Metabolically engineered cells for the production of resveratrol or an oligomeric or glycosidically-bound derivative thereof

Patent metadata

Field	Value
Publication number	US-9834793-B2
Application number	US-201514690542-A
Country	US
Kind code	B2
Filing date	Apr 20, 2015
Priority date	Feb 22, 2005
Publication date	Dec 5, 2017
Grant date	Dec 5, 2017

Abstract

A recombinant micro-organism producing resveratrol by a pathway in which phenylalanine ammonia lyase (PAL) produces trans-cinnamic acid from phenylalanine, cinnamate 4-hydroxylase (C4H) produces 4-coumaric acid from said trans-cinnamic acid, 4-coumarate-CoA ligase (4CL) produces 4-coumaroyl CoA from said 4-coumaric acid, and resveratrol synthase (VST) produces said resveratrol from said 4-coumaroyl CoA, or in which L-phenylalanine- or tyrosine-ammonia lyase (PAL/TAL) produces 4-coumaric acid, 4-coumarate-CoA ligase (4CL) produces 4-coumaroyl CoA from said 4-coumaric acid, and resveratrol synthase (VST) produces said resveratrol from said 4-coumaroyl CoA. The micro-organism may be a yeast, fungus or bacterium including Saccharomyces cerevisiae, E. coli, Lactococcus lactis, Aspergillus niger , or Aspergillus oryzae.

First claim

The invention claimed is:

1. A genetically engineered micro-organism comprising an engineered operative metabolic pathway producing at least 0.44 to 0.53 micrograms of resveratrol per gram on a dry weight basis of the genetically engineered micro-organism when cultured for a time and under conditions wherein the micro-organism produces resveratrol, wherein the engineered operative metabolic pathway produces:
a) 4-coumaric acid from L-phenylalanine catalysed by a phenylalanine ammonia lyase and a cinnamate 4-hydroxylase expressed in the micro-organism or from tyrosine catalysed by a phenylalanine ammonia lyase or a tyrosine ammonia lyase expressed in said micro-organism;
b) 4-coumaroyl-CoA from 4-coumaric acid catalysed by a 4-coumarate-CoA ligase expressed in the micro-organism; and
c) resveratrol is produced from the 4-coumaroyl-CoA by a resveratrol synthase expressed in the micro-organism;
wherein the micro-organism is Saccharomyces cerevisiae that is cultured in a media with a carbon substrate and does not require an external source of phenylalanine, tyrosine, cinnamic acid or coumaric acid, and further comprises a more than a native expression level of an acetyl coenzymeA carboxylase (ACC1) enzyme.

2. The micro-organism of claim 1 , wherein the more than native expression level of the ACC1 enzyme has been provided by replacing a native promoter of a gene expressing the ACC1 enzyme with a promoter providing a higher level of expression.

3. The micro-organism of claim 2 , wherein the native promoter is replaced with a strong constitutive yeast promoter.

4. The micro-organism of claim 3 , wherein the strong constitutive promoter is a yeast promoter selected from the promoters for yeast genes:
triosephoshosphate dehydrogenase 3 (TDH3), alcohol dehydrogenase 1 (ADH1), triose phosphate isomerase 1 (TPI1) 1 actin (ACTT), glyceraldehyde-3-phosphate dehydrogenase (GPD) and phosphoglucose isomerase (PGI).

5. The micro-organism of claim 1 , wherein the more than native expression level of the ACC1 enzyme has been provided by recombinantly introducing into the micro-organism at least one copy of an exogenous genetic sequence encoding the ACC1 enzyme.

6. The micro-organism of claim 1 , wherein the ACC1 enzyme is from Saccharomyces cerevisiae.

7. The micro-organism of claim 1 , further comprising a more than a native expression level of a NADPH:cytochrome P450 reductase (CPR) enzyme.

8. The micro-organism of claim 7 , wherein the more than native expression level of the CPR enzyme has been provided by replacing a native promoter of a gene expressing the CPR enzyme with a promoter providing a higher level of expression.

9. The micro-organism of claim 8 , wherein the native promoter is replaced with a strong constitutive yeast promoter.

10. The micro-organism of claim 9 , wherein the strong constitutive promoter is a yeast promoter selected from the promoters for yeast genes: triosephoshosphate dehydrogenase 3 (TDH3), alcohol dehydrogenase 1 (ADH1), triose phosphate isomerase 1 (TPI1) 1 actin (ACTT), glyceraldehyde-3-phosphate dehydrogenase (GPD) and phosphoglucose isomerase (PGI).

11. The micro-organism of claim 7 , wherein the more than native expression level of the CPR enzyme has been provided by recombinantly introducing into the micro-organism at least one copy of an exogenous genetic sequence encoding the CPR enzyme.

12. The micro-organism of claim 7 , wherein the CPR enzyme is from Saccharomyces cerevisiae.

13. The micro-organism of claim 1 , wherein the micro-organism produces at least 1.83 milligrams of pinosylvin per gram on a dry weight basis.

14. A method for producing resveratrol or an oligomeric or glycosidically-bound derivative thereof, comprising:
a) cultivating the genetically engineered micro-organism of claim 1 ; and
b) recovering the resveratrol, or the oligomeric or glycosidically-bound derivative thereof, from the culture media.

Assignees

• Evolva Sa

Inventors

• Katz Michael
• Smits Hans Peter
• Forster Jochen
• Nielsen Jens Bredal

Classifications

• C12N9/1037

  Naringenin-chalcone synthase (2.3.1.74), i.e. chalcone synthase · CPC title

• C12P7/22 Primary

  aromatic · CPC title

• C12Y203/01074

  Naringenin-chalcone synthase (2.3.1.74), i.e. chalcone synthase · CPC title

• C12Y114/13011

  Trans-cinnamate 4-monooxygenase (1.14.13.11) · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title