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

Patent metadata

Field	Value
Publication number	US-9957527-B2
Application number	US-201615208099-A
Country	US
Kind code	B2
Filing date	Jul 12, 2016
Priority date	Nov 10, 2009
Publication date	May 1, 2018
Grant date	May 1, 2018

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 valencene or a derivative thereof, 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 valencene through a recombinantly expressed downstream synthesis pathway comprising Farnesyl Diphosphate Synthase and Valencene Synthase; and
culturing the E. coli to produce the valencene or derivative thereof, wherein the accumulation of indole in the culture is controlled to below 100 mg/L to thereby increase production of valencene or derivative; and
incorporating the valencene or derivative into a product.

2. The method of claim 1 , wherein the product is a food product, food additive, beverage, chewing gum, candy, oral care product.

3. The method of claim 1 , wherein the product is a fragrance product, a cosmetic, a cleaning product, or a soap.

4. The method of claim 1 , wherein the product is an insecticide, pesticide, or pest control agent.

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

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

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

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

9. The method of claim 1 , wherein the valencene or derivative is produced at 10 mg/L or more.

10. The method of claim 1 , wherein the valencene or derivative is produced at 100 mg/L or more.

11. 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.

12. The method of claim 11 , wherein the E. coli has a heterologous dxs, idi, ispDF operon.

13. The method of claim 1 , wherein the E. coli further expresses a P450 enzyme.

14. The method of claim 1 , wherein the E. coli produces the valencene derivative nootkatone.

15. A method for making valencene or a derivative thereof, 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 valencene or derivative through a recombinantly expressed downstream synthesis pathway comprising Farnesyl Diphosphate Synthase and Valencene Synthase; and culturing the E. coli to produce the valencene or derivative, wherein the accumulation of indole in the culture is controlled to below 100 mg/L to thereby increase terpenoid production; and recovering the valencene or derivative.

16. The method of claim 15 , wherein accumulation of indole in the culture is controlled by balancing the upstream MEP pathway with the downstream terpenoid synthesis pathway.

17. The method of claim 15 , further comprising, measuring the amount or concentration of indole continuously or intermittently.

18. The method of claim 15 , wherein accumulation of indole in the culture is maintained to below 50 mg/L.

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

20. The method of claim 15 , wherein the valencene or derivative is produced at 10 mg/L or more.

21. The method of claim 15 , wherein the valencene or derivative is produced at 100 mg/L or more.

22. The method of claim 15 , wherein the E. coli has additional copies of one or more of the dxs, idi, ispD and ispF genes of the MEP pathway.

23. The method of claim 15 , wherein the E. coli has a heterologous dxs, idi, ispDF operon.

24. The method of claim 15 , wherein the E. coli further expresses a P450 enzyme.

25. The method of claim 15 , wherein the E. coli produces the valencene derivative nootkatone.

Assignees

• Massachusetts Inst Technology
• Nat Univ Singapore

Inventors

• Ajikumar Parayil K
• Stephanopoulos Gregory
• Too Heng Phon

Classifications

• C12P7/42

  Hydroxy-carboxylic acids · CPC title

• 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

• C12P17/02

  Oxygen as only ring hetero atoms · CPC title

• C12Q1/689

  for bacteria · CPC title

• C12P5/007 Primary

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