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 heterologous genes encoding a respective enzyme involved in synthesis of acetyl-CoA, with the proviso that the heterologous genes are not a heterologous gene encoding a pyruvate formate lyase, wherein the heterologous genes comprise:
at least one heterologous gene encoding a cytosolic pyruvate dehydrogenase complex selected from the group consisting of genes encoding an Escherichia coli cytosolic pyruvate dehydrogenase complex, genes encoding a Saccharomyces cerevisiae pyruvate dehydrogenase complex but lacking mitochondrial target signal (MTS), genes encoding an Azotobacter vinelandii pyruvate dehydrogenase complex, and genes encoding an Enterococcus faecalis pyruvate dehydrogenase complex; and
at least one heterologous gene encoding respective enzymes involved in attachment and activation of lipoyl groups to said cytosolic pyruvate dehydrogenase complex selected from the group consisting of genes encoding Escherichia coli lipoic acid synthetase and/or lipoate-protein ligase, and genes encoding Saccharomyces cerevisiae LIP2, LIP3, LIP5 and GCV3, each of which is lacking MTS.
2. The yeast according to claim 1 , wherein the genes encoding an Escherichia coli cytosolic pyruvate dehydrogenase complex are aceE, aceF, and/or lpd, the genes encoding a Saccharomyces cerevisiae pyruvate dehydrogenase complex but lacking mitochondrial target signal (MTS) are PDA1, PDB1, LAT1, LPD1, and/or PDX1, each of which lack MTS, the genes encoding an Azotobacter vinelandii pyruvate dehydrogenase complex are aceE, aceF, and/or lpdA, the genes encoding an Enterococcus faecalis pyruvate dehydrogenase complex are pdhA, pdhB, aceF and/or lpdA, and the genes encoding Escherichia coli lipoic acid synthetase and/or lipoate-protein ligase are lipA, lipB, and/or lplA.
3. The yeast according to claim 1 , wherein the yeast lacks any endogenous gene encoding pyruvate decarboxylase or comprises a disrupted gene or genes encoding pyruvate decarboxylase.
4. The yeast according to claim 3 , 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.
5. 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.
6. The method according to claim 5 , 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.
7. A method of producing acetyl-Coenzyme A (CoA) comprising culturing a yeast according to claim 3 culture conditions suitable for production of cytosolic acetyl-CoA from said yeast.
8. 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 a respective 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:
at least one heterologous gene encoding a cytosolic pyruvate dehydrogenase complex selected from the group consisting of genes encoding an Escherichia coli cytosolic pyruvate dehydrogenase complex, genes encoding a Saccharomyces cerevisiae pyruvate dehydrogenase complex but lacking mitochondrial target signal (MTS), genes encoding an Azotobacter vinelandii pyruvate dehydrogenase complex, and genes encoding an Enterococcus faecalis pyruvate dehydrogenase complex; and
at least one heterologous gene encoding respective enzymes involved in attachment and activation of lipoyl groups to said cytosolic pyruvate dehydrogenase complex selected from the group consisting of genes encoding Escherichia coli lipoic acid synthetase and/or lipoate-protein ligase, and genes encoding Saccharomyces cerevisiae LIP2, LIP3, LIP5 and GCV3, each of which is lacking MTS.
9. The method of claim 8 , further comprising deleting or disrupting any endogenous gene encoding pyruvate decarboxylase in said yeast.