Production of metabolites

Patent metadata

Field	Value
Publication number	US-9822374-B2
Application number	US-201113700011-A
Country	US
Kind code	B2
Filing date	May 24, 2011
Priority date	May 26, 2010
Publication date	Nov 21, 2017
Grant date	Nov 21, 2017

Abstract

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.

First claim

The invention claimed is:

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 .

Assignees

• Katz Michael Patrik
• Durhuus Thomas
• Smits Hans Peter
• Förster Jochen
• Evolva Sa

Inventors

• Katz Michael Patrik
• Durhuus Thomas
• Smits Hans Peter
• Förster Jochen

Classifications

• C12N15/81 Primary

  for yeasts · CPC title

• C12N9/93

  Ligases (6) · CPC title

• C12N1/18

  Baker's yeast; Brewer's yeast · CPC title

• C07K14/395

  from Saccharomyces · CPC title

• C12P7/22

  aromatic · CPC title