Co-production of a sesquiterpene and a carotenoid

1. A method for co-production of two or more isoprenoids, the method comprising:

(a) culturing, in a culture medium, a host cell genetically modified with one or more heterologous nucleic acids encoding one or more enzymes in a first biosynthetic pathway to produce a first isoprenoid and with one or more heterologous nucleic acids encoding one or more enzymes in a second biosynthetic pathway to produce a second isoprenoid, which has a molecular weight that is different from the first isoprenoid wherein the first isoprenoid is produced and secreted into the culture medium, and wherein the second isoprenoid is produced and remains associated with the host cell, and wherein the host cell co-produces about 2.5 g/L to about 200 g/L of the first isoprenoid compound and about 1 mg/L to about 4000 mg/L of the second isoprenoid compound;

(b) recovering the first isoprenoid from the culture medium; and

(c) recovering the second isoprenoid from the host cell.

2. The method of claim 1 wherein the host cell is not genetically modified to produce a target compound for recovery other than a compound derived from isopentenyl pyrophosphate (IPP).

3. The method for co-production of isoprenoids of claim 1 , wherein the first isoprenoid and second isoprenoid are produced concurrently during a fermentation run from a single inoculum.

4. The method for co-production of isoprenoids of claim 1 , wherein the first isoprenoid and second isoprenoid are produced sequentially from a single inoculum comprising the host cell, or sequentially using a genetic switch.

5. The method for co-production of isoprenoids of claim 1 , wherein the culturing and recovering comprise:

(a) culturing the single inoculum comprising the host cell to build a population of host cells;

(b) culturing the population of host cells under conditions to produce the first isoprenoid from the population of host cells, wherein the conditions do not activate production of the second isoprenoid;

(c) recovering the first isoprenoid from the population;

(d) after separating the first isoprenoid, culturing the population or a subpopulation of the host cells under conditions to activate production of the second isoprenoid; and

(e) recovering the second isoprenoid.

6. The method for co-production of isoprenoids of claim 1 , wherein the first isoprenoid is predominantly released from the host cell into the culture medium and the second isoprenoid predominantly remains intracellularly.

7. The method for co-production of isoprenoids of claim 1 , wherein the second isoprenoid predominantly remains intracellularly and is recovered together with the host cell mass.

8. The method for co-production of claim 1 , wherein the first isoprenoid is a C5, C10, C15, or C20 isoprenoid, and wherein the second isoprenoid is a C30, C35, C40, or higher carbon isoprenoid.

9. The method for co-production of isoprenoids of claim 1 , wherein the first isoprenoid is a C15 isoprenoid and the second isoprenoid is a C40 isoprenoid.

10. The method for co-production of isoprenoids of claim 1 , wherein the first isoprenoid is farnesene and the second isoprenoid is a carotenoid.

11. The method for co-production of isoprenoids of claim 1 , wherein the second isoprenoid is astaxanthin, xanthophyll, or ketocarotenoid; or the second isoprenoid is one or more of astaxanthin, canthaxanthin, zeaxanthin, β-carotene, lycopene, and lutein.

12. The method for co-production of isoprenoids of claim 1 , wherein the first isoprenoid is a sesquiterpene and the second isoprenoid is one or more carotenoids; wherein the ratio of the one or more carotenoids to the sesquiterpene is between about 0.001 to 2% by weight or between about 0.1 to about 1% by weight.

13. The method for co-production of isoprenoids of claim 1 , wherein the host cell co-produces about 100 g to about 5000 g of the first isoprenoid and about 1 g to about 50 g of the second isoprenoid, each per kilogram of dry cell mass.

14. The method for co-production of isoprenoids of claim 1 , wherein the amount of the first isoprenoid produced during co-production with the second isoprenoid is at least about 90% of the amount of the first isoprenoid produced by a parent host cell, wherein said parent host cell is genetically modified to produce the first isoprenoid but not the second isoprenoid.

15. The method for co-production of isoprenoids of claim 1 , wherein the amount of the first isoprenoid produced during co-production with the second isoprenoid is at least about 95% of the amount of the first isoprenoid produced under the same culture conditions but without co-production of the second isoprenoid.

16. The method for co-production of isoprenoids of claim 1 , wherein a cell density of the host cell during co-production of the first isoprenoid and the second isoprenoid is at least about 90% or 95% of a cell density of a parent host cell which is genetically modified to produce the first isoprenoid but not the second isoprenoid.

17. The method for co-production of isoprenoids of claim 1 , wherein the first isoprenoid is a sesquiterpene and the second isoprenoid is a carotenoid, wherein a carbon flux towards the production of carotenoid is reduced compared to a carbon flux towards the production of sesquiterpene.

18. The method for co-production of isoprenoids of claim 1 , wherein the second isoprenoid is lycopene, and the host cell comprises:

(a) a heterologous nucleic acid encoding a phytoene synthase; and

(b) a heterologous nucleic acid encoding a phytoene desaturase.

19. The method for co-production of isoprenoids of claim 1 , wherein the second isoprenoid is β-carotene, and the host cell comprises:

(a) at least one of:

(i) a heterologous nucleic acid encoding a phytoene synthase;

(ii) a heterologous nucleic acid encoding a lycopene cyclase; or

(iii) a heterologous nucleic acid encoding a bifunctional enzyme having phytoene synthase and lycopene cyclase activities; and

(b) a heterologous nucleic acid encoding a phytoene desaturase.

20. The method for co-production of isoprenoids of claim 1 , wherein the second isoprenoid is cantaxanthin, and the host cell comprises:

(a) at least one of:

(i) a heterologous nucleic acid encoding a phytoene synthase;

(ii) a heterologous nucleic acid encoding a lycopene cyclase; or

(iii) a heterologous nucleic acid encoding a bifunctional enzyme having phytoene synthase and lycopene cyclase activities;

(b) a heterologous nucleic acid encoding a phytoene desaturase; and

(c) a heterologous nucleic acid encoding a β-carotene ketolase.

21. The method for co-production of isoprenoids of claim 1 , wherein the second isoprenoid is zeaxanthin, and the host cell comprises:

(a) at least one of:

(i) a heterologous nucleic acid encoding a phytoene synthase;

(ii) a heterologous nucleic acid encoding a lycopene cyclase; or

(iii) a heterologous nucleic acid encoding a bifunctional enzyme having phytoene synthase and lycopene cyclase activities;

(b) a heterologous nucleic acid encoding a phytoene desaturase; and

(c) a heterologous nucleic acid encoding β-carotene hydroxylase.

22. The method for co-production of claim 1 , wherein the second isoprenoid is astaxanthin, and the host cell comprises:

(a) at least one of:

(i) a heterologous nucleic acid encoding a phytoene synthase;

(ii) a heterologous nucleic acid encoding a lycopene cyclase; or

(iii) a heterologous nucleic acid encoding a bifunctional enzyme having phytoene synthase and lycopene cyclase activities;

(b) a heterologous nucleic acid encoding a phytoene desaturase;

(c) at least one of:

(i) a heterologous nucleic acid encoding a β-carotene ketolase;

(ii) a heterologous nucleic acid encoding a β-carotene hydroxylase;

(iii) a heterologous nucleic acid encoding a cytochrome p450 hydroxylase and ketolase capable of converting β-carotene to echinenone and subsequently to β-cryptoxanthin and to astaxanthin; or

(iv) a heterologous nucleic acid encoding a cytochrome p450 reductase which interacts with the cytochrome p450 hydroxylase and ketolase.

23. The method for co-production of isoprenoids of claim 1 , wherein the second isoprenoid is lutein, and the host cell comprises:

(a) a heterologous nucleic acid encoding a lycopene cyclase;

(b) a heterologous nucleic acid encoding a δ-carotene β-cyclase;

(c) a heterologous nucleic acid encoding β-ring hydroxylase; and

(d) a heterologous nucleic acid encoding a carotene ε-monooxygenase.

24. The method for co-production of isoprenoids of claim 1 , wherein the host cell further comprises a heterologous nucleic acid encoding a polyprenyl synthase for producing a polyprenyl diphosphate.

25. The method for co-production of isoprenoids of claim 24 , wherein the host cell further comprises a heterologous nucleic acid encoding a farnesyl diphosphate (FPP) synthase, or a heterologous nucleic acid encoding a geranyl pyrophosphate (GGPP) synthase, or both.

26. The method for co-production of isoprenoids of claim 24 , wherein the host cell further comprises a heterologous nucleic acid encoding a farnesyl diphosphate (FPP) synthase and comprises an endogenous nucleic acid encoding a geranyl pyrophosphate (GGPP) synthase but does not comprise a heterologous nucleic acid encoding a GGPP synthase.

27. The method for co-production of isoprenoids of claim 1 , wherein the host cell further comprises one or more heterologous nucleic acids encoding one or more or all of the enzymes of the mevalonte pathway.

28. The method for co-production of isoprenoids of claim 1 , wherein the host cell is a microbial cell.

29. The method for co-production of isoprenoids of claim 28 , wherein the microbial host cell is a yeast.

30. The method for co-production of isoprenoids of claim 1 , wherein the host cell is Saccharomyces cerevisiae.

31. The method for co-production of isoprenoids of claim 1 , the method further comprising extracting and purifying the carotenoids.

32. A composition comprising purified carotenoids produced by the method of claim 1 .

33. An animal feed comprising the microbial cell and the second isoprenoid produced from the method of claim 1 .

34. A host cell comprising:

(a) one or more heterologous nucleic acids encoding enzymes of a mevalonate pathway;

(b) a heterologous nucleic acid encoding a biosynthetic pathway enzyme for production of sesquiterpene; and

(c) a heterologous nucleic acid encoding a biosynthetic pathway enzyme for production of a carotenoid.

35. The host cell of claim 34 wherein the biosynthetic pathway enzyme for production of sesquiterpene is a synthase or synthetase.

36. The host cell of claim 34 that comprises one or more heterologous nucleic acids encoding enzymes of a pathway for production of the carotenoid.

37. A host cell comprising:

(a) a heterologous nucleic acid encoding a phytoene synthase;

(b) a heterologous nucleic acid encoding a phytoene desaturase;

or comprising:

(a) at least one of:

(i) a heterologous nucleic acid encoding a phytoene synthase;

(ii) a heterologous nucleic acid encoding a lycopene cyclase; or

(iii) a heterologous nucleic acid encoding a bifunctional enzyme having phytoene synthase and lycopene cyclase activities; and

(b) a heterologous nucleic acid encoding a phytoene desaturase;

or comprising:

(a) at least one of:

(i) a heterologous nucleic acid encoding a phytoene synthase;

(ii) a heterologous nucleic acid encoding a lycopene cyclase; or

(iii) a heterologous nucleic acid encoding a bifunctional enzyme having phytoene synthase and lycopene cyclase activities;

(b) a heterologous nucleic acid encoding a phytoene desaturase; and

(c) a heterologous nucleic acid encoding a β-carotene ketolase;

or comprising:

(a) at least one of:

(i) a heterologous nucleic acid encoding a phytoene synthase;

(ii) a heterologous nucleic acid encoding a lycopene cyclase; or

(iii) a heterologous nucleic acid encoding a bifunctional enzyme having phytoene synthase and lycopene cyclase activities;

(b) a heterologous nucleic acid encoding a phytoene desaturase; and

(c) a heterologous nucleic acid encoding β-carotene hydroxylase;

or comprising:

(a) at least one of:

(i) a heterologous nucleic acid encoding a phytoene synthase;

(ii) a heterologous nucleic acid encoding a lycopene cyclase; or

(iii) a heterologous nucleic acid encoding a bifunctional enzyme having phytoene synthase and lycopene cyclase activities;

(b) a heterologous nucleic acid encoding a phytoene desaturase;

(c) at least one of:

(i) a heterologous nucleic acid encoding a β-carotene ketolase;

(ii) a heterologous nucleic acid encoding a β-carotene hydroxylase;

(iii) a heterologous nucleic acid encoding a cytochrome p450 hydroxylase and ketolase capable of converting β-carotene to echinenone and subsequently to β-cryptoxanthin and to astaxanthin; or

(iv) a heterologous nucleic acid encoding a cytochrome p450 reductase

which interacts with the cytochrome p450 hydroxylase and ketolase;

or comprising:

(a) a heterologous nucleic acid encoding a lycopene cyclase;

(b) a heterologous nucleic acid encoding a δ-carotene β-cyclase;

(c) a heterologous nucleic acid encoding β-ring hydroxylase; and

(d) a heterologous nucleic acid encoding a carotene ε-monooxygenase;

the host cell optionally further comprising:

a heterologous nucleic acid encoding a polyprenyl synthase for producing a polyprenyl diphosphate; or

a heterologous nucleic acid encoding a FPP synthase, or a heterologous nucleic acid encoding a GGPP synthase, or both; or

one or more heterologous nucleic acids encoding one or more or all of the enzymes of the mevalonte pathway.

38. The method of claim 1 , wherein at least 70%, 75%, 80%, 85%, 90%, or 95% of the first isoprenoid produced is found in the culture medium.

39. The method of claim 1 , wherein at least 70%, 75%, 80%, 85%, 90%, or 95% of the second isoprenoid produced is associated with the host cell.

40. A method for co-production of two or more isoprenoids, the method comprising:

(a) culturing, in a culture medium, a host cell genetically modified with one or more heterologous nucleic acids encoding one or more enzymes in a first biosynthetic pathway to produce a first isoprenoid, and with one or more heterologous nucleic acids encoding one or more enzymes in a second biosynthetic pathway to produce a second isoprenoid, which has a molecular weight that is different from the first isoprenoid wherein the first isoprenoid is produced and secreted into the culture medium, and wherein the second isoprenoid is produced and remains associated with the host cell, wherein the first isoprenoid is a sesquiterpene and the second isoprenoid is one or more carotenoids; wherein the ratio of the one or more carotenoids to the sesquiterpene is between about 0.001 to 2% by weight or between about 0.1 to about 1% by weight;

(b) recovering the first isoprenoid from the culture medium; and

(c) recovering the second isoprenoid from the host cell.