Heterologous and in Vitro Reconstitution of Fuscanodin, a Lasso Peptide from Thermobifida fusca

Abstract

Lasso peptides are a class of ribosomally-derived natural products typified by their threaded rotaxane structure. The conversion of a linear precursor peptide into a lasso peptide structure requires two enzymatic activities: cleavage of the precursor via a cysteine protease and cyclization via isopeptide bond formation. In vitro studies of lasso peptide enzymology have been hampered by difficulties in obtaining pure, soluble enzymes. We reasoned that thermophilic bacteria would be a good source for well-behaved lasso peptide biosynthetic enzymes. The genome of the thermophilic actinobacterium Thermobifida fusca encodes for a lasso peptide with an unprecedented Trp residue at its N-terminus, a peptide we have named fuscanodin. Here we reconstitute fuscanodin biosynthesis in vitro with purified components, establishing a minimal fuscanodin synthetase. These experiments have allowed us to probe the kinetics of lasso peptide biosynthesis for the first time, and we report initial rates of fuscanodin biosynthesis. The fuscanodin biosynthetic enzymes are insensitive to substrate concentration and operate in a near single-turnover regime in vitro. While lasso peptides are often touted for their stability to both chaotropic and thermal challenges, fuscanodin is found to undergo a conformational change consistent with lasso peptide unthreading in organic solvents at room temperature.

Steric complementarity directs sequence promiscuous leader binding in RiPP biosynthesis

“…S3), despite the lack of extensive sequence conservation among the leader sequences of the TbiAα and TbiAβ precursor peptides. Lastly, although ATP is a required cosubstrate for processing by the fused B proteins (14), proteolysis by the therbactin system is consistent with other split B proteins, as it does not require ATP (15,16,20).…”

“…Binding studies of proteins from different split lasso peptide biosynthetic clusters demonstrate that the B1 proteins from each cluster bind to the cognate leader peptides with submicromolar affinity (7,19,20). Coincubation studies with heterologously purified lasso peptide biosynthetic proteins from the paeninodin and fuscanodin/fusilassin clusters demonstrates that proteolysis of the precursor peptide by the B2 protein only occurs in the presence of the corresponding B1 protein, indicating that leader recognition precedes and dictates peptide processing (15,16,20).…”

“…In some lasso peptide biosynthetic clusters, the B protein is a single polypeptide, but, in others, such as those that produce lassomycin (17), lariatin (18), and fuscanodin/fusilassin (15,16), this gene is split into 2 open reading frames (ORFs), B1 and B2. These 2 genes are similar to the N and C termini, respectively, of full-length B. Bioinformatics analyses of B-protein sequences identifies that the B1 protein (and the corresponding N-terminal region of fused B proteins) contains hallmarks of an RRE (7).…”

“…The minimal set of genes required for the biosynthesis of lasso peptides encodes the precursor peptide (A protein), a transglutaminase-like protease (B protein), and an asparagine synthase-like protein (C protein). In vitro reconstitution of the lasso peptides microcin J25 (13,14) and fuscanodin/ fusilassin (15,16) provides a route for understanding lasso peptides biosynthesis. First, the B-protein protease binds to and cleaves the leader peptide from the A-protein precursor peptide to yield the core peptide.…”

Lasso Peptides: Heterologous Production and Potential Medical Application

“…The heterologous production of lasso peptide brevunsin from Brevundimonas diminuta was achieved with a yield of 10.2 mg/L in Sphingomonas subterranea (Kodani et al, 2018). The genes involved in the biosynthesis and posttranslational modification have become accessible with the development of the functional study of lasso peptides (Pan et al, 2012a;Burkhart et al, 2015;Li et al, 2015;Zhu et al, 2016b;Hegemann et al, 2018;Chekan et al, 2019;DiCaprio et al, 2019;Koos and Link, 2019;Sumida et al, 2019). The heterologous expression of lasso peptides has gained increasing attention, and significant progress has been made in the production, especially based on the E. coli and Streptomyces systems.…”