Engineered biosynthetic pathways for production of (6E)-8-hydroxygeraniol by fermentation

Patent metadata

Field	Value
Publication number	US-10662415-B2
Application number	US-201916450780-A
Country	US
Kind code	B2
Filing date	Jun 24, 2019
Priority date	Dec 7, 2017
Publication date	May 26, 2020
Grant date	May 26, 2020

Abstract

The present disclosure describes the engineering of microbial cells for fermentative production of (6E)-8-hydroxygeraniol and provides novel engineered microbial cells and cultures, as well as related (6E)-8-hydroxygeraniol production method.

First claim

What is claimed is:

1. An engineered microbial cell, wherein the engineered microbial cell expresses:
(a) a non-native geranyl diphosphate diphosphatase (geraniol synthase); and
(b) a non-native geraniol-8-hydroxylase;
wherein the engineered microbial cell comprises increased activity of one or more upstream (6E)-8-hydroxygeraniol pathway enzyme(s), said increased activity being increased relative to a control cell, the one or more upstream pathway enzymes being selected from the group consisting of ATP-citrate synthase, an acetyl-CoA synthetase, a thiolase, a hydroxymethylglutaryl coenzyme A synthase (HMG-CoA synthase), a hydroxymethylglutaryl coenzyme A reductase (HMG-CoA reductase), a mevalonate kinase, a phosphomevalonate kinase, a diphosphomevalonate decarboxylase, an isopentenyl-diphosphate delta-isomerase, and a geranyl diphosphate synthase; and
wherein, when cultured, the engineered microbial cell produces (6E)-8-hydroxygeraniol at a level greater than 10 mg/L of culture medium.

2. The engineered microbial cell of claim 1 , wherein the one or more upstream (6E)-8-hydroxygeraniol pathway enzyme(s) comprise the isopentenyl-diphosphate delta-isomerase.

3. The engineered microbial cell of claim 1 , wherein the engineered microbial cell comprises reduced activity of one or more enzyme(s) that consume one or more (6E)-8-hydroxygeraniol pathway precursors, said reduced activity being reduced relative to a control cell, the one or more enzyme(s) that consume one or more (6E)-8-hydroxygeraniol pathway precursors comprising a bifunctional (2E,6E)-farnesyl diphosphate synthase/dimethylallyltranstransferase and/or a geranyl pyrophosphate synthase.

4. The engineered microbial cell of claim 1 , wherein the engineered microbial cell additionally expresses a feedback-deregulated HMG-CoA reductase.

5. The engineered microbial cell of claim 1 , wherein the engineered microbial cell over-expresses an ATP-citrate synthase and/or an acetyl-CoA synthetase to increase availability of acetyl-CoA, relative to a control cell, and/or comprises an inactivated or deleted gene selected from the group consisting of Pdc5, Pdc6, and Pdc1 to reduce the rate of acetyl-CoA consumption, relative to a control cell.

6. The engineered microbial cell of claim 1 , wherein the engineered microbial cell comprises a fungal cell.

7. The engineered microbial cell of claim 1 , wherein the non-native geraniol synthase comprises a geraniol synthase having at least 70% amino acid sequence identity with a geraniol synthase from Perilla setoyensis comprising SEQ ID NO:5.

8. The engineered microbial cell of claim 1 , wherein the non-native geraniol synthase comprises a geraniol synthase having at least 70% amino acid sequence identity with a geraniol synthase from Vitis vinifera comprising SEQ ID NO:97.

9. The engineered microbial cell of claim 1 , wherein the non-native geraniol-8-hydroxylase comprises a geraniol-8-hydroxylase having at least 70% amino acid sequence identity with a geraniol-8-hydroxylase from Phaseolus angularis comprising SEQ ID NO:11.

10. The engineered microbial cell of claim 2 , wherein the increased activity of the isopentenyl-diphosphate delta-isomerase is achieved by heterologously expressing an isopentenyl-diphosphate delta-isomerase.

11. The engineered microbial cell of claim 10 , wherein the heterologous isopentenyl-diphosphate delta-isomerase comprises an isopentenyl-diphosphate delta-isomerase having at least 70% amino acid sequence identity with an isopentenyl-diphosphate delta-isomerase from Saccharomyces cerevisiae comprising SEQ ID NO:25.

12. The engineered microbial cell of claim 3 , wherein the one or more enzyme(s) that consume one or more (6E)-8-hydroxygeraniol pathway precursors comprise a bifunctional (2E,6E)-farnesyl diphosphate synthase/dimethylallyltranstransferase.

13. The engineered microbial cell of claim 12 , wherein the bifunctional (2E,6E)-farnesyl diphosphate synthase/dimethylallyltranstransferase dimethylallyltranstransferase comprises amino acid substitution S80F and has at least 70% amino acid sequence identity with a bifunctional (2E,6E)-farnesyl diphosphate synthase/dimethylallyltranstransferase from Escherichia coli comprising SEQ ID NO:85.

14. The engineered microbial cell of claim 1 , wherein, when cultured, the engineered microbial cell produces (6E)-8-hydroxygeraniol at a level greater than 50 mg/L of culture medium.

15. A culture of engineered microbial cells according to claim 1 .

16. The culture of claim 15 , wherein the culture comprises (6E)-8-hydroxygeraniol at a level greater than 10 mg/L of culture medium.

17. A method of culturing engineered microbial cells according to claim 1 , the method comprising culturing the cells under conditions suitable for producing (6E)-8-hydroxygeraniol.

18. The method of claim 17 , wherein the method additionally comprises recovering (6E)-8-hydroxygeraniol from the culture.

19. The engineered microbial cell of claim 1 , wherein the one or more upstream pathway enzymes comprises ATP-citrate synthase.

20. The engineered microbial cell of claim 1 , wherein the one or more upstream pathway enzymes comprises an acetyl-CoA synthetase.

21. The engineered microbial cell of claim 1 , wherein the one or more upstream pathway enzymes comprises a thiolase.

22. The engineered microbial cell of claim 1 , wherein the one or more upstream pathway enzymes comprises a hydroxymethylglutaryl coenzyme A synthase (HMG-CoA synthase).

23. The engineered microbial cell of claim 1 , wherein the one or more upstream pathway enzymes comprises a hydroxymethylglutaryl coenzyme A reductase (HMG-CoA reductase).

24. The engineered microbial cell of claim 1 , wherein the one or more upstream pathway enzymes comprises a mevalonate kinase.

25. The engineered microbial cell of claim 1 , wherein the one or more upstream pathway enzymes comprises a phosphomevalonate kinase.

26. The engineered microbial cell of claim 1 , wherein the one or more upstream pathway enzymes comprises a diphosphomevalonate decarboxylase.

27. The engineered microbial cell of claim 1 , wherein the one or more upstream pathway enzymes comprises a geranyl diphosphate synthase.

Assignees

• Zymergen Inc

Inventors

• Tracewell Cara Ann
• Edgar Steven M
• Tymoshenko Stepan
• Shearer Alexander Glennon

Classifications

• C12N9/90

  Isomerases (5.) · CPC title

• C12Y205/01001

  Dimethylallyltranstransferase (2.5.1.1) · CPC title

• C12N9/16 Primary

  acting on ester bonds (3.1) · CPC title

• C12N9/1085

  transferring alkyl or aryl groups other than methyl groups (2.5) · CPC title

• C12Y503/03002

  Isopentenyl-diphosphate DELTA-isomerase (5.3.3.2) · CPC title