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

Abstract

Non-ribosomal peptide synthetases (NRPSs) are the origin of aw ide range of natural products,i ncluding many clinically used drugs.Efficient engineering of these often giant biosynthetic machineries to produce novel non-ribosomal peptides (NRPs) is an ongoing challenge.H ere we describe ac loning 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 À1 .E xtending 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.T hese synthetic type of NRPS (type S) enables easier access to engineering,o vercomes cloning limitations,a nd provides as imple and rapid approach to building peptide libraries via the combination of different NRPS subunits.

Evolution-inspired engineering of nonribosomal peptide synthetases

“…The absolute production titers were calculated as previously described ( 71 ). Therefore, calibration curves based on pure 1a (for quantification of 1 , 2 and 3 ), 4 (for 4 , 5 , 15 , 17 and 18 ), 10 ( 6 , 7 , 8 , 9 , 10 , 11 and 16 ), 67 ( 67 , 68 , 69 and 70 ), 26a (for 26 , 27 , 38 , 39 ), 28b (for 28 , 29 , 36 , 37 , 40 and 41 ), 34 (for 34 and 35 ), 42a (for 30 , 31 , 42 , 43 ), 44 (for 32 , 33 , 44 , 45 ) and 47 (for 46 , 47 and 48 ), were prepared.…”

“…The absolute production titers were calculated as previously described (71). Therefore, calibration curves based on pure 1a (for quantification of 1, 2 and 3), 4 (for 4, 5, 15, 17 and 18), 10 (6, 7, 8, 9, 10, 11 and 16), 67 ( 45) and 47 (for 46, 47 and 48), were prepared.…”

Recent progress in the reprogramming of nonribosomal peptide synthetases

“…After developing the XU concept, Bozhüyük et al introduced SYNZIPs into NRPS recombination as COM/DD domain surrogates to bypass cloning and protein size issues in heterologous NRPS expression systems. 47 The authors incorporated the anti-parallel interacting SYNZIPs 17 and 18 into two subunits between consecutive XUs at the conserved WNATE motif in the C-A linker. 31 The XtpSand GxpS-NRPS 37,39 were divided into two subunits in between XUs 2 and 3 using SYNZIPs 17 and 18, producing xenotetrapeptide (3) (structure is shown in Figure 3E) with 30% yield (28 mg/l) and gameX-Peptides (4-7) with 2.9-64% yield (0.2-4.9 mg/l), respectively, compared to XtpS-and GxpS-NRPS (wt) (Figure 4A).…”