Production of carotenoids in oleaginous yeast and fungi

• US 8,691,555 B2
• Filed: 09/28/2007
• Issued: 04/08/2014
• Est. Priority Date: 09/28/2006
• Status: Active Grant

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Inventors: Bailey, Richard B., Madden, Kevin T., Trueheart, Joshua, Doten, Reed, Mayorga, Maria, Dunn, Joshua Griffin, Dueppen, Dan
• Primary Examiner(s): Joike, Michele K
• Application Number:
  US12/443,362
• Publication Number:
  US 20110039299A1
• Time in Patent Office: 2,384 Days
• Field of Search: None
• US Class Current: 435/254.2
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12P 23/00 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

Abstract

The present invention provides systems for producing engineered oleaginous yeast or fungi that express carotenoids.

Claims

1. An engineered Y. lipolytica strain that produces at least one retinolic compound selected from the group consisting of retinol, retinal, retinoic acid, retinyl palmitate and combinations thereof, the strain comprising at least one retinologenic modification selected from the group consisting of increased expression or activity of a beta-carotene 15,15′

-monooxygenase polypeptide, increased expression or activity of a retinol dehydrogenase polypeptide, and combinations thereof;

wherein the engineered strain can accumulate lipid to at least about 20% of its dry cell weight; and

wherein as a result of genetic engineering, the engineered strain produces the at least one retinolic compound to a level at least about 1% of its dry cell weight.

2. The engineered Y. lipolytica strain of claim 1, wherein the strain does not naturally produce the at least one retinolic compound.

3. The engineered Y. lipolytica strain of claim 1, further comprising at least one oleaginic modification.

4. The engineered Y. lipolytica strain of claim 3, wherein the at least one oleaginic modification increases or decreases expression or activity of at least one oleaginic polypeptide, selected from the group consisting of acetyl-CoA carboxylase polypeptide, pyruvate decarboxylase polypeptide, isocitrate dehydrogenase polypeptide, ATP-citrate lyase polypeptide, malic enzyme polypeptide, AMP deaminase polypeptide, malate dehydrogenase polypeptide, glucose-6-phosphate dehydrogenase polypeptide, 6-phosphogluconate dehydrogenase polypeptide, fructose 1,6 bisphosphatase polypeptide, NADH kinase polypeptide, transhydrogenase polypeptide, acyl-CoA:

diacylglycerol acyltransferase polypeptide, phospholipid;

diacylglycerol acyltransferase polypeptide, acyl-CoA;

cholesterol acyltransferase polypeptide, triglyceride lipase polypeptide, acyl-coenzyme A oxidase polypeptide and combinations thereof.

5. The engineered Y. lipolytica strain of claim 3, wherein the at least one oleaginic modification increases or decreases expression or activity of at least one oleaginic polypeptide selected from the group consisting of a polypeptide in any one of Tables 1 through 6 or 31-47.

6. The engineered Y. lipolytica strain of claim 1, wherein the at least one retinologenic modification confers to the engineered strain the ability to produce the at least one retinolic compound to a level at least about 1% of its dry cell weight.

7. The engineered Y. lipolytica strain of claim 1, wherein the at least one retinologenic modification confers to the engineered strain the ability to produce at least one retinolic compound which a non-engineered strain does not naturally produce.

8. The engineered Y. lipolytica strain of claim 1, wherein the at least one retinologenic modification increases expression or activity of beta-carotene 15,15′

-monoxygenase or beta carotene retinol dehydrogenase.

9. The engineered Y. lipolytica strain of claim 1, wherein the beta-carotene 15,15′

-monooxygenase polypeptide is selected from the group consisting of a polypeptide in Table 67.

10. The engineered Y. lipolytica strain of claim 1, wherein the at least one retinologenic modification further comprises decreasing the expression or activity of one or more components of the SAGA histone acetyltransferase complex.

11. The engineered Y. lipolytica strain of claim 10, wherein the at least one retinologenic modification comprises disruption of the endogenous SPT8 gene.

12. The engineered Y. lipolytica strain of claim 10 wherein the one or more components of the SAGA histone acetyltransferase complex are selected from the polypeptides listed in Table 69.

13. The engineered Y. lipolytica strain of claim 1, wherein the retinologenic modification further comprises altering the expression or activity of one or more carotenoid biosynthesis polypeptides.

14. The engineered Y. lipolytica strain of claim 13, wherein the one or more carotenoid biosynthesis polypeptides are selected from the group consisting of phytoene synthase, phytoene dehydrogenase, lycopene cyclase, carotene ketolase, carotene hydroxylase, astaxanthin synthase, carotenoid epsilon hydroxylase, lycopene cyclase, carotenoid glucosyltransferase, acyl CoA:

diacyglycerol acyltransferase, geranylgeranylpyrophosphate synthase, and truncated HMG-CoA reductase.

15. The engineered Y. lipolytica strain of claim 13, wherein the one or more carotenoid biosynthesis polypeptides are selected from the sequences provided in Tables 17a-25.

16. A method of producing a retinolic compound, the method comprising steps of:

a. cultivating the fungus of claim 1 under conditions that allow production of the retinolic compound;

b. and isolating the produced retinolic compound.

17. The engineered Y. lipolytica strain of claim 1, wherein the at least one retinologenic modification comprises introduction of a gene encoding a heterologous beta-carotene 15,15′

-monooxygenase polypeptide, a heterologous retinol dehydrogenase polypeptide, or combinations thereof.

18. The engineered Y. lipolytica strain of claim 1, wherein the retinol dehydrogenase polypeptide is selected from the group consisting of a polypeptide in Table 68.

19. An engineered Y. lipolytica strain that produces at least one retinolic compound selected from the group consisting of retinol, retinal, retinoic acid, retinyl palmitate and combinations thereof, the strain comprising at least one genetic modification selected from the group consisting of:

increased expression or activity of a beta-carotene 15,15′

-monooxygenase polypeptide;

increased expression or activity of a retinol dehydrogenase polypeptide;

increased expression or activity of phytoene synthase;

increased expression or activity of lycopene cyclase;

increased expression or activity of phytoene dehydrogenease;

increased expression or activity of geranylgeranylpyrophosphate synthase;

increased expression or activity of truncated HMG-CoA reductase; and

combinations thereof;

wherein the engineered strain can accumulate lipid to at least about 20% of its dry cell weight; and

wherein as a result of genetic engineering, the engineered strain produces the at least one retinolic compound to a level at least about 1% of its dry cell weight.

20. The engineered Y. lipolytica strain of claim 19, wherein the genetic modification comprises introduction of one or more heterologous genes encoding metabolic enzymes selected from the group consisting of:

beta-carotene 15,15′

-monooxygenase, phytoene synthase, lycopene cyclase, phytoene dehydrogenease, geranylgeranylpyrophosphate synthase, truncated HMG-CoA reductase, and combinations thereof.