IP Library Granted Patent US 12,297,475
Granted Patent B2
US 12,297,475 · App. 16/962,017 · Granted May 13, 2025

System for the assembly and modification of non-ribosomal peptide synthases

Inventors: Helge B. Bode (Oberursel, DE); Kenan Bozhüyük (Frankfurt am Main, DE); Annabell Linck (Frankfurt am Main, DE)
Assignee: Johann Wolfgang Goethe-Universität Frankfurt am Main
C12P21/02C12N9/93
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Quick Facts
Patent No.
US 12,297,475
App. No.
16/962,017
Granted
May 13, 2025
Kind
B2
Abstract

The present invention pertains to a system for the assembly and modification of non-ribosomal peptide synthases (NRPS). The system uses novel well defined building blocks (units) comprising condensation subdomains. This strategy allows for the efficient combination of assembly units referred to as eXchange Units (XU2.0) independent on their natural occurring specificity for the subsequent NRPS adenylation domain. The system of the invention allows for the easy assembly of NRPS having any amino acid sequence of choice, without any restrictions due to natural occurring NRPS units. The system also allows the exchange of natural NRPS building blocks with the inventive XU2.0 leading to the production of modified peptides. The invention provides the system, their individual exchange units, nucleic acids encoding these units, as well as methods and uses thereof.

Claims (33)

1. A library of non-ribosomal peptide synthetase (NRPS) eXchange Units (XU 2.0 ) for assembling an NRPS, wherein the library comprises an XU 2.0 , and wherein the XU 2.0 is selected from the group consisting of:

C Dsub -A-T-C Asub ,

C Dsub -A-T-C/E Asub ,

C/E Dsub -A-T-C/E Asub , and

C/E Dsub -A-T-C Asub ,

wherein A is an adenylation domain, T is a thiolation domain, C Asub is an incomplete condensation-domain comprising only an acceptor site subdomain, C/E Asub is an incomplete condensation/epimerization-domain comprising only an acceptor site subdomain, C Dsub is an incomplete condensation-domain comprising only a donor site subdomain, and C/E Dsub is an incomplete condensation/epimerization-domain comprising only a donor site subdomain

wherein the XU 2.0 does not comprise a full-length C- or C/E-domain comprising both acceptor site subdomain and donor site subdomain.

2. The library according to claim 1 , comprising at least two XU 2.0 of which each has a different amino acid specificity.

3. The library according to claim 1 , the library further comprising a XU 2.0 termination and/or initiation unit, wherein the XU 2.0 initiation unit comprises only a C or C/E domain donor subdomain, a domain structure C-A X -T-C Dsub X or C-A X -T-C/E Dsub X , specific for the incorporation of acyl units as starting units, and wherein the termination unit comprises any one of a terminal condensation domain (Cterm), an internal condensation (C) domain, an internal condensation and epimerization (C/E)-didomain, a cyclization (Cy) domain, an epimerization (E) domain, a reduction (R) domain, an oxidation (Ox) domain, or a thioesterase (TE) domain.

4. The library according to claim 1 , the library further comprising a XU 2.0 initiation unit.

5. The library according to claim 1 , wherein the library comprises at least two XU 2.0 and the at least two XU 2.0 when put into sequence provide the NRPS.

6. The library according to claim 1 , further comprising at least one XU 2.0 having a modification domain.

7. A method for producing a non-ribosomal peptide synthetase (NRPS), the method comprising a step of assembling at least two NRPS eXchange Units (XU 2.0 ), wherein each XU 2.0 is selected from the group consisting of:

C Dsub -A-T-C Asub ,

C Dsub -A-T-C/E Asub ,

C/E Dsub -A-T-C/E Asub , and

C/E Dsub -A-T-C Asub ,

wherein A is an adenylation domain, T is a thiolation domain, C Asub is an incomplete condensation-domain comprising only an acceptor site subdomain, C/E Asub is an incomplete condensation/epimerization-domain comprising only an acceptor site subdomain, C Dsub is an incomplete condensation-domain comprising only a donor site subdomain and C/E Dsub is an incomplete condensation/epimerization-domain comprising only a donor site subdomain;

wherein the XU2.0 does not comprise a full-length C- or C/E-domain comprising both acceptor site subdomain and donor site subdomain.

8. A method for the production of non-ribosomal peptides having a specific sequence, the method comprising assembling a NRPS according to the method accordingly to claim 7 , wherein the NRPS is composed of a sequence of at least three XU 2.0 having specificity according to the non-ribosomal peptide to be produced.

9. The library according to claim 2 , wherein each different amino acid specificity is selected from a specificity to any one or two, three, four or more natural or non-natural amino acid.

10. The library according to claim 3 , wherein the acyl units comprise fatty acids or derivatives thereof.

11. The library according to claim 4 , wherein the XU 2.0 initiation unit is C Asub -A X -T-C Dsub X , C/E Asub -A X -T-C Dsub X , C Asub -A X -T-C/E Dsub X , or C/E Asub -A X -T-C/E Dsub X .

12. The library according to claim 5 , wherein the library comprises three, four, or more XU 2.0 put into sequence.

13. The library according to claim 6 , wherein the modification domain is an E, MT, or Ox modification domain.

14. The library accordingly to claim 1 , comprising a first XU 2.0 and a second XU 2.0 each selected from the group consisting of:

C Dsub -A-T-C Asub ,

C Dsub -A-T-C/E Asub ,

C/E Dsub -A-T-C/E Asub , and

C/E Dsub -A-T-C Asub ,

wherein the first XU 2.0 and the second XU 2.0 , when connected to each other, form a functional assembled C or C/E domain composed of one partial C or C/E domain of the first XU 2.0 and one partial C or C/E domain of the second XU 2.0 .

15. The library accordingly to claim 1 , wherein the library comprises at least two XU 2.0 each specific for a different or identical amino acid X.

16. The library accordingly to claim 1 , wherein the C Asub , C/E Asub , C Dsub , and C/E Dsub are specific for a given amino acid X.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 3, 2020
From: BODE, HELGE B.; BOZHÜYÜK, KENAN; LINCK, ANNABELL
To: JOHANN WOLFGANG GOETHE-UNIVERSITÄT FRANKFURT AM MAIN
Reel/Frame 053387/0760 →
Priority Claims (1)
EP 18151588 · Jan 15, 2018 · regional
Continuity (1)
Related Publication 20200347428A1 · Nov 5, 2020
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