Roads to Rome: Role of Multiple Cassettes in Cyanobactin RiPP Biosynthesis

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are ubiquitous natural products. Bioactive RiPPs are produced from a precursor peptide, which is modified by enzymes. Usually, a single product is encoded in a precursor peptide. However, in cyanobactins and several other RiPP pathways, a single precursor peptide encodes multiple bioactive products flanking with recognition sequences known as “cassettes”. The role of multiple cassettes in one peptide is mysterious, but in general their presence is a marker of biosynthetic plasticity. Here, we show that in cyanobactin biosynthesis the presence of multiple cassettes confers distributive enzyme processing to multiple steps of the pathway, a feature we propose to be a hallmark of multi-cassette RiPPs. TruD heterocyclase is stochastic and distributive. Although a canonical biosynthetic route is favored with certain substrates, every conceivable biosynthetic route is accepted. Together, these factors afford greater plasticity to the biosynthetic pathway by equalizing the processing of each cassette, enabling access to chemical diversity.

Translation-Targeting RiPPs and Where to Find Them

“…As a result, in a single ORF, there are two putative core sequences rich in Ser and Cys residues separated by an "internal" leader (another leader is N-terminally located) ( Figure 8C). A similar cassette-like arrangement of core peptides has been described for several different groups of RiPPs including cyanobactins (Gu et al, 2018), thiovarsolines (Santos-Aberturas et al, 2019), orbitides (Shim et al, 2015), and dikaritins (Ding et al, 2016); but in all these cases, precursors are composed of a single leader, followed by several core peptides, interspersed by signal sequences required for the cleavage of each core at Cand N-termini by dedicated peptidases (Figure 8B shows, as an example, the sequence of TruE1 -the precursor of patellins 2 and 3, representatives of cyanobactins).…”

“…These different genome arrangements from the closely related species provide a glimpse on how the genes of cassette-containing peptides may originate from an independent single short ORF through gene duplication [ Figure 8D(1)], fusion [ Figure 8D(2)], and a subsequent reduction of the role of the internal leader to that of a recognition sequence of proteases [ Figure 8D(4)]. Further multiplication of cassette-containing precursor genes may lead to arrangements found in several cyanobactin clusters [ Figure 8D(5); Gu et al, 2018]. Strikingly, only the BGCs with fused precursors contain an additional gene, which is a predicted protease (Figure 8A, orange).…”

“…The precursor peptide sequence of cyanobactins patellins 2 and 3 (truE1 gene product) is shown for comparison on the gray background. Functional parts of the peptide including leader (yellow), two cores (red), and recognition sequences of peptidases (RS1 and RS2) are shown(Gu et al, 2018). (C) Sequences of cassette-containing precursor peptides of flavazolicins showing the conserved positions in two leader sequences.…”

Mechanisms and Evolution of Diversity-Generating RiPP Biosynthesis

“…This decrease in efficiency is offset by the acceptance of the increasingly structurally diversified substrate pool. Further facilitating diversity generation, these pathways do not follow a strict order of posttranslational modifications [107], which would decrease the diversity of products that can be formed. It has been suggested that these may be general features of diversity-generating biosynthetic pathways [6,108].…”

Characterization of a Dehydratase and Methyltransferase in the Biosynthesis of Ribosomally Synthesized and Post‐translationally Modified Peptides in Lachnospiraceae

“…A strict sequence of PTM installation has been observed previously for some RiPP biosynthetic pathways such as the thiopeptides, select lanthipeptides, microcin C7, and the bottromycins . However, in other systems such as the cyanobactins, the biosynthetic enzymes have demonstrated much plasticity and can act in a variety of orders . Although we cannot rule out the possibility of strict order for the lah system, we find it unlikely that LahM1/M2 would act on a large number of previously post‐translationally modified LahA peptides.…”