Synthetic Zippers as an Enabling Tool for Engineering of Non‐Ribosomal Peptide Synthetases

Abstract Non‐ribosomal peptide synthetases (NRPSs) are the origin of a wide range of natural products, including many clinically used drugs. Efficient engineering of these often giant biosynthetic machineries to produce novel non‐ribosomal peptides (NRPs) is an ongoing challenge. Here we describe a cloning and co‐expression strategy to functionally combine NRPS fragments of Gram‐negative and ‐positive origin, synthesising novel peptides at titres up to 220 mg L−1. Extending from the recently introduced definition of eXchange Units (XUs), we inserted synthetic zippers (SZs) to split single protein NRPSs into independently expressed and translated polypeptide chains. These synthetic type of NRPS (type S) enables easier access to engineering, overcomes cloning limitations, and provides a simple and rapid approach to building peptide libraries via the combination of different NRPS subunits.

S-3 1 Material and methods

Cultivation of strains
All E. coli, Xenorhabdus and Photorhabdus strains were cultivated in liquid or on solid LB-medium (pH 7.5, 10 g/L tryptone, 5 g/L yeast extract and 5 g/L NaCl). Solid media contained 1% (w/v) agar. Kanamycin (50 μg/ml) and chloramphenicol (34 μg/ml) were used as selection markers. All E. coli cultures were cultivated at 37 °C and at 22 °C for peptide production purposes. Xenorhabdus and Photorhabdus strains were grown at 30 °C.

Cloning of biosynthetic gene clusters
Genomic DNA of selected Xenorhabdus and Photorhabdus strains were isolated using the Qiagen Gentra Puregene Yeast/Bact Kit. All PCRs were performed with oligonucleotides obtained from Eurofins Genomics (Supplementary Table 4). NRPS fragments for Hot Fusion cloning [1] were amplified with primers coding for the respective homology arms (20-30 bp) in a two-step PCR program. The coding sequences for the SYNZIPs were also attached upstream or downstream to the NRPS genes by PCR. In the following, the cloning procedure for the basic vectors is explained. pJW61/62 was obtained by the following steps: First, the SYNZIP17/18 coding sequences (pENTR-SYNZIP17/18 [2] were a gift from Amy Keating, Addgene plasmids #80671/80672; RRID:Addgene_80671/80672) were inserted into the plasmids pCOLA_ara/tacI and pCK_0402 by oligonucleotides KB-pACYC-FW/RV or KB-pCOLA-FW/RV in two-step polymerase chain reactions (PCRs) combined with Hot Fusion Cloning [1] . Second, these plasmids were linearized by single-step PCRs with the help of the oligonucleotides KB-pCOLA-II-FW/RV or KB-pACYC-II-FW/RV, which further allowed us to introduce NRPS fragments by Hot Fusion cloning. Therefore, the respective NRPS coding sequences were amplified again in two-step PCRs, using oligonucleotides with additional coding regions for homology arms (20-30 bp). where the SYNZIP coding sequences were located, confirmed the correct plasmid construction. (Bruker) was used to evaluate the measurements.

Peptide quantification
The absolute production titers of selected peptides were calculated with calibration curves based on pure synthetic 1, 2 (for quantification of 2-5), 6 (for quantification of were synthesized in-house [3,4] and the further pure synthetic 15 was produced by Synpeptide.

Chemical synthesis
Chemical synthesis of all peptides was performed as described previously [3] .

Figure S17.
A schematic representation of all bipartite type S NRPSs (NRPS-7, -9, -11, -13, -15 --28) using subunit 1 building blocks from GxpS, XtpS, BacA, AmbS XldS, SzeS and BicA combined with subunit 2 building blocks from GxpS, XtpS and RtpS. All constructed subunits 1 and 2 with attached synthetic zippers were functional, as at least one functional combination could be observed in each case. Co-expression of two subunits each led to detectable peptide amounts in 9 out of 18 cases. Nonproductive type S NRPS combinations involved subunits 2, either from XtpS (type S NRPSs: 7, 13, 21, 22, 23, and 24) or RtpS (type S NRPSs: 25 -28). From these non-producing type S NRPSs it can be deduced that: (I) the TE domain from XtpS has a very narrow substrate range, at least when it comes to positions 1 and 2 of the synthesised peptides; and (II) that subunits of Gram-positive and -negative origin can be functionally combined in trans, if the additive negative effect of introduced impairments is not too great, i.e. the substrate specificity of involved TE and C domains as well as the formed chimeric C-A interface. In conclusion, especially for subunits of only distantly related bacteria it is imperative to keep these caveats in mind. Figure S18. (a) Production of D/L-tripeptides exemplary of NRPS-5 (Fig. 3). The tripeptide production is related to the unpaired activity of GxpS subunit 2 resulted in the production of peptides 22/23 and 24/25. The different epimers could be identified by their retention times. (b) Tripeptide 22/23 and 24/25 amounts and yields (determined in triplicates (n=3)) are given for all NRPS systems shown in Fig. 3. The colour code of the NRPS subunits is depicted at the bottom of the figures. The domain assignment is as described in Fig. 3 and 4.