Microbial engineering for the production of chemical and pharmaceutical products from the isoprenoid pathway

Patent metadata

Field	Value
Publication number	US-9404130-B2
Application number	US-201414552676-A
Country	US
Kind code	B2
Filing date	Nov 25, 2014
Priority date	Nov 10, 2009
Publication date	Aug 2, 2016
Grant date	Aug 2, 2016

Abstract

The invention relates to the production of one or more terpenoids through microbial engineering, and relates to the manufacture of products comprising terpenoids.

First claim

What is claimed is:

1. A method for making a product containing steviol or steviol glycoside, the method comprising:
providing an Escherichia coli ( E. coli ) that produces isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) through an upstream methylerythritol pathway (MEP) and converts the IPP and DMAPP to steviol or steviol glycoside through a recombinantly expressed downstream synthesis pathway comprising geranylgeranylpyrophosphate synthase, ent-copalyl diphosphate synthase, ent-kaurene synthase, ent-kaurene oxidase, and ent-kaurenoic acid 13-hydroxylase, and optionally one or more UDP glycosyltransferases;
culturing the E. coli to produce the steviol or steviol glycoside, wherein the accumulation of indole in the culture is controlled to below 100 mg/L to thereby increase steviol or steviol glycoside production; and
incorporating the steviol or steviol glycoside into a food product, food additive, beverage, chewing gum, candy, or oral care product.

2. The method of claim 1 , wherein accumulation of indole in the culture is controlled by balancing the upstream MEP pathway with the downstream synthesis pathway.

3. The method of claim 1 , further comprising, measuring the amount of concentration of indole continuously or intermittently.

4. The method of claim 1 , wherein accumulation of indole in the culture is maintained to below 50 mg/L.

5. The method of claim 1 , wherein accumulation of indole in the culture is maintained to below 10 mg/L.

6. The method of claim 1 , wherein the steviol or steviol glycoside is produced at 10 mg/L or more.

7. The method of claim 1 , wherein the E. coli has additional copies of one or more of the dxs, idi, ispD and ispF genes of the MEP pathway.

8. The method of claim 7 , wherein the E. coli has an additional dxs-idi-ispD-ispF module.

9. The method of claim 1 , wherein the steviol or steviol glycoside is recovered from the culture in an organic layer.

10. The method of claim 1 , wherein the product is a flavor enhancer or sweetener.

11. A method for making steviol or steviol glycoside, the method comprising:
providing an Escherichia coli ( E. coli ) that produces isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) through an upstream methylerythritol pathway (MEP) and converts the IPP and DMAPP to steviol or steviol glycoside through a recombinantly expressed downstream synthesis pathway comprising geranylgeranylpyrophosphate synthase, ent-copalyl diphosphate synthase, ent-kaurene synthase, ent-kaurene oxidase, and ent-kaurenoic acid 13-hydroxylase, and optionally one or more UDP glycosyltransferases;
culturing the E. coli to produce the steviol or steviol glycoside, wherein the accumulation of indole in the culture is controlled to below 100 mg/L to thereby increase steviol or steviol glycoside production.

12. The method of claim 11 , wherein accumulation of indole in the culture is controlled by balancing the upstream MEP pathway with the downstream synthesis pathway.

13. The method of claim 11 , further comprising, measuring the amount of concentration of indole continuously or intermittently.

14. The method of claim 11 , wherein accumulation of indole in the culture is maintained to below 50 mg/L.

15. The method of claim 11 , wherein accumulation of indole in the culture is maintained to below 10 mg/L.

16. The method of claim 11 , wherein the steviol or steviol glycoside is produced at 10 mg/L or more.

17. The method of claim 11 , wherein the E. coli has additional copies of one or more of the dxs, idi, ispD and ispF genes of the MEP pathway.

18. The method of claim 17 , wherein the E. coli has an additional dxs-idi-ispD-ispF module.

19. The method of claim 11 , wherein the steviol or steviol glycoside is recovered from the culture in an organic layer.

Assignees

• Massachusetts Inst Technology
• Nat Univ Singapore

Inventors

• Ajikumar Parayil K
• Stephanopoulos Gregory
• Too Heng Phon

Classifications

• C12P23/00

  Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes (containing heterorings C12P17/00) · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12P7/42 Primary

  Hydroxy-carboxylic acids · CPC title

• C12P15/00

  Preparation of compounds containing at least three condensed carbocyclic rings {(gibbanes C12P27/00; naphthacenes C12P29/00)} · CPC title

• C12P5/007 Primary

  containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00) · CPC title