Production of metabolites
A recombinant micro-organism such as Saccharomyces cerevisiae which produces and excretes into culture medium a stilbenoid metabolite product when grown under stilbenoid production conditions, which expresses in above native levels a ABC transporter which transports said stilbenoid out of said micro-organism cells to the culture medium. The genome of the Saccharomyces cerevisiae produces an auxotrophic phenotype which is compensated by a plasmid which also expresses one or more of said enzymes constituting said metabolic pathway producing said stilbenoid, an expression product of the plasmid is genetically modified to include a ubiquitination tag sequence. Expression of an enzyme participating in catabolism of phenylalanine by the Ehrlich pathway is optionally reduced compared to its native expression level.
1. A recombinant Saccharomyces cerevisiae micro-organism which produces and excretes into culture medium resveratrol, wherein the micro-organism comprises:
(a) a resveratrol producing metabolic pathway comprising a phenylalanine ammonia lyase (PAL) or a tyrosine ammonia lysase (TAL), a cinnamate 4-hydroxylase (C4H), a 4-coumarate-CoA ligase (4CL) and a stilbene synthase;
(b) a greater than native expression level of SNQ2,
(c) a functionally disabled or deleted ARO10, and
(d) a greater than native expression level of an acetyl coenzymeA carboxylase (ACC1) enzyme, and
wherein the micro-organism is capable of producing above 4,000 mg/L of resveratrol when cultured for a time and under conditions wherein the recombinant micro-organism produces resveratrol.
2. The recombinant micro-organism of claim 1 , wherein said SNQ2 is an expression product of the gene SNQ2 of Saccharomyces cerevisiae .
3. The recombinant micro-organism of claim 1 , wherein a gene expressing said SNQ2 is endogenous and is present in a higher copy number than in the native micro-organism.
4. The recombinant micro-organism of claim 1 , wherein the stilbene synthase is a resveratrol synthase.
5. The recombinant micro-organism of claim 4 , wherein the resveratrol synthase is from Vitis pseudoreticulata .
6. The recombinant micro-organism of claim 4 , wherein the resveratrol synthase is from Vitis vinifera .
7. The recombinant micro-organism of claim 1 , in which the gene products of the Saccharomyces cerevisiae genes Aro4 and Aro7 are expressed at levels in excess of those produced in the wild type of the micro-organism by replacing a native promoter of the Aro4 and Aro7 genes with a strong promoter providing a higher level of expression.
8. The recombinant micro-organism of claim 7 , wherein the strong promoter providing a higher level of expression is TDH3, TEF1, TPI1, ADH1 or TEF2.
9. The recombinant micro-organism of claim 7 , wherein the Aro4 gene is encoded by SEQ ID NO.:138 and the Aro7 gene is encoded by SEQ ID NO.:139.
10. The recombinant micro-organism of claim 1 , wherein the micro-organism is capable of producing over 5,000 mg/L of resveratrol when cultured for a time and under conditions wherein the recombinant micro-organism produces resveratrol.
11. The recombinant micro-organism of claim 1 , wherein the micro-organism is capable of producing at least about 4,000 mg/L of resveratrol to about 5,000 mg/L of resveratrol when cultured for a time and under conditions wherein the recombinant micro-organism produces resveratrol.
12. The recombinant micro-organism of claim 1 , wherein said SNQ2 gene is exogenous or is endogenous to said micro-organism and is expressed at a level higher than the native expression level in the micro-organism by replacing a native promoter of a gene expressing said SNQ2 gene with a strong promoter providing a higher level of expression in the micro-organism.
13. The recombinant micro-organism of claim 12 , wherein the strong promoter providing a higher level of expression is TDH3, TEF1, TPI1, ADH1 or TEF2.
14. A dried biomass of the recombinant micro-organism of claim 1 .
15. An animal feed comprising the recombinant micro-organism of claim 1 or the dried biomass of claim 14 .