase (EPT1).
  • CPT1 choline phosphotransferase
  • EPT1 ethanolamine phosphotransferase
  • the Yarrowia comprises mutations that disrupt or delete the CPT1 and/or EPT1 genes.
  • the activity of phosphatidylcholine: diacylglycerol cholinephosphotransferase (PDCT) is reduced or eliminated in the Yarrowia.
  • the genes encoding PDCTs are disrupted or deleted.
  • the Yarrowia is modified to reduce or eliminate the activity of beta-ketoacyl-ACP synthase.
  • the Yarrowia can have a mutation disrupting or deleting a gene encoding beta-ketoacyl-ACP synthase (e.g., FAB1).
  • the Yarrowia is modified to reduce or eliminate the activity of a fatty acid elongase.
  • the Yarrowia can have a mutation disrupting or deleting a gene encoding fatty acid elongase (e.g.. FAE1).
  • the Yarrowia is modified to reduce or eliminate the activity of a fatty acid desaturase (e.g., fatty acid desaturase 2).
  • the Yarrowia can have a mutation disrupting or deleting a gene encoding the fatty 7 acid desaturase 2 gene (FAD2)
  • the Yarrowia can comprise any combination of the foregoing genetic modifications.
  • the Yarrowia comprises (a) a heterologous nucleic acid encoding a thioesterase, and, optionally, (b) a heterologous nucleic acid encoding a sucrose invertase, optionally (c) a heterologous nucleic acid encoding RAD51 or RAD52, and optionally (d) a mutation disrupting or deleting KU70 and/or KU80.
  • the engineered Yarrowia comprises (a) a heterologous nucleic acid encoding a thioesterase, and, optionally, (b) a heterologous nucleic acid encoding a sucrose invertase, optionally (c) a heterologous nucleic acid encoding RAD51 or RAD52, but has an intact native KU70 and/or KU80 gene (i.e., does not have a KU70 or KU80 disruption or deletion).
  • any yeast from the Yarrowia clade can be used. This may include, without limitation, Candida (C.) alimentaria, Y. deformans, C. galli, C. hispaniensis, C. hollandica, C. oslonensis. C. phangngensis , Y. yakushimensis, and Y. lipolytica.
  • the yeast is Yarrowia lipolytica.
  • the yeast is a wild-ty pe Yarrowia lipolytica.
  • the yeast is a Yarrowia that does not comprise a genoty pe with one or more (or all) of the following modifications: MatA. Ieu2-27O, ura3-302, xpr2- 322, axp-2.
  • heterologous nucleic acids e.g., genes
  • a vector such as a plasmid
  • the nucleic acids encoding desired proteins can be under the control of a suitable promoter, which can be heterologous or endogenous to the Yarrowia.
  • the vector can express the genes without integration into the Yarrowia genome (e.g., self-propagating plasmids), or the vector can integrate the target genes into the Yarrowia genome.
  • the vector can comprise an expression cassette comprising the heterologous nucleic acid of interest along with any desired regulatory sequences (e.g., promotors, transcription termination sequences, etc.) and flanking sequences that will integrate into a target region of the Yarrowia chromosome by homologous recombination).
  • the heterologous nucleic acid to be expressed will be under control of a promoter.
  • Promoters and other regulatory 7 sequences can be native or non-native, and can be any ty pically used in yeast.
  • Native promoters include, for instance. TEF promoter, GPAT promoter, GPD promoter, GPM promoter, FBA promoter, FBAIN promoter, GPDIN promoter.
  • Termination regions also can be native or non-native, and can be any typically used in yeast, for instance, approximately 100 bp of the 3' end of the Yarrowia lipolytica extracellular protease (XPR; GenBank Accession No. Ml 7741); the acyl-coA oxidase (Aco3: GenBank Accession No. AJ001301 and No. CAA04661; Pox3: GenBank Accession No.
  • Transformation can be effected by any of several routine methods, such as lithium acetate, heat shock, or electroporation.
  • the heterologous nucleic acids can be introduced into any gene locus that does not affect the growth of the Yarrowia and does not adversely affect production or accumulation of TAGs to any significant degree.
  • the heterologous nucleic acids are introduced at the A08 locus.
  • the expression cassette comprising the heterologous nucleic acid to be inserted at A08 comprises flanking sequences (homology arms) identical to sequences in the AOS gene locus to facilitate homologous recombination.
  • genes can be disrupted or deleted in the Yarrowia by any of several known techniques. For instance, genes can be disrupted or deleted by inserting a non-native nucleic acid (e.g., a selectable marker) into the native gene to interrupt the coding sequence and thereby functionally deactivate the gene. Deletion cassettes for this purpose can be inserted into the gene targeted for deletion using, for example, homologous recombination. Other techniques for gene disruption also can be used, such as RNA-guided endonuclease (CRISPR/CAS) systems. Disruption or deletion of a given gene is accomplished if the expression of the gene after the modification is less than the expression of the gene before the modification (or as compared to the same microorganism without the modification).
  • CRISPR/CAS RNA-guided endonuclease
  • the engineered Yarrowia can be used to produce triacylglycerol (TAG) with enhanced amounts of C 16:0 at the sn-2 position by culturing the Yarrowia in a culture media comprising a carbon source.
  • TAG triacylglycerol
  • 40% w/w or more (e.g., 70 % w/w or more) of the TAGs produced in the engineered Yarrowia have a C16:0 fatty acyl group at the sn-2 position.
  • 40% w/w or more (e.g., 70 % w/w or more) of the TAGs produced in the engineered Yarrowia is l,3-dioleoyl-2-palmitoyl glycerol and/or 1 -oleoyl-2-palmitoyl-3-linoleoyl-rac-glycerol.
  • the engineered Yarrowia provides a total lipid titer of 20g/L or more (e.g., 60 g/L or more).
  • the engineered Y arrowia provides a total lipid content (% lipid of cell weight) of 40% or more (e.g., 60% or more).
  • TAG profiles can be analyzed using LC-MS/MS.
  • TAG composition can also be analyzed by GC-MS to determine fatty acid methyl ester composition, and lipase digestion followed by GC-MS analysis for sn-2 vs sn-1/3 ratios.
  • the carbon source ty pically comprises a sugar.
  • the carbon source includes one or more fermentable sugars, such as xylose, lactose, cellulose, glucose, fructose, sucrose, or hydrolysed lignocellulose.
  • the carbon source further comprises one or more fatty acids and/or fatty acid esters, such as Cl 6:0 fatty acids or fatty acid esters.
  • the carbon source includes at least 30% w/w Cl 6:0 fatty acids and/or fatty acid esters.
  • the carbon source further comprises one or more vegetable oils, such as palm oil.
  • the carbon source comprises sucrose or glycerol.
  • the carbon source comprises acetate or methanol, or a CCh-derived feedstock, such as C Ch-derived acetate or methanol.
  • the culture media can comprise any suitable amount of the carbon source(s).
  • the culture media comprises 10 to 60 g/L of a sugar and/or glycerol.
  • the culture mediate comprises 1 to 5 g/L of fatty acids or fatty acid esters.
  • the culture media comprises 1 to 10 g/L of a vegetable oil.
  • a suitable pH range for the fermentation is typically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.0 is preferred as the range for the initial growth conditions.
  • the fermentation process includes media that may have a carbon composition typically ranging from 5g/L to 1 OOg/L, and a C/N ratio between 25 and 250.
  • the fermentation may be conducted under aerobic or anaerobic conditions.
  • a two-stage fermentation process may be used in desired, whereby the first stage of the fermentation is dedicated to the generation and accumulation of cell mass and is characterized by rapid cell growth and cell division, and the second stage of the fermentation utilizes conditions (e.g., nitrogen deprivation) that promotes high levels of lipid accumulation.
  • Production of lipid from a recombinant microbial host may be produced by a batch, fed-batch or continuous fermentation process.
  • TAGs are isolated and purified from the yeast culture.
  • TAG can be isolated and purified from the culture by any suitable method, such as by using organic solvents, sonication, supercritical fluid extraction (e.g., using carbon dioxide), saponification and physical means such as presses, or combinations thereof.
  • isolation is performed using solvents.
  • the TAG produced by the engineered Yarrowia is believed to be useful in food compositions for humans or animals, especially imitation dairy products, dietary supplements, and particularly human milk fat substitute compositions such as infant formula.
  • a food composition, especially an infant formula composition comprising the TAG, optionally combined with carbohydrates and/or proteins.
  • Such a composition will typically comprise 3-5 wt.% TAGs in liquid form, or 25-30 wt.% in dry powder form.
  • the TAGs produced by the engineered Yarrowia can be isolated prior to combining with the other components of the food composition, or the TAGs can be used without isolation from the engineered Yarrowia, which can be used as a “whole cell” composition (optionally lysed) or fraction thereof including the TAGs and the other cellular components.
  • Embodiment 1 An engineered Yarrowia yeast comprising a heterologous gene encoding a lysophosphatidic acid acyltransferase specific for C 16:0-Coenzyme A (C16:0 LPAAT).
  • Embodiment 2 The engineered yeast of embodiment 1. wherein the heterologous gene encoding the Cl 6:0 LPAAT does not disrupt gsyl.
  • Embodiment 3 The engineered yeast of embodiment 1 or 2 further comprising a heterologous gene encoding DGAT or GPAT.
  • Embodiment 4 The engineered yeast of any of embodiments 1-3, wherein the Cl 6:0 LPAAT is at the A08 locus of the yeast genome.
  • Embodiment 5 The engineered yeast of any of embodiments 1-4, wherein the C16:0 LPAAT is CrLPAAT2, BnLPATl, RoLPATl, RoLPAT2, HsAGPATl.
  • Embodiment 6 The engineered yeast of any of embodiments 1-5, further comprising a heterologous gene encoding a thioesterase.
  • Embodiment 7 The engineered yeast of embodiment 6, wherein the thioesterase is a plant thioesterase.
  • Embodiment 8 The engineered yeast of embodiment 6 or 7, wherein the thioesterase is AthFATAl. ChFATBl, or JcFATBl.
  • Embodiment 9 The engineered yeast of any of embodiments 6-8, wherein the thioesterase is located at the A08 locus of the yeast genome.
  • Embodiment 10 The engineered yeast of any of embodiments 1-9, further comprising a heterologous RAD52 gene.
  • Embodiment 11 The engineered yeast of embodiment 10, wherein the RAD52 gene is at the A08 locus of the yeast genome.
  • Embodiment 12 The engineered yeast of any of embodiments 1-11, further comprising a heterologous gene encoding an invertase.
  • Embodiment 13 The engineered yeast of embodiment 12, wherein the gene encoding the invertase is SUC2. optionally ScSUC2.
  • Embodiment 14 The engineered yeast of any of embodiments 1-13, wherein the yeast is Yarrowia lipolyticci.
  • Embodiment 15 The engineered yeast of any of embodiments 1-13, wherein the yeast does not comprise one or more (or does not comprise any) of the following genetic modifications: MatA, leu2-270, ura3-302, xpr2-322, or axp-2.
  • Embodiment 16 A triacylglycerol (TAG) composition made by the engineered yeast of any of embodiments 1-15.
  • TAG triacylglycerol
  • Embodiment 17 A food composition comprising the TAG composition of embodiment 16 and added protein and/or carbohydrates.
  • Embodiment 18 The food composition of embodiment 17, wherein the food composition is infant formula.
  • Embodiment 19 A method of preparing an engineered yeast according to any of embodiments 1-15, the method comprising introducing a heterologous gene encoding a C16:0 LPAAT into a Yarrowia yeast
  • Embodiment 20 The method of embodiment 19, wherein the heterologous gene is introduced by random integration.
  • Embodiment 21 The method of embodiment 19 or 20. wherein yeast is a wildtype Yarrowia yeast.
  • Embodiment 22 The method of any of embodiments 19-21, wherein the yeast does not comprise MatA, leu2-270, ura3-302, xpr2-322, and/or axp-2 modifications.
  • the following procedure was used to transform all Y. lipolytica transgenic strains unless otherwise noted: the base strains to be transformed were grown overnight in 50-mL conical tube at 250 rpm at 28 °C in YPD medium. The cells were transferred to 20 mL of fresh YPD medium at OD 0.5 in a 125-mL flask, growing at 28 °C for 3 hours. Hydroxyurea was then added to the culture at 50 mM. After 2 hours. 2 OD of cells (about 10 A 8 cells) were harvested by centrifuging at 5000 rpm. The supernatant was removed. The cells were washed with 1 mL of sterile water and centrifuged again.
  • fatty acid methyl ester (FAME) analysis was performed in accordance with AOCS Official Method Ce lj-07 ( American Oil Chemists’ Society (2017), AOCS Official Method Ce lj-07: cis-, trans-, Saturated, Monounsaturated and Polyunsaturated Fatty Acids in Extracted Fats by Capillary GLC, Official Methods and Recommended Practices of the AOCS, 7th ed. AOAC International).
  • This example illustrates the production of engineered Y. lipolytica.
  • Plasmid vector is constructed comprising a ScRAD52 gene and TEF2 promoter (Fig. 1). Wild type Y. lipolytica is transformed with the plasmid to generate a new base strain. The new strain is optionally further modified to knock-out KU70 and/or KU80.
  • Additional vectors comprising LPAATs and various thioesterases are generated with flanking sequences identical to regions of A08 (e.g., Figs. 2-5) to facilitate insertion in the A08 locus by homologous recombination, and the base engineered Y. lipolytica is transformed with two or more of the vectors to produce engineered strains.
  • One or more of the plasmid vectors includes Suc2 allowing the engineered Yarrowia to utilize sucrose as a carbon source.
  • This example illustrates the production of engineered Y. lipolytica expressing heterologous C16:0 LPAATs.
  • CrLP AAT2 or HsAGPAT 1 was introduced into a wild-type Y. lipolytica strain
  • cassettes 1-3 are illustrated in Figs. 6, 7, and 8, respectively.
  • Cassette 6 was designed for targeted integration of HsAGPATl at the Gsyl locus
  • Cassettes 7 and 8 were designed for random integration of CrLPAAT2 or HsAGPATl.
  • Table 1 shows the OPO content (as a percentage of total TAGs measured in LC- MS-MS) of 5 transgenic strains generated using cassettes 1-3.
  • Y. lipolytica base strain Y005 is shown as a non-transgenic control. Strains were grown for 72 hours in 50-mL conical tube at 250 rpm at 28 °C in lipid production medium. Biomass was harvested and pelleted. The sample preparation and TAG analysis was performed and TAG profile data generated using LC-MC-MC method.
  • FIG. 9 This example illustrates the production of engineered Y. lipolytica expressing heterologous C16:0 LPAATs.
  • CrLPAAT2 was introduced into a wild-type K lipolytica strain (designated Y005) with different promoters.
  • the expression cassettes 4-6 are illustrated in FIGs 9, 10, and 1 1 , respectfully.
  • Cassette 4 and 5 were designed for targeted integration at the Gsy 1 locus, and Cassette 6 is designed for random integration.
  • Table 2 shows the OPO content (as a percentage of total TAGs measured in LC- MS-MS) of 4 transgenic strains generated using cassette 4-6. Strains generated with cassette 3 (Example 2) is provided for comparison, and Y. lipolytica base strain Y005 is shown as a non-transgenic control. Strains were grown for 72 hours in 50-mL conical tube at 250 rpm at 28 °C in lipid production medium. Biomass was harvested and pelleted. The sample preparation and TAG analysis was performed and the TAG profile data was generated using LC-MC-MC method.
  • Y. lipolytica can be engineered with heterologous thioesterases to increase C 16 fatty acid levels.
  • Y005 wild-type Y. lipolytica was transformed with pY088(AthFATAl), pY089(ChFATBl), pY090 (JcFATBl).
  • the cassettes used in the transformants are provided in FIGs. 12, 13, and 14,
  • the Y.lipolylica base strain Y005 was used as a non-transgenic control. Strains were grown for 72 hours in 50-mL conical tube at 200 rpm at 28°C in lipid production medium. Biomass was harvested, and subjected to direct transesterification to generate FAMEs for subsequent quantitation and characterization by GC/FID.
  • the C 16 fatty acid levels (% of total TAGs) are shown in Table 3.

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Abstract

La présente invention concerne une Yarrowia modifiée comprenant un gène hétérologue codant pour une acyltransférase d'acide lysophosphatidique spécifique de la coenzyme A C16:0 (LPAAT 16:0), et son utilisation pour la production de triacylglycérol.
PCT/US2024/030037 2023-05-17 2024-05-17 Levure modifiée et utilisation pour la production de triacylglycérol Pending WO2024238964A1 (fr)

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