Cell-Free Biosynthesis to Evaluate Lasso Peptide Formation and Enzyme-Substrate Tolerance

Abstract

Lasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique lariat-like structure. Owing to a rigid topology, lasso peptides are unusually stable towards heat and proteolytic degradation. Some lasso peptides have been shown to bind human cell-surface receptors and exhibit anticancer properties, while others display antibacterial or antiviral activities. Known lasso peptides are produced by bacteria and genome-mining studies indicate that lasso peptides are a relatively prevalent RiPP class; however, the discovery, isolation, and characterization of lasso peptides are constrained by the lack of an efficient production system. In this study, we employ a cell-free biosynthesis (CFB) strategy to address the longstanding challenges associated with lasso peptide production. We report the successful formation of a diverse array of lasso peptides that include known examples as well as a new predicted lasso peptide from Thermobifida halotolerans. We further demonstrate the utility of CFB to rapidly generate and characterize multisite precursor peptide variants in order to evaluate the substrate tolerance of the biosynthetic pathway. We show that the lasso-forming cyclase from the fusilassin pathway can produce millions of sequence-diverse lasso peptides via CFB with an extraordinary level of sequence permissiveness within the ring region of the lasso peptide. These data lay a firm foundation for the creation of large lasso peptide libraries using CFB to identify new variants with unique properties.

Cell-free mutant analysis combined with structure prediction of a lasso peptide biosynthetic protein B2

“…We chose the lasso peptide biosynthetic system from T. fusca , as the structure of the B1 protein (TfuB1) was successfully determined 5 and in vitro synthesis of the lasso peptide (fusilassin/fuscanodin) in the PURE system was reported. 21 Synthetic genes of TfuB1, TfuB2, and the precursor peptide fused with sfGFP (TfuA_sfGFP) were amplified by PCR using primers containing a T7 promoter and an appropriately positioned ribosome binding site (Supplementary Tables 1–3). Proteins encoded by resulting DNA molecules were co-expressed in the PURE system at 37 ºC for 1 hour and then incubated at 50 ºC, the temperature for the TfuB2 activity test.…”

Site-specific nonenzymatic peptide S/O–glutamylation reveals the extent of substrate promiscuity in glutamate elimination domains

“…The role of hydrophobic enzymesubstrate interactions in RiPP biosynthesis is well-documented, but most studies on the topic to date have focused on the LP-enzyme interactions (vide infra). The preference for hydrophobic amino acids in substrate CPs for promiscuous biosynthetic enzymes has also been noted, 44,45,[47][48][49] but to our knowledge, never clearly articulated. Thorough biophysical studies on LazF and other RiPP enzymes will be needed to further evaluate this idea.…”

Biocatalytic synthesis of peptidic natural products and related analogues

“…We will not discuss these types of cyclization, as their biocatalytic applications have been developed to a less extent, in comparison with lanthipeptides and microviridins. On the other hand, lasso peptides consist of a macrolactam formed between the N -terminus of the peptide and a carboxylate sidechain in an ATP-dependent manner, and the production of these peptides with synthetic and biocatalytic methods has been reported previously ( Cheng and Hua, 2020 ; Hegemann et al., 2015 ; Maksimov et al., 2012 ; Si et al., 2020 ).…”