IP Library › Granted Patent US 12,188,073
Granted Patent B2
US 12,188,073 · App. 17/845,842 · Granted Jan 7, 2025

Yeast cells and methods for producing fatty alcohols

Inventors: Leopold D'Espaux (San Francisco, CA); Jay D. Keasling (Berkeley, CA)
Assignee: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
C12P7/04C12N9/0008C12N9/1029C12N9/93C12N15/52C12Y101/01001C12Y102/01084C12Y203/0102C12Y203/01085C12Y604/01002
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Quick Facts
Patent No.
US 12,188,073
App. No.
17/845,842
Granted
Jan 7, 2025
Kind
B2
Abstract

The present invention provides for a genetically modified yeast cell comprising at least six or more of the following modifications: increased expression of Mus musculus fatty acid reductase, acetyl-CoA carboxylase, fatty acid synthase 1, fatty acid synthase 2, a mutant of the bottleneck enzyme encoded by ACC1 insensitive to post-transcriptional and post-translational repression, and/or a desaturase encoded by OLE1, and reduced expression of DGA1, HFD1, ADH6, and/or GDH1. The present invention provides a method for constructing the genetically modified yeast cell, and a method for producing a fatty alcohol from the genetically modified yeast cell.

Claims (18)

1. A genetically modified yeast cell comprising the following modifications: (a) a first nucleic acid encoding a Mus musculus fatty acid reductase (FAR)(MmFAR1) or Tyto alba FAR1 (TaFAR1) operatively linked to a promoter, and (b) a second nucleic acid encoding acetyl-CoA carboxylase (ACC1) operatively linked to a promoter and (c) (i) a reduced expression of or knocked out for a homolog of fatty acid aldehyde (HFD1), (ii) a reduced expression of or knocked out for an alcohol dehydrogenase (ADH6), (iii) a nucleic acid encoding a mutant of a bottleneck enzyme encoded by ACC1 insensitive to post-transcriptional and post-translational repression (ACC1**) operatively linked to a promoter, (iv) a reduced expression of or knocked out for a glutamate dehydrogenase (GDH1), or (v) a nucleic acid encoding a fatty acid-desaturase (OLE1) operatively linked to a promoter; wherein the MmFAR1 or TaFAR1, and ACC1 have an increased expression compared to expression of each corresponding MmFAR1 or TaFAR1, and ACC1 in an unmodified yeast cell, and the genetically modified yeast cell, when grown in 1% yeast extract, 2% peptone, and 2% dextrose (YPD) medium, is capable of producing at least 1.0 g of fatty alcohol per liter of medium.

2. A method of constructing a genetically modified yeast cell comprising: (a) introducing a first nucleic acid encoding Mus musculus fatty acid reductase (FAR) MmFAR1 or Tyto alba FAR1 (TaFAR1) operatively linked to a promoter capable of expressing the MmFAR1 or TaFAR1 gene product in the yeast cell; (b) introducing a second nucleic acid encoding acetyl-CoA carboxylase (ACC1) operatively linked to a promoter capable of expressing the ACC1 gene product in the yeast cell, or replacing the native promoter of ACC1 with a promoter with a higher transcription activity; and (c)(i) reducing expression of or knocking out for a homolog of fatty acid aldehyde (HFD1), (ii) reducing expression of or knocking out for an alcohol dehydrogenase (ADH6), (iii) introducing a nucleic acid encoding a mutant of a bottleneck enzyme encoded by ACC1 insensitive to post-transcriptional and post-translational repression (ACC1**) operatively linked to a promoter, (iv) reducing expression of or knocking out for a glutamate dehydrogenase (GDH1), or (v) introducing a nucleic acid encoding a fatty acid-desaturase (OLE1) operatively linked to a promoter;

wherein the higher transcription activity is higher than each corresponding MmFAR1 or TaFAR1, and ACC1 in an unmodified yeast cell, and the genetically modified yeast cell, when grown in 1% yeast extract, 2% peptone, and 2% dextrose (YPD) medium, is capable of producing at least 1.0 g of fatty alcohol per liter of medium.

3. A method of producing a fatty alcohol from a genetically modified yeast cell comprising: (a) providing a genetically modified yeast cell of claim 1 , and (b) growing or culturing the genetically modified yeast cell in a medium such that the genetically modified yeast cell produces at least 1.0 g of one or more fatty alcohols, or a mixture thereof, per liter of the medium.

4. The genetically modified yeast cell of claim 1 , comprising the following modifications: a third nucleic acid encoding fatty acid synthase 1 (FAS1) operatively linked to a promoter, and a fourth nucleic acid encoding fatty acid synthase 2 (FAS2) operatively linked to a promoter; wherein the FAS1 and FAS2 have an increased expression compared to expression of each corresponding FAS1 and FAS2 in an unmodified yeast cell.

5. The genetically modified yeast cell of claim 4 , comprising a reduced expression of or knocked out for a diacylglycerol O-acyltransferase (DGA1).

6. The genetically modified yeast cell of claim 4 , comprising a reduced expression of or knocked out for a homolog of fatty acid aldehyde (HFD1).

7. The genetically modified yeast cell of claim 4 , comprising a reduced expression of or knocked out for an alcohol dehydrogenase (ADH6).

8. The genetically modified yeast cell of claim 4 , comprising an increased expression of a mutant of a bottleneck enzyme encoded by ACC1 insensitive to post-transcriptional and post-translational repression (ACC1**).

9. The genetically modified yeast cell of claim 4 , comprising a reduced expression of or knocked out for a glutamate dehydrogenase (GDH1).

10. The genetically modified yeast cell of claim 4 , comprising and an increased expression of a fatty acid-desaturase (OLE1).

11. The genetically modified yeast cell of claim 10 , comprising a reduced expression of or knocked out for a diacylglycerol O-acyltransferase (DGA1), a reduced expression of or knocked out for a homolog of fatty acid aldehyde (HFD1), a reduced expression of or knocked out for an alcohol dehydrogenase (ADH6), an increased expression of a mutant of a bottleneck enzyme encoded by ACC1 insensitive to post-transcriptional and post-translational repression (ACC1**), and a reduced expression of or knocked out for a glutamate dehydrogenase (GDH1).

12. The genetically modified yeast cell of claim 1 ; wherein the first nucleic acid encoding MmFAR1 or TaFAR1 is operatively linked to a GAL1 or TEF1 promoter.

13. The genetically modified yeast cell of claim 1 , wherein the genetically modified yeast cell is capable of producing at least 5.0 g of fatty alcohol per liter of medium.

14. The genetically modified yeast cell of claim 13 , wherein the genetically modified yeast cell is capable of producing at least 10.0 g of fatty alcohol per liter of medium.

15. The genetically modified yeast cell of claim 1 , wherein the genetically modified yeast cell is a Saccharomyces cell.

16. The genetically modified yeast cell of claim 15 , wherein the Saccharomyces cell is a Saccharomyces cerevisiae cell.

17. The method of claim 3 , wherein the growing or culturing step is growing or culturing in a continuous culture.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 2, 2022
From: D'ESPAUX, LEOPOLD; KEASLING, JAY D
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 060979/0706 →
Continuity (4)
Continuation 16786558 · Feb 10, 2020
Continuation 15979144 · May 14, 2018
Provisional Application 62505725 · May 12, 2017
Related Publication 20230039548A1 · Feb 9, 2023
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