IP Library Granted Patent US 11,046,981
Granted Patent B2
US 11,046,981 · App. 16/212,515 · Granted Jun 29, 2021

Host cell modified to produce lactams

Inventors: Jingwei Zhang (Fullerton, CA); Jay D. Keasling (Berkeley, CA)
Assignee: The Regents of the University of California
C12P13/02C12N9/1048C12N15/52
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Quick Facts
Patent No.
US 11,046,981
App. No.
16/212,515
Granted
Jun 29, 2021
Kind
B2
Abstract

The present invention provides for a genetically modified host cell capable of producing a lactam comprising a 2-pyrrolidone synthase, or an enzymatically active fragment thereof, heterologous to the host cell.

Claims (19)

1. A genetically modified host cell comprising a 2-pyrrolidone synthase heterologous to the host cell, or thereof linked to a saccharide binding protein, wherein the 2-pyrrolidone synthase catalyzes the following reaction:

or reaction (2):

wherein Compound 3 is a “Substrate” and Compound 4 is a “Product” as indicated in Table 2 herein; wherein the genetically modified host cell is a yeast or prokaryotic cell, and comprises 5-aminovaleric acid (5-AVA), 6-aminohexanoic acid (6-AHA), 4-quanidinobutyric acid, valeric acid, 4-methyl-hexanoic acid, 6-aminocaproic acid, 6-quanidinohexanoic acid, or 2-aminobutyric acid.

2. The genetically modified host cell of claim 1 , wherein the 2-pyrrolidone synthase comprises an amino acid sequence having at least 70% identity with SEQ ID NO:1, wherein the amino acid sequence comprises one or more of the following conserved amino acid motifs or sites: acyl-activating enzyme (AAE) consensus motif (residues 137, 140-145, and 147-148), acyl-activating enzyme (AAE) consensus motif (residues 140, 257-258, 279-284, 357, 369, 372, 382, and 458), AMP binding site (residues 140, 180-181, 227, 229-230, 233, 257-258, 279-284, 357, 369, 372, 379-382, and 439), and CoA binding site (residues 180, 229-230, 233, 257, 379-381, 433, and 439).

3. The genetically modified host cell of claim 2 , wherein host cell is capable of synthesizing Compound 1 or uptaking Compound 1 from the environment or culture.

4. The genetically modified host cell of claim 3 , wherein host cell further comprises one or more enzymes of a pathway for synthesizing Compound 1 from a carbon source.

5. The genetically modified host cell of claim 4 , wherein pathway for synthesizing Compound 1 from a carbon source that is native to the host cell.

6. The genetically modified host cell of claim 4 , wherein pathway for synthesizing Compound 1 from a carbon source that is heterologous to the host cell.

7. The genetically modified host cell of claim 1 , wherein host cell lacks betaine-CoA ligase.

8. The genetically modified host cell of claim 2 , wherein n is an integer from 1 to 20.

9. The genetically modified host cell of claim 8 , wherein n is an integer from 1 to 10.

10. The genetically modified host cell of claim 9 , wherein n is an integer from 1 to 7.

11. The genetically modified host cell of claim 10 , wherein n is an integer from 1 to 3.

12. A method of producing a Compound 2 in a genetically modified host cell, comprising: (a) providing the genetically modified host cell of claim 1 , (b) culturing the genetically modified host cell in a medium under a suitable condition such that the culturing results in the genetically modified host cell producing a Compound 2.

13. The method of claim 12 , further comprising introducing one or more nucleic acid(s) into the host cell encoding the enzyme operably linked to a suitable promoter capable of transcription in the host cell, and optionally encoding the one or more enzyme(s) of a pathway for synthesizing Compound 1 from a carbon source, wherein the introducing step is prior to the culturing step.

14. The method of claim 12 , further comprising separating Compound 2 from the host cell and/or the medium, wherein the separating step is subsequent, concurrent or partially concurrent with the culturing step.

15. The genetically modified host cell of claim 1 , wherein the genetically modified host cell comprises 5-aminovaleric acid (5-AVA) or 6-aminohexanoic acid (6-AHA).

16. The genetically modified host cell of claim 1 , wherein the genetically modified host cell is a Yarrowia, Candida, Bebaromyces, Saccharomyces, Schizosaccharomyces or Pichia cell.

17. The genetically modified host cell of claim 1 , wherein the genetically modified host cell is a Escherichia, Bacillus, Salmonella, Klebsiella, Enterobacter, Pseudomonas, Streptomyces, Cynechocystis, Cynechococcus, Sinorhizobium , and Caulobacter cell.

Assignments (2)
CONFIRMATORY LICENSE Recorded Dec 19, 2019
From: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
To: UNITED STATES DEPARTMENT OF ENERGY
Reel/Frame 051332/0853 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jan 2, 2019
From: ZHANG, JINGWEI; KEASLING, JAY D.
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 048000/0744 →
Continuity (3)
Continuation PCTUS2017036168 · Jun 6, 2017
Provisional Application 62346294 · Jun 6, 2016
Related Publication 20190338321A1 · Nov 7, 2019