Methods of producing lipid-derived compounds and host cells thereof

Patent metadata

Field	Value
Publication number	US-11560576-B1
Application number	US-202117323195-A
Country	US
Kind code	B1
Filing date	May 18, 2021
Priority date	May 18, 2021
Publication date	Jan 24, 2023
Grant date	Jan 24, 2023

Abstract

The present disclosure relates to genetically engineered host cells and methods of producing a lipid-derived compound by employing such host cells. In particular embodiments, the host cell includes a first mutant gene encoding a cytoplasmic tRNA thiolation protein. Optionally, the host cell can include other mutant genes for decreasing fatty alcohol catabolism, decreasing re-importation of secreted fatty alcohol, or displaying other useful characteristics, as described herein.

First claim

The invention claimed is:

1. An isolated, genetically engineered host cell comprising:
a first mutant gene that is a deletion of a gene encoding a cytoplasmic tRNA thiolation protein; and
one or more expressed nucleic acids encoding 1) a fatty acyl-CoA reductase, or 2) a thioesterase, a carboxylicacid reductase and an aldehyde reductase.

2. The host cell of claim 1 , wherein the cytoplasmic tRNA thiolation protein is cytoplasmic tRNA 2-thiolation protein 2.

3. The host cell of claim 1 wherein the cytoplasmic tRNA thiolation protein comprises a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs: 1, 3-8, 10-16 and 18-20.

4. The host cell of claim 1 , wherein the engineered host cell further comprises a second mutant gene comprising insertion of a nucleic acid encoding an acetyl-CoA carboxylase, thereby providing overexpression of the acetyl-CoA carboxylase.

5. The host cell of claim 4 , wherein the acetyl-CoA carboxylase comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:30.

6. The host cell of claim 1 , wherein the engineered host cell further comprises a second mutant gene comprising deletion of a nucleic acid encoding a lysophospholipid acyltransferase, a fatty alcohol oxidase, an aldehyde dehydrogenase, an isocitrate dehydrogenase, or a pyruvate decarboxylase.

7. The host cell of claim 6 , wherein the lysophospholipid acyltransferase comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 31; the fatty alcohol oxidase comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 32; the aldehyde dehydrogenase comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 36; the isocitrate dehydrogenase comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 34; or the pyruvate decarboxylase comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 35.

8. A method of producing a fatty alcohol, the method comprising:
incubating an isolated, genetically engineered host cell in a culture; and
isolating one or more fatty alcohols from the culture,
wherein the genetically engineered host cell comprises a first mutant gene that is a deletion of a gene encoding a cytoplasmic tRNA thiolation protein, and further one or more expressed nucleic acids encoding 1) a fatty acyl-CoA reductase, or 2) a thioesterase, a carboxylic acid reductase and an aldehyde reductase.

9. The method of claim 8 , said incubating comprises 0 to 2 μM of zinc, 0 to 20 μM of cobalt, 0 to 20 μM of copper and/or 0.5 to 5 g/L ammonium in the culture.

10. The method of claim 8 , wherein the host cell provides an increased amount of the one or more fatty alcohols, as compared to a corresponding control cell lacking deletion of the first mutant gene.

11. The method of claim 8 , wherein the cytoplasmic tRNA thiolation protein is cytoplasmic tRNA 2-thiolation protein 2.

12. The method of claim 8 , wherein the cytoplasmic tRNA thiolation protein comprises a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs: 1, 3-8, 10-16 and 18-20.

13. The method of claim 8 , wherein the host cell further comprises a second mutant gene comprising insertion of a nucleic acid encoding an acetyl-CoA carboxylase or a fatty alcohol reductase, thereby providing overexpression of the acetyl-CoA carboxylase or overexpression of the fatty alcohol reductase.

14. The method of claim 8 , wherein the host cell further comprises a second mutant gene comprising deletion of a nucleic acid encoding a lysophospholipid acyltransferase, a fatty alcohol oxidase, an aldehyde dehydrogenase, an isocitrate dehydrogenase, or a pyruvate decarboxylase.

15. The method of claim 8 , wherein the fatty alcohol comprises a structure of R′OH, in which R′ is a C4-32 aliphatic that is optionally substituted.

Assignees

• Nat Tech & Eng Solutions Sandia Llc

Inventors

• Coradetti Samuel
• Gladden John Michael
• Liu Di
• Geiselman Gina Marie

Classifications

• C12N9/93 Primary

  Ligases (6) · CPC title

• C07K14/39

  from yeasts · CPC title

• C12P7/6436

  Fatty acid esters · CPC title

• C12Y203/01023

  1-Acylglycerophosphocholine O-acyltransferase (2.3.1.23), i.e. lysophosphatidylcholine acyltransferase or LPCAT · CPC title

• C12Y102/01003

  Aldehyde dehydrogenase (NAD+) (1.2.1.3) · CPC title