Microbial production of natural sweeteners, diterpenoid steviol glycosides

Patent metadata

Field	Value
Publication number	US-9284570-B2
Application number	US-201113306633-A
Country	US
Kind code	B2
Filing date	Nov 29, 2011
Priority date	Nov 30, 2010
Publication date	Mar 15, 2016
Grant date	Mar 15, 2016

Abstract

The invention relates to recombinant expression of a steviol or steviol glycosides biosynthetic pathway enzymes in cells and the production of steviol or steviol glycosides.

First claim

What is claimed is:

1. A method for producing steviol or steviol glycoside comprising:
culturing an E. coli strain having balanced expression of (1) an upstream methylerythritol pathway (MEP) that produces isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), with respect to (2) a downstream pathway that produces steviol or steviol glycoside from said IPP and DMAPP, the downstream pathway comprising a recombinantly expressed copalyl diphosphate synthase (CPS), kaurene synthase (KS), a geranylgeranyl diphosphate synthase (GGPPS) kaurenoic acid 13-hydroxylase (KAH) and kaurene oxidase (KO), and optionally one or more Stevia UDP glycosyl transferase enzymes;
wherein said balanced expression is obtained by increasing or decreasing the expression level of a downstream pathway module and increasing or decreasing the expression level of an upstream pathway module together in E. coli , and identifying an E. coli strain with higher production of steviol or steviol glycoside and/or lower accumulation of indole as having balanced expression.

2. The method of claim 1 , wherein the copalyl diphosphate synthase (CPS) enzyme is a Stevia enzyme.

3. The method of claim 1 , wherein the kaurene synthase (KS) enzyme is a Stevia enzyme.

4. The method of claim 1 , wherein the GGPPS enzyme is a Taxus enzyme or a Stevia enzyme.

5. The method of claim 1 , wherein the upstream pathway module comprises dxs, idi, ispD, and ispF genes of the MEP pathway.

6. The method of claim 5 , wherein the upstream pathway module comprises dxs, idi, ispD and ispF genes of the MEP pathway expressed as the operon dxs-idi-ispD-ispF.

7. The method of claim 1 , wherein the downstream module comprises the gene encoding the copalyl diphosphate synthase (CPS) enzyme, the gene encoding the kaurene synthase (KS) enzyme and the gene encoding the GGPPS enzyme co-expressed on an operon.

8. The method of claim 1 , wherein the downstream module further comprises kaurene oxidase (KO) and kaurenoic acid 13-hydroxylase (KAH) enzymes co-expressed on an operon, optionally each as fusions with a cytochrome P450 reductase.

9. The method of claim 1 , wherein the expression of the upstream pathway module and the expression of the downstream pathway module are balanced by one or more of: increasing or decreasing promoter strengths, increasing or decreasing gene or operon copy number, and changing the position of genes within the module.

10. The method of claim 9 , wherein one or more operons is integrated into the E. coli genome.

11. The method of claim 1 , wherein the KAH and KO are Stevia enzymes.

12. The method of claim 11 , wherein the KAH and/or KO comprise catalytically active portions fused to a Stevia cytochrome P450 reductase enzyme.

13. The method of claim 12 , wherein the KAH and KO enzymes have an N-terminal truncation and contain the N-terminal peptide sequence MALLLAVF (SEQ ID NO: 16).

14. The method of claim 1 , further comprising recovering the steviol or steviol glycoside.

15. The method of claim 14 , wherein the steviol or steviol glycoside is recovered from the gas phase of the culture by adding an organic layer.

Assignees

• Stephanopoulos Gregory
• Ajikumar Parayil K
• Massachusetts Inst Technology

Inventors

• Stephanopoulos Gregory
• Ajikumar Parayil K

Classifications

• C12Y505/01012

  Copalyl diphosphate synthase (5.5.1.12) · CPC title

• C12P7/42

  Hydroxy-carboxylic acids · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12Y114/13078

  Ent-kaurene oxidase (1.14.13.78) · CPC title

• A01H13/00

  Algae (unicellular algae C12N1/12) · CPC title