Compositions and methods for producing lipids and other biomaterials from grain ethanol stillage and stillage derivatives
Lipogenic yeasts bioengineered to overexpress genes for lipid production, and methods of use thereof. The yeasts are modified to express, constitutively express, or overexpress an acetyl-CoA carboxylase, an alpha-amylase, an ATP citrate lyase, a diacylglycerol acyltransferase, a fatty acid synthase, a glycerol kinase, a 6-phosphogluconate dehydrogenase, a glycerol-3-phosphate dehydrogenase, a malic enzyme, a fatty acyl-CoA reductase, a delta-9 acyl-CoA desaturase, a glycerol-3-phosphate acyltransferase, a lysophosphatidate acyltransferase, a glucose-6-phosphate dehydrogenase, a beta-glucosidase, a hexose transporter, a glycerol transporter, a glycoside hydrolase enzyme, an auxiliary activity family 9 enzyme, or combinations thereof. The yeasts in some cases are also modified to reduce or ablate activity of certain proteins. The methods include cultivating the yeast to convert low value soluble organic stillage byproducts into lipids suitable for biodiesel production and other higher value uses.
1. A recombinant yeast comprising one or more recombinant genes, wherein the one or more recombinant genes are configured to express:
a diacylglycerol acyltransferase;
a malic enzyme; and
a glycerol-3-phosphate acyltransferase, wherein the glycerol-3-phosphate acyltransferase comprises a sequence at least about 90% identical to SEQ ID NO:40.
2. The recombinant yeast of claim 1 , wherein the yeast is a recombinant lipogenic yeast.
3. The recombinant yeast of claim 1 , wherein the yeast is a recombinant Lipomyces starkeyi.
4. The recombinant yeast of claim 1 , wherein the diacylglycerol acyltransferase comprises a sequence at least about 90% identical to SEQ ID NO:14.
5. The recombinant yeast of claim 4 , wherein the one or more recombinant genes are further configured to express a second diacylglycerol acyltransferase, wherein the second diacylglycerol acyltransferase comprises a sequence at least about 90% identical to SEQ ID NO:58.
6. The recombinant yeast of claim 1 , wherein the malic enzyme comprises a sequence at least about 90% identical to SEQ ID NO:34.
7. The recombinant yeast of claim 1 , wherein:
the diacylglycerol acyltransferase comprises a sequence at least about 90% identical to SEQ ID NO:14;
the one or more recombinant genes are further configured to express a second diacylglycerol acyltransferase, wherein the second diacylglycerol acyltransferase comprises a sequence at least about 90% identical to SEQ ID NO:58; and
the malic enzyme comprises a sequence at least about 90% identical to SEQ ID NO:34.
8. The recombinant yeast of claim 7 , wherein the yeast is a recombinant lipogenic yeast.
9. The recombinant yeast of claim 7 , wherein the yeast is a recombinant Lipomyces starkeyi.
10. The recombinant yeast of claim 1 , wherein the recombinant genes are further configured to express a glycerol-3-phosphate dehydrogenase.
11. A recombinant yeast comprising one or more recombinant genes, wherein the one or more recombinant genes are configured to express:
a diacylglycerol acyltransferase;
a malic enzyme;
a glycerol-3-phosphate acyltransferase; and
a glycerol-3-phosphate dehydrogenase, wherein the glycerol-3 -phosphate dehydrogenase comprises a sequence at least about 90% identical to SEQ ID NO:32.
12. The recombinant yeast of claim 11 , wherein the one or more recombinant genes are further configured to express a second glycerol-3-phosphate dehydrogenase, wherein the second glycerol-3-phosphate dehydrogenase comprises a sequence at least about 90% identical to SEQ ID NO:56.
13. The recombinant yeast of claim 11 , wherein the one or more recombinant genes are further configured to express a glycerol kinase.
14. A recombinant yeast comprising one or more recombinant genes, wherein the one or more recombinant genes are configured to express:
a diacylglycerol acyltransferase;
a malic enzyme;
a glycerol-3-phosphate acyltransferase;
a glycerol-3-phosphate dehydrogenase; and
a glycerol kinase, wherein the glycerol kinase comprises a sequence at least about 90% identical to SEQ ID NO:26.
15. The recombinant yeast of claim 14 , wherein:
the diacylglycerol acyltransferase comprises a sequence at least about 90% identical to SEQ ID NO:14;
the one or more recombinant genes are further configured to express a second diacylglycerol acyltransferase, wherein the second diacylglycerol acyltransferase comprises a sequence at least about 90% identical to SEQ ID NO:58;
the malic enzyme comprises a sequence at least about 90% identical to SEQ ID NO:34;
the glycerol-3-phosphate acyltransferase comprises a sequence at least about 90% identical to SEQ ID NO:40;
the glycerol-3-phosphate dehydrogenase comprises a sequence at least about 90% identical to SEQ ID NO:32; and
the one or more recombinant genes are further configured to express a second glycerol-3- phosphate dehydrogenase, wherein the second glycerol-3-phosphate dehydrogenase comprises a sequence at least about 90% identical to SEQ ID NO:56.
16. The recombinant yeast of claim 15 , wherein the yeast is a recombinant lipogenic yeast.
17. The recombinant yeast of claim 15 , wherein the yeast is a recombinant Lipomyces starkeyi.
18. The recombinant yeast of claim 10 , wherein the one or more recombinant genes are further configured to express an acetyl-CoA carboxylase.
19. The recombinant yeast of claim 18 , wherein the acetyl-CoA carboxylase comprises a sequence at least about 90% identical to SEQ ID NO:2.
20. A recombinant yeast comprising one or more recombinant genes, wherein the one or more recombinant genes are configured to express:
a diacylglycerol acyltransferase;
a malic enzyme;
a glycerol-3-phosphate acyltransferase;
a glycerol-3-phosphate dehydrogenase;
an acetyl-CoA carboxylase, wherein the acetyl-CoA carboxylase comprises a sequence at least about 90% identical to SEQ ID NO:2 and the acetyl-CoA carboxylase comprises a residue other than serine and threonine at a position corresponding to position 1146 of SEQ ID NO:2.
21. The recombinant yeast of claim 18 , wherein:
the diacylglycerol acyltransferase comprises a sequence at least about 90% identical to SEQ ID NO:14;
the one or more recombinant genes are further configured to express a second diacylglycerol acyltransferase, wherein the second diacylglycerol acyltransferase comprises a sequence at least about 90% identical to SEQ ID NO:58;
the malic enzyme comprises a sequence at least about 90% identical to SEQ ID NO:34;
the glycerol-3-phosphate dehydrogenase comprises a sequence at least about 90% identical to SEQ ID NO:32; and
the acetyl-CoA carboxylase comprises a sequence at least about 90% identical to SEQ ID NO:2 and the acetyl-CoA carboxylase comprises a residue other than serine and threonine at a position corresponding to position 1146 of SEQ ID NO:2.
22. The recombinant yeast of claim 21 , wherein the yeast is a recombinant lipogenic yeast.
23. The recombinant yeast of claim 21 , wherein the yeast is a recombinant Lipomyces starkeyi.