Heterologous Production of Microbial Ribosomally Synthesized and Post-translationally Modified Peptides

Abstract

Ribosomally synthesized and post-translationally modified peptides, or RiPPs, which have mainly isolated from microbes as well as plants and animals, are an ever-expanding group of peptidic natural products with diverse chemical structures and biological activities. They have emerged as a major category of secondary metabolites partly due to a myriad of microbial genome sequencing endeavors and the availability of genome mining software in the past two decades. Heterologous expression of RiPP gene clusters mined from microbial genomes, which are often silent in native producers, in surrogate hosts such as Escherichia coli and Streptomyces strains can be an effective way to elucidate encoded peptides and produce novel derivatives. Emerging strategies have been developed to facilitate the success of the heterologous expression by targeting multiple synthetic biology levels, including individual proteins, pathways, metabolic flux and hosts. This review describes recent advances in heterologous production of RiPPs, mainly from microbes, with a focus on E. coli and Streptomyces strains as the surrogate hosts.

Keywords: E. coli; RiPPs; Streptomyces; heterologous expression; precursor peptide; processing enzymes; synthetic biology.

Figure 1

(A) Representative structures of five select RiPP families with diverse bioactivities. Post-translational modification(s) on each structure are highlighted in red. (B) A schematic depiction of RiPP biosynthesis. Precursor peptide typically contains the leader peptide (in green) followed by the core peptide (in blue). Modifications of the core peptides (in brown) are guided by the leader peptides that interact with processing enzymes. Proteolytic release of the leader peptides then gives rise to mature RiPPs (in yellow).

Figure 2

A summary of multiple emerging strategies that target on manipulating individual proteins, pathways, metabolic flux or hosts to improve the success of heterologous expression of RiPPs. All of these strategies will be discussed below with select recent examples.

Figure 3

High throughput discovery of functional microcin J25 variants with multiple amino acid substitutions was enabled by an orthogonally inducible system which separately controls the production and export/immunity of mature RiPPs. More specifically, the expression of the precursor gene mcjA and the transporter gene mcjD was independently induced by IPTG and arabinose, respectively. In the noninduced state, leaky expression leads to the low levels of both McjA and McjD (left). When IPTG and glucose are added, the expression of mcjA mutants is highly induced, but not mcjD, resulting in cytoplasmic accumulation of McjAs. If McjAs are processed into mature MccJ25 variants with antibacterial activity, accumulated lasso peptides will inhibit the growth of the host cell (top right). The poor growth of these cells will be salvaged by the addition of arabinose to overexpress McjD. By contrast, inactive MccJ25 variants will have no inhibitory effect on the cell growth (bottom right).

Figure 4

A chimeric leader peptide strategy to produce unnatural RiPP hybrids. By properly designing the concatenated leader peptides, recognition and processing by multiple enzymes from unrelated RiPP pathways could be realized. By using this method, a thiazoline-forming cyclodehydratase was combined with biosynthetic enzymes from the sactipeptide and lanthipeptide families to create new-to-nature hybrid RiPPs, demonstrating the feasibility of the strategy.

Figure 5

Structures of select RiPPs produced by uncommon surrogate hosts exemplified by Streptomyces avermitilis SUKA17 (A), Nonomuraea sp. ATCC 39727 (B) and Aspergillus oryzae (C).