Generation of acyl amino acids

• US 9,970,036 B2
• Filed: 03/14/2014
• Issued: 05/15/2018
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Modular Genetics, Inc.
• Original Assignee: Modular Genetics, Inc.
• Inventors: Jarrell, Kevin A., Reznik, Gabriel O., Vishwanath, Prashanth, Pynn, Michelle
• Primary Examiner(s): Saidha, Tekchand
• Application Number:
  US14/776,805
• Publication Number:
  US 20160076065A1
• Time in Patent Office: 1,523 Days
• Field of Search: 435183, 43525231
• CPC Class Codes: A01N 37/36 containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids
• CPC Class Codes: C07C 235/06 having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
• CPC Class Codes: C12N 9/80 acting on amide bonds in linear amides (3.5.1)
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 13/02 Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes peptides C12P21/00 or C07K
• CPC Class Codes: C12Y 603/02 Acid—amino-acid ligases (peptide synthases)(6.3.2)

Abstract

Engineered polypeptides useful in synthesizing acyl amino acids are provided. Also provided are methods of making acyl amino acids using engineered polypeptides.

Claims

1. A Bacillus subtilis cell engineered to express at least one engineered peptide synthetase lacking a thioesterase domain and lacking a reductase domain, which peptide synthetase synthesizes an acyl amino acid.

2. The Bacillus subtilis cell of claim 1, wherein the engineered peptide synthetase includes an adenylation (A) domain, a thiolation (T) domain, and a condensation (C) domain.

3. The Bacillus subtilis cell of claim 1, wherein the engineered peptide synthetase contains only a single peptide synthetase domain.

4. The Bacillus subtilis cell of claim 1, wherein the engineered peptide synthetase is or comprises a peptide synthetase domain found in a peptide synthetase complex as a first domain in a peptide synthetase that synthesizes a lipopeptide.

5. The Bacillus subtilis cell of claim 1, wherein acyl amino acid composition includes, as a prominent component, an acyl amino acid whose amino acid moiety is from an amino acid selected from the group consisting of glycine or glutamate and whose acyl moiety is from a fatty acid selected from the group consisting of myristic acid and lauric acid.

6. The Bacillus subtilis cell of claim 4, wherein the peptide synthetase domain is or comprises Module 1 of a surfactin (srf) synthetase.

7. The Bacillus subtilis cell of claim 6, wherein the srf synthetase is srfAA.

8. The Bacillus subtilis cell of claim 4, wherein the peptide synthetase domain is or comprises Module 1 of a mycosubtilisin synthetase.

9. The Bacillus subtilis cell of claim 8, wherein the mycosubtilisin synthetase is or comprises MycA.

10. The Bacillus subtilis cell of claim 4, wherein the peptide synthetase domain is or comprises Module 1 of a daptomycin synthetase.

11. The Bacillus subtilis cell of claim 10, wherein the daptomycin synthetase is or comprises dptA1.

12. The Bacillus subtilis cell engineered of claim 1, wherein the engineered peptide synthetase acylates an amino acid selected from the group consisting of 2,4-diaminobutyric acid, (2S)-2,3-diaminobutyric acid, 2,3-diaminopropionic acid, ornithine and lysine.

13. A method of making an acyl amino acid composition by culturing the engineered cell of claim 1 under conditions and for a time sufficient for an acyl amino acid composition to be made.

14. A method of claim 13, further comprising steps of:

enriching the acyl amino acid composition for a particular acyl amino acid; and

combining the enriched acyl amino acid composition with at least one other component to produce a product.