Engineering of acetyl-CoA metabolism in yeast
The invention relates to engineering of acetyl-CoA metabolism in yeast and in particular to production of acetyl-CoA in a non-ethanol producing yeast lacking endogenous gene(s) encoding pyruvate decarboxylase and comprising a heterologous pathway for synthesis of cytosolic acetyl-CoA.
1. A yeast comprising at least one heterologous pathway for synthesis of cytosolic acetyl-Coenzyme A (CoA), the at least one heterologous pathway comprising at least one heterologous gene encoding a respective enzyme involved in synthesis of acetyl-CoA, wherein the yeast lacks any endogenous gene encoding pyruvate decarboxylase or comprises disrupted gene or genes encoding pyruvate decarboxylase, and the at least one heterologous gene comprises:
a heterologous gene encoding a pyruvate ferredoxin oxidoreductase;
a heterologous gene encoding a ferredoxin/flavodoxin reductase; and
a heterologous gene encoding a ferredoxin/flavodoxin reductase substrate, wherein the ferredoxin/flavodoxin reductase substrate is ferredoxin and/or flavodoxin.
2. The yeast according to claim 1 , wherein
said heterologous gene encoding said pyruvate ferredoxin oxidoreductase is a gene encoding a Desulfovibrio africanus pyruvate ferredoxin oxidoreductase;
said heterologous gene encoding said ferredoxin/flavodoxin reductase is a gene encoding an Escherichia coli flavodoxin/ferredoxin reductase; and
said heterologous gene encoding said ferredoxin/flavodoxin reductase substrate is a heterologous E. coli gene encoding said flavodoxin/ferredoxin reductase substrate.
3. The yeast according to claim 2 , wherein the Desulfovibrio africanus pyruvate ferredoxin oxidoreductase is D. africanus pfor.
4. The yeast according to claim 2 , wherein the Escherichia coli ferredoxin/flavodoxin reductase is E. coli fpr.
5. The yeast according to claim 2 , wherein E. coli gene encoding said ferredoxin/flavodoxin reductase substrate is E. coli fdx or E. coli fldA.
6. The yeast according to claim 1 , wherein said yeast lacks endogenous genes encoding PDC1, PDC5 and/or PDC6 or comprises disrupted genes encoding PDC1, PDC5 and/or PDC6 to block conversion of pyruvate to ethanol.
7. A method of producing acetyl-Coenzyme A (CoA) comprising culturing a yeast according to claim 1 in culture conditions suitable for production of cytosolic acetyl-CoA from said yeast.
8. The method according to claim 7 , further comprising collecting said acetyl-CoA or a compound generated by said yeast from said acetyl-CoA from a culture medium in which said yeast is cultured or from said yeast, wherein said compound is fatty acids, 3-hydroxypropionic acid, isoprenoids, polyhydroxyalkanoates, 1-butanol and polyketides, or any combination thereof.
9. A method of producing a yeast suitable for the production of cytosolic acetyl-Coenzyme A (CoA) comprising:
introducing into said yeast at least one heterologous pathway for synthesis of cytosolic acetyl-CoA, said at least one heterologous pathway comprising heterologous genes encoding an enzyme involved in synthesis of acetyl-CoA, with the proviso that said heterologous genes are not a heterologous gene encoding a pyruvate formate lyase,
wherein the heterologous genes comprise:
a heterologous gene encoding a pyruvate ferredoxin oxidoreductase;
a heterologous gene encoding a ferredoxin/flavodoxin reductase; and/or
a heterologous gene encoding a ferredoxin/flavodoxin reductase substrate, wherein the ferredoxin/flavodoxin reductase substrate is ferredoxin and/or flavodoxin; and deleting or disrupting any endogenous gene encoding pyruvate decarboxylase (PDC) in said yeast.
10. The method according to claim 9 , wherein
said heterologous gene encoding said pyruvate ferredoxin oxidoreductase is a gene encoding a Desulfovibrio africanus pyruvate ferredoxin oxidoreductase;
said heterologous gene encoding said ferredoxin/flavodoxin reductase is a gene encoding an Escherichia coli ferredoxin/flavodoxin reductase; and
said heterologous gene encoding said ferredoxin/flavodoxin reductase substrate is a heterologous E. coli gene encoding said ferredoxin/flavodoxin reductase substrate.
11. The method according to claim 10 , wherein the Desulfovibrio africanus pyruvate ferredoxin oxidoreductase is D. africanus pfor.
12. The method according to claim 10 , wherein the Escherichia coli ferredoxin/flavodoxin reductase is E. coli fpr.
13. The method according to claim 10 , wherein E. coli gene encoding said ferredoxin/flavodoxin reductase substrate is E. coli fdx or E. coli fldA.
14. The method according to claim 9 , wherein the endogenous gene encoding pyruvate decarboxylase that is deleted or disrupted is PDC1, PDC5 and/or PDC6.
15. The method according to claim 9 , further comprising collecting said acetyl-CoA or a compound generated by said yeast from said acetyl-CoA from a culture medium in which said yeast is cultured and/or from said yeast, wherein said compound is selected from a group consisting of fatty acids, 3-hydroxypropionic acid, isoprenoids, polyhydroxyalkanoates, 1-butanol and polyketides.