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, threaded conformation. Owing to a locked three-dimensional structure, lasso peptides can be unusually stable toward 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. All known lasso peptides are produced by bacteria and genome-mining studies indicate that lasso peptides are a relatively prevalent class of RiPPs; 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 longstanding challenges associated with lasso peptide production. We report the successful use of CFB for the formation of an array of sequence-diverse 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 to evaluate the substrate tolerance of the biosynthetic pathway. By evaluating more than 1000 randomly chosen variants, we show that the lasso-forming cyclase from the fusilassin pathway is capable of producing millions of sequence-diverse lasso peptides via CFB. These data lay a firm foundation for the creation of large lasso peptide libraries using CFB to identify new variants with unique properties.

Figure 1.. In vitro lasso peptide production through CFB.

(A) The biosynthetic gene clusters provided in the CFB reaction for the production of burhizin, capistruin, fusilassin, and cellulassin respectively. RRE: RiPP leader peptide Recognition Element. MBP: maltose binding protein. (B) Endpoint MALDI-TOF-MS assay of burhizin (m/z 1848), capistruin (m/z 2046), fusilassin (m/z 2269), and cellulassin (m/z 2277) produced from CFB. Blue, macrolactam ring residues; Red, acceptor site. The mass label corresponds to the [M+H]⁺ ion of the lasso peptides.

Figure 2.. Chimeric substrate strategy to generate diverse lasso peptides.

(A) The chimeric substrate principle fuses diverse (non-cognate) core peptides to the leader peptide sequence of FusA (residues −1 to −22). This permits a single set of biosynthetic proteins (FusB, C, and E) to produce various lasso peptides. (B) Endpoint MALDI-TOF-MS assay of CFB reactions with fusilassin as the positive control; FusA_LP-CelA_CP (cellulassin, 2277 Da; uncyclized core 2295 Da) and FusA_LP-HalA_CP (halolassin, 2047 Da) as the chimeric substrates. Precursor peptides were produced from CFB and reacted with purified MBP-FusB, MBP-FusC, and MBP-FusE to form the mature lasso peptide. Blue, macrolactam ring residues; Red, acceptor site. The mass label corresponds to the [M+H]⁺ ion of the lasso peptide. * indicates the [M+K]⁺ ion for the lasso peptide.

Figure 3.. Evaluating FusC substrate tolerance through Ala-substitution.

The sequence of wildtype fusilassin is provided at the top with endpoint MALDI-TOF-MS assay data for CFB-produced fusilassin variants shown below. The Glu9 acceptor site is intolerant to substitution. The mass label denotes the lasso peptide [M+H]⁺ ion. * indicates the lasso peptide [M+K]⁺ ion.

Figure 4.. Substrate compatibility evaluation of FusA ring.

(A) Experimental workflow for fusilassin ring library screening. Positions 2–6 of the FusA core region were diversified using 5 NNK codons (orange, library size of 3.2 M unique peptide sequences). After library cloning and transformation into E. coli, colony PCR was performed in 96-well plates. The linear DNA products were then used in CFB reactions to produce lasso peptides. (B) MALDI-TOF mass spectra of 10 representative fusilassin ring variants. The sequences of the varied region are orange. (C) Heatmap analysis on confirmed FusC substrates (lasso-compatible sequences) showing the percent difference between the occurrence of a residue by core position and the expected frequency based on the NNK codon. The analysis was conducted from 280 substrates. (D) MALDI-TOF mass spectra showing the lasso peptide formation of five predicted FusC substrates from the heatmap. At least four residues in designed sequences are predicted to be favored in FusC substrates. The precursor peptides were synthesized through CFB and reacted with heterologously expressed and purified FusB, C, and E. The mass labels denote the [M+H]⁺ ion of the lasso peptides. ^ indicates the uncyclized linear core peptide. * indicates the [M+K]⁺ ion for the lasso peptide.

Figure 5.. Substrate compatibility evaluation of FusA loop.

(A) Experimental workflow for fusilassin loop library evaluation. Positions 10–14 of the FusA core region were diversified using 5 contiguous NNK codons (orange, theoretical library size of 3.2 M unique peptide sequences). After library cloning and transformation into E. coli, colony PCR (cPCR) was performed in 96-well plates. The linear DNA products were then used in CFB reactions to produce lasso peptides. (B) From 57 randomly chosen clones, lasso peptide formation was only detected for three loop variants. Orange depicts the varied sequence at positions 10–14 of the FusA loop region. The mass labels denote to the [M+H]⁺ ion of the lasso peptide. * indicates [M+K]⁺ ion for the lasso peptide.