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