Engineered microbes and methods for microbial oil overproduction from cellulosic materials

Patent metadata

Field	Value
Publication number	US-9096876-B2
Application number	US-201313923607-A
Country	US
Kind code	B2
Filing date	Jun 21, 2013
Priority date	Jun 22, 2012
Publication date	Aug 4, 2015
Grant date	Aug 4, 2015

Abstract

The invention relates to engineering microbial cells for utilization of cellulosic materials as a carbon source, including xylose.

First claim

The invention claimed is:

1. An isolated oleaginous cell comprising a nucleic acid construct that increases expression of:
a xylose reductase (XYL1) gene product and a xylitol dehydrogenase (XYL2) gene product;
wherein the nucleic acid construct comprises an intron and
(a) an expression cassette comprising a nucleic acid sequence encoding the XYL1 and XYL2 gene products under the control of a suitable homologous or heterologous promoter, and/or
(b) a nucleic acid sequence that modulates the level of expression of the XYL1 and XYL2 gene products when inserted into the genome of the cell.

2. The isolated oleaginous cell of claim 1 , further comprising a genetic modification that increases expression of a xylulokinase (XYL3) gene product.

3. The isolated oleaginous cell of claim 1 , further comprising a genetic modification that increases expression of a diacylglycerol acyltransferase (DGA) gene product, an acetyl-coA carboxylase (ACC) gene product, a stearoyl-CoA-desaturase (SCD) gene product, and/or an ATP-citrate lyase (ACL) gene product.

4. The isolated oleaginous cell of claim 1 , wherein the intron is downstream of a transcription initiation site of the nucleic acid sequence encoding one or more of the gene products.

5. The isolated oleaginous cell of claim 4 , wherein the intron is within the nucleic acid sequence encoding one or more of the XYL1 and XYL2 gene products.

6. The isolated oleaginous cell of claim 1 , wherein the nucleic acid construct inhibits or disrupts the natural regulation of a native gene encoding the XYL1 and XYL2 gene products resulting in overexpression of the native gene.

7. The isolated oleaginous cell of claim 1 , wherein the increased expression of the XYL1 and XYL2 gene products confers a beneficial phenotype for the conversion of a carbon source to a fatty acid, fatty acid derivative and/or triacylglycerol (TAG) to the cell.

8. The isolated oleaginous cell of claim 7 , wherein the beneficial phenotype is a modified fatty acid profile, a modified TAG profile, an increased fatty acid and/or triacylglycerol synthesis rate, an increase conversion yield, an increased triacylglycerol accumulation in the cell, and/or an increased triacylglycerol accumulation in a lipid body of the cell.

9. The isolated oleaginous cell of claim 8 , wherein the synthesis rate, yield or accumulation of a fatty acid or a TAG of the cell is at least 2-fold increased as compared to unmodified cells of the same cell type.

10. The isolated oleaginous cell of claim 7 , wherein the cell converts a carbon source to a fatty acid or a TAG at a conversion rate within the range of about 0.025 g/g to about 0.32g/g (g TAG produced/g Glucose consumed).

11. The isolated oleaginous cell of claim 1 , wherein the cell is an oleaginous yeast cell.

12. The isolated oleaginous cell of claim 1 , wherein the cell is a Yarrowia lipolytica cell, a Hansenula polymorpha cell, a Pichia pastoris cell, a Saccharomyces cerevisiae cell, a S. bayanus cell, a S. K. lactis cell, a Waltomyces lipofer cell, a Mortierella alpine cell, a Mortierella isabellina cell, a Hansenula polymorpha cell, a Mucor rouxii cell, a Trichosporon cutaneu cell, a Rhodotorula glutinis cell, a Saccharomyces diastasicus cell, a Schwanniomyces occidentalis cell, a S. cerevisiae cell, a Pichia stipitis cell, or a Schizosaccharomyces pombe cell.

13. A culture, comprising the oleaginous cell of claim 1 .

14. The culture of claim 13 , further comprising a carbon source.

15. The culture of claim 14 , wherein the carbon source comprises a fermentable sugar.

16. A method, comprising
contacting a carbon source with an isolated oleaginous cell of claim 1 , and
incubating the carbon source contacted with the cell under conditions suitable for at least partial conversion of the carbon source into a fatty acid or a triacylglycerol by the cell.

17. The method of claim 16 , wherein the carbon source is a fermentable sugar.

18. The method of claim 17 , wherein the fermentable sugar is a C5 and/or a C6 sugar.

19. A method for increasing production of fatty acid or triacylglycerol by an oleaginous cell, comprising
culturing the oleaginous cell of claim 1 with at least two types of carbon sources,
wherein the first type of carbon source contains or is xylose, and
wherein the second type of carbon source is a carbon source other than xylose,
whereby the production of fatty acid or triacylglycerol by the oleaginous cell is improved relative to culturing the cell or the culture without the second type of carbon source.

20. The method of claim 19 , wherein the second type of carbon source contains or is a C2 carbon source, a C3 carbon source, a CS carbon source other than xylose or a C6 carbon source.

Assignees

• Massachusetts Inst Technology

Inventors

• Stephanopoulos Gregory
• Tai Mitchell

Classifications

• C12Y101/01307

  D-Xylose reductase (1.1.1.307) · CPC title

• Y02E50/13

  Cross-Sectional Technologies · mapped topic

• C12Y101/0101

  L-Xylulose reductase (1.1.1.10) · CPC title

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12P2203/00

  Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source · CPC title