Cell-free biosynthesis and engineering of ribosomally synthesized lanthipeptides

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with diverse chemical structures and potent biological activities. A vast majority of RiPP gene clusters remain unexplored in microbial genomes, which is partially due to the lack of rapid and efficient heterologous expression systems for RiPP characterization and biosynthesis. Here, we report a unified biocatalysis (UniBioCat) system based on cell-free gene expression for rapid biosynthesis and engineering of RiPPs. We demonstrate UniBioCat by reconstituting a full biosynthetic pathway for de novo biosynthesis of salivaricin B, a lanthipeptide RiPP. Next, we delete several protease/peptidase genes from the source strain to enhance the performance of UniBioCat, which then can synthesize and screen salivaricin B variants with enhanced antimicrobial activity. Finally, we show that UniBioCat is generalizable by synthesizing and evaluating the bioactivity of ten uncharacterized lanthipeptides. We expect UniBioCat to accelerate the discovery, characterization, and synthesis of RiPPs.

Plasmids constructed in this study.

Plasmid
Antibiotic Application pJL1_sboA 50 μg/mL kanamycin Templates used for cellfree gene expression pET28a-sboM pET28a-sboT pJL1-sboA_Q6/L20 (a total of 53 mutated plasmids) pRSFDuet-1-sboA-sboM Plasmids used for in vivo  In the alkylation assay, NEM is added to the thiol (-SH) group of the cysteine residue and the derivatives show a molecular mass increase of 125 Da per reaction.The partially modified SboA_A-1R was a peptide mixture purified from the in vivo co-expression of sboA_A-1R and sboM for 1 h at 30°C, in which serine and threonine residues were not completely dehydrated to form the thioether crosslinks leaving 1-3 cysteine residues to react with NEM.When NEM was added, the peptide molecular mass increased accordingly.For the fully modified peptide, no NEM was added and thus the peptide molecular mass was not changed as observed in (b) for both salivaricin B and salivaricin B-1.The consistent molecular weights indicate the successful modification of the thioether crosslinks in the mature peptides.

Supplementary Figure 4 .Supplementary Figure 5 Supplementary Figure 6 .Supplementary Figure 13 .
Effects of endogenous potential proteases on salivaricin B synthesis.(a)Generation of salivaricin B analogs by non-specific protease degradation both in vivo and in vitro.The red and green stars indicate salivaricin B and salivaricin B-1, respectively.Analogs detected in vitro were products from cell-free co-expression of sboA, sboM, and sboT.Analogs detected in vivo were products from coexpression of sboA and sboM.Harvested strains were divided into three parts undergoing different treatments.Sample 1 was suspended in LanA I buffer containing 4 M guanidine hydrochloride and then sonicated for cell lysis, in which the endogenous proteases were inactivated by guanidine hydrochloride.Sample 2 was suspended , sonicated, and then immediately placed on ice.Sample 3 was suspended in lysis buffer and incubated at 30°C for 3 h after sonication, in which the endogenous proteases conducted catalysis.All of samples were then centrifuged and the supernatants were desalted by C18 ZipTip and analyszed by MALDI-TOF-MS with linear positive mode.(b) Products from cell-free reactions (SboA and SboM) with increased concentrations of protein inhibitor cocktail in the UniBioCat reactions.(c) Products from cell-free reactions (SboA, SboM, and SboT) with increased concentrations of protein inhibitor cocktail in the UniBioCat reactions.The catalytic activities of endogenous proteases/peptidases and SboT were inhibited by a high concentration of protein inhibitor (5x).Strain engineering to reduce non-specific protease/peptidase degradation in UniBioCat reactions.(a) Schematic diagram of protease/peptidase deletion for the preparation of enhanced cell extracts for UniBioCat reactions.Created with BioRender.com.(b) Observed products by non-specific degradation with endogenous proteases and/or peptidases.(c) MALDI-TOF-MS analysis of salivaricin B (highlighted in red), salivaricin B-1 (highlighted in blue), and other non-specifically degraded products.Cell extracts used for UniBioCat were prepared from the wild-type (WT) strain E. coli BL21 Star (DE3) and three other engineered strains with protease (degP) and/or peptidase (pepN) gene(s) deleted.Note that SboM was pre-expressed in each engineered strain for the preparation of SboM-enriched cell extracts, which were then used for cell-free co-expression of SboA and SboT.Reduction of the non-specific protease/peptidase degradation in UniBioCat reactions.Top, observed products by non-specific degradation with endogenous proteases/peptidases. Bottom, MALDI-TOF-MS analysis of salivaricin B (red star), salivaricin B-1 (blue star), and other non-specifically degraded products.Cell extracts used for UniBioCat were prepared from the wild-type (WT) strain E. coli BL21 Star (DE3) and four engineered strains with protease or peptidase deleted.Alignment of homologous peptides based on the salivaricin B precursor peptide.Amino acid sequences of the homologous peptides were aligned by using MEGA.Q6 and L20 were selected for the mutation.Evaluating substrate tolerance of SboT (cleavage site "GAG" is highlighted).(a) SboM modified precursors (SboA) serving as substrates for SboT.The position of A-1 was selected for saturation mutagenesis.The position of G-2 was mutated to seven other amino acids (i.e., W, V, K, H, E, C, and A).(b) Effect of specific-site mutation (A-1 or G-2) on the cleavage site of SboT.Heatmap analysis indicates the relative intensity (%) of mutated products to wild-type peptides (the percent values are calculated according to the mass peaks observed by MALDI-TOF-MS, see Supplementary Figure14).m, mutated amino acids at the position of -1 or -2; WT, wild-type SboA without mutation.Source data are provided as a Source Data file.

b
Supplementary Figure 14.MALDI-TOF-MS analysis of peptides matured by SboT.Products were identified by MALDI-TOF-MS with linear positive mode.Red lines indicate salivaricin B resulted from cleavage site of mutant-1/G and A-1/G.Green stars indicate products resulted from cleavage site of G-2/mutant.Green line indicates salivaricin B-1 resulted from cleavage site of mutant-2/A-1.See Supplementary Figure13for the cleavage site "GAG" of SboT and mutated positions.

Table 2
Primers used in this study (F, forward primer; R, reverse primer; Bold letters, mutated codon).

Table 4
Proteases and peptidases deleted in the genome of E. coli BL21 Star (DE3). 5Supplementary

Table 5
The yields of salivaricin B variants synthesized in UniBioCat reactions.Minimal inhibitory concentrations (MIC) and IC50 of salivaricin B and variants.
a MW, molecular weight.Supplementary Table6