Genetically modified fungal cells for overexpression of an acetyl-CoA carboxylase and a pyruvate carboxylase
A fungal cell is capable of producing high levels of fatty acids and fatty acid-derived products. The fungal cell comprises at least one modification to the endogenous fatty acid metabolism.
1. A fungal cell for the production of fatty acids and/or fatty acid-derived products, wherein said fungal cell is genetically modified for overexpression of an endogenous acetyl-CoA carboxylase and an endogenous pyruvate carboxylase, and wherein said fungal cell has an increased production of fatty acids and/or fatty acid-derived products over a wild-type fungal cell that is not genetically modified for overexpression of said endogenous acetyl-CoA carboxylase and said endogenous pyruvate carboxylase.
2. The fungal cell according to claim 1 , wherein said endogenous acetyl-CoA carboxylase is ACC1 and said endogenous pyruvate carboxylase is PYC1.
3. The fungal cell according to claim 1 , wherein said fungal cell is further genetically modified for overexpression of at least one protein selected from the group consisting of an endogenous mitochondrial pyruvate carrier, a citrate synthase selected from the group consisting of Rhodosporidium toruloides citrate synthase RtCIT1 and Saccharomyces cerevisiae citrate synthase ScCIT1, and an endogenous citrate and oxoglutarate carrier protein.
4. The fungal cell according to claim 3 , wherein said endogenous mitochondrial pyruvate carrier is selected from the group consisting of MPC1 and MPC3.
5. The fungal cell according to claim 3 , wherein said endogenous citrate and oxoglutarate carrier protein is YHM2.
6. The fungal cell according to claim 1 , wherein said fungal cell is further modified for overexpression of at least one protein selected from the group consisting of an endogenous cytosolic isocitrate dehydrogenase and Aspergillus nidulans ATP-citrate lyase AnACL.
7. The fungal cell according to claim 6 , wherein said endogenous cytosolic isocitrate dehydrogenase is IDP2.
8. The fungal cell according to claim 1 , wherein said fungal cell is further genetically modified for downregulation of at least one endogenous protein selected from the group consisting of an endogenous mitochondrial NAD+-dependent isocitrate dehydrogenase and an endogenous phosphoglucose isomerase.
9. The fungal cell according to claim 8 , wherein said endogenous mitochondrial NAD+-dependent isocitrate dehydrogenase is encoded by IDH2.
10. The fungal cell according to claim 8 , wherein said endogenous phosphoglucose isomerase is encoded by PGI1.
11. The fungal cell according to claim 8 , wherein said fungal cell has a native promoter of said endogenous protein replaced by a weaker promoter.
12. The fungal cell according to claim 11 , wherein said weaker promoter is selected from the group consisting of pINH1, pSDH4, pATP5, pGSY2, pGSP2, and pRBK1.
13. The fungal cell according to claim 1 , wherein a native promoter of at least one essential gene is replaced by a carbon-source-dependent promoter.
14. The fungal cell according to claim 13 , wherein said at least one essential gene is selected from the group consisting of ERG9 and LEU2.
15. The fungal cell according to claim 1 , wherein pyruvate decarboxylase activity in said fungal cells is downregulated by the deletion of at least one gene selected from the group consisting of PDC1, PDC5 and PDC6.
16. The fungal cell according to claim 1 , wherein said fungal cell is further genetically modified for downregulation of an endogenous fructose-1,6-bisphosphate (FBP)-sensitive pyruvate kinase PYK1.
17. The fungal cell according to claim 1 , wherein said fungal cell is further genetically modified for overexpression of an endogenous fructose-1,6-bisphosphate(FBP)-insensitive pyruvate kinase PYK2.
18. The fungal cell according to claim 1 , wherein said fungal cell is genetically modified for overexpression of a fatty acid transport protein selected from the group consisting of an endogenous ATP-binding cassette (ABC) protein, an endogenous lipid transfer protein (LTP), an endogenous fatty acid transporter protein (FATP) selected from the group consisting of Homo sapiens FATP1, Homo sapiens FATP4, and Saccharomyces cerevisiae FAT1, and a plant wax ester transporter selected from the group consisting of Arabidopsis ABCG11, ABCG12, LTPG1 and LTPG2.
19. The fungal cell according to claim 1 , wherein said fungal cell is genetically modified for overexpression of a gene selected from the group consisting of MPP6, ACP1, EPT1, FAA1, GEP4, GGA2, IDP3, INP54, LPP1, MCR1, ORM1, RTC3, SPO7, TGL1 and YFT2.
20. The fungal cell according to claim 19 , wherein said fungal cell is genetically modified for overexpression of GGA2.
21. The fungal cell according to claim 1 , wherein said fungal cell is a fungal cell selected from the group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Candida, Hansenula, Torulopsis, Kloeckera, Pichia, Schizosaccharomyces, Trigonopsis, Brettanomyces, Debaromyces, Nadsonia, Lipomyces, Cryptococcus, Aureobasidium, Trichosporon, Lipomyces, Rhodotorula, Yarrowia, Rhodosporidium, Phaffia, Schwanniomyces, Aspergillus , and Ashbya.
22. The fungal cell according to claim 21 , wherein said fungal cell is a fungal cell selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Ashbya gossypii, Saccharomyces boulardii, Zygosaccharomyces bailii, Kluyveromyces lactis, Rhodosporidium toruloides and Yarrowia lipolytica.
23. A method for producing a fatty acid comprising:
culturing a fungal cell according to claim 1 in a culture medium and in culture conditions suitable for production of said fatty acid by said fungal cell, and
collecting said fatty acid from said culture medium and/or said fungal cell.
24. A method for producing a fatty acid derived-product comprising:
culturing a fungal cell according claim 1 in a culture medium and in culture conditions suitable for production of said fatty acid-derived product by said fungal cell; and
collecting said fatty acid-derived product from said culture medium and/or said fungal cell, wherein said fatty acid-derived product is selected from the group consisting of a hydrocarbon, a triacylglyceride, a phospholipid, a lactone, a fatty alcohol, a fatty aldehyde, a fatty acid ester, and a mixture thereof.