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

The system according to any one of claims 1 to 7, the system further comprising a XU2.0 termination and/or initiation unit, wherein the XU2.0 initiation unit comprises only a C or C/E domain donor subdomain, a domain structure C-AX-T-CDsubX or C-AX-T-C/EDsubX, specific for the incorporation of acyl units (fatty acids and their derivatives) as starting units, and wherein the termination module 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), an oxidation (Ox) or a thioesterase (TE) domain.

The system according to any one of claims 1 to 7, the system further comprising a XU2.0 initiation unit, preferably selected from the formulas:

• CAsub-AX-T-CDsubX, C/EAsub-AX-CDsubX, CAsub-AX-T-E/EDsubX, or C/EAsub-AX-T-C/EDsubX

The system according to any one of claims 1 to 9, wherein at least two, preferably three, four, or more, XU2.0 when put into sequence provide the NRPS.

An isolated nucleic acid, comprising a partial non-ribosomal peptide synthases (NRPS) condensation (C)-domain sequence or partial NRPS condensation/epimerization (C/E)-domain sequence, wherein said sequence encodes for at least a partial C or C/E domain comprising a C or C/E domain donor or acceptor site, but does not encode for a partial or full-length C or C/E domain comprising a functionally connected donor and acceptor site.

A library of nucleic acid molecules, wherein the library comprises at least two or more nucleic acid constructs each encoding a NRPS eXchange Unit (XU2.0) having an amino acid specificity X, and wherein the XU2.0 comprises at least one partial condensation (C)- or partial condensation/epimerization (C/E)-domain selected from the group consisting of a condensation-domain acceptor site subdomain (CAsub) having an amino acid specificity X, a condensation/epimerization-domain acceptor site subdomain (C/EAsub) having an amino acid specificity X, a condensation-domain donor site sub-domain (CDsub) having an amino acid specificity X and a condensation/epimerization-domain donor site subdomain (C/EDsub) having an amino acid specificity X.

A method for producing a NRPS, the method comprising a step of assembling at least two NRPS eXchange Units (XU2.0) each having an identical or different amino acid specificity X, and wherein the XU2.0 comprises at least one partial condensation (C)- or partial condensation/epimerization (C/E)-domain selected from the group consisting of a condensation-domain acceptor site subdomain (CAsub) having an amino acid specificity X, a condensation/epimerization-domain acceptor site subdomain (C/EAsub) having an amino acid specificity X, a condensation-domain donor site subdomain (CDsub) having an amino acid specificity X and a condensation/epimerization-domain donor site subdomain (C/EDsub) having an amino acid specificity X.