GENERATION OF ACYL AMINO ACIDS

• US 20180340198A1
• Filed: 05/14/2018
• Published: 11/29/2018
• 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
• Granted Patent: US 10,640,799 B2
• Time in Patent Office: Days
• 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

In certain embodiments, the present invention comprises compositions and methods useful in the generation of acyl amino acids. In certain embodiments, the present invention provides an engineered polypeptide comprising a peptide synthetase domain; in some such embodiments, the engineered polypeptide comprises only a single peptide synthetase domain. In some embodiments, the present invention provides an engineered peptide synthetase that is substantially free of a thioesterase domain, and/or a reductase domain. In certain embodiments, the present invention provides an acyl amino acid composition comprising a plurality of different forms of an acyl amino acid. In some such compositions, substantially all of the acyl amino acids within the composition contain the same amino acid moiety and differ with respect to acyl moiety. We also described populations where the fatty acid si for example 95% one length (C14, myristic).

Claims

1. A method of making an acyl amino acid composition by contacting an engineered peptide synthetase with an amino acid substrate and an acyl entity substrate for the engineered peptide synthetase, under conditions and for a time sufficient for an acyl amino acid composition to be made.

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

1A1. A1. The method of claim 1 or claim 1A, wherein the engineered peptide synthetase lacks thioesterase domain, and/or a reductase domain.

1A1a. A1a. The method of claim 1 or 1A1, wherein the engineered peptide synthetase contains only a single peptide synthetase domain.

1A2. A2. The method of claim 1 or 1A1a, wherein the engineered peptide synthetase is or comprises a peptide synthetase domain found in as a first domain in a peptide synthetase that synthesizes a lipopeptide.

1B. B. The method of claim 1 or claim 1A, 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 or lauric acid.

1C. C. The method of any one of the preceding claims, wherein the step of contacting comprises providing a cell engineered to express at least one engineered peptide synthetase.

5A2. A2. The method of any one of claims 1A, 1A1, or 1A1a, wherein the step of contacting comprises contacting the cell with the substrates.

2. A cell engineered to express at least one engineered peptide synthetase that synthesizes an acyl amino acid.

3. An acyl amino acid composition produced by an engineered peptide synthetase.

3B. B. The composition of claim 3, wherein substantially all of the acyl amino acids in the composition contain the same amino acid component.

3C. C. The composition of any one of claims 3-3B, wherein acyl amino acids in the composition comprise different acyl moieties.

4. A method of preparing a product comprising:

providing or obtaining an acyl amino acid composition prepared in an engineered microbial cell;

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

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

5. A method comprising steps of:

contacting an engineered peptide synthetase polypeptide that comprises a single peptide synthetase domain and lacks a thioesterase domain, and/or a reductase domain with;

an amino acid substrate of the peptide synthetase polypeptide; and

an acyl moiety substrate of the peptide synthetase polypeptide,

the contacting being performed under conditions and for a time sufficient that the engineered peptide synthetase polypeptide covalently links the acyl moiety from the acyl moiety substrate to the amino acid so that an acyl amino acid is generated.

5A. A. The method of claim 5, wherein the engineered peptide synthetase polypeptide is produced by a cell.

5A1. A1. The method of claim 5A, wherein the cell is a microbial cell.

5A1a. A1a. The method of claim 5A1, wherein the cell is a bacterial cell.

5B. B. The method of claim 5, wherein the cell is an engineered cell.

1C-1. C. The method of claim 5B, wherein the cell is engineered in that the peptide synthetase polypeptide is an engineered peptide synthetase polypeptide.