Microbial production of small peptide: pathway engineering and synthetic biology

Abstract

Small peptides are a group of natural products with low molecular weights and complex structures. The diverse structures of small peptides endow them with broad bioactivities and suggest their potential therapeutic use in the medical field. The remaining challenge is methods to address the main limitations, namely (i) the low amount of available small peptides from natural sources, and (ii) complex processes required for traditional chemical synthesis. Therefore, harnessing microbial cells as workhorse appears to be a promising approach to synthesize these bioactive peptides. As an emerging engineering technology, synthetic biology aims to create standard, well-characterized and controllable synthetic systems for the biosynthesis of natural products. In this review, we describe the recent developments in the microbial production of small peptides. More importantly, synthetic biology approaches are considered for the production of small peptides, with an emphasis on chassis cells, the evolution of biosynthetic pathways, strain improvements and fermentation.

Fig. 1

Schematic representation of biosynthetic mechanisms of RiPPs and NRPs. A. The biosynthesis of RiPPs undergoes the ribosome and post‐translational modification machinery. The precursor peptide usually consists of an N‐terminal leader peptide (for recognition by PTM enzymes and for export) and a core peptide (harbouring various PTM sites). In some cases, the C‐terminal follower peptide serves as a leader peptide or transcriptional factor. Following the completion of PTMs, the leader peptide and follower peptide are removed by proteolysis. Examples of different small peptides belong to the RiPPs family. B. Model of an NRPS assembly line showing the typical linear. (A)‐adenylation domain, (T)‐thiolation domain, (C)‐condensation domains, (TE)‐thioesterase domain. The module of NRPS may contain additional domains including epimerization (E), N‐methylation (M) and cyclization (Cy) domains. Examples of different small peptides produced by NRPS assembly lines.

Fig. 2

Evolution of artificial pathways. A. Mining BGCs, the BGCs encoding the biosynthesis of small peptides are identified based on advanced computational tools. B. Refactor BGCs, including the remove of native regulation elements, fine‐tuning of gene expression using diverse synthetic biology tools. C. Assembly BGCs, the fusion of different genetic fragments based on various assembly strategies, and further clone the BGCs into suitable vectors. D. Scheme of semi‐in vitro biosynthesis. The prepeptide of RiPP is synthetized and modified within the cell, and the mature peptide can subsequently be released by removing the leader peptide through the in vitro cleavage system.

Fig. 3

Metabolic engineering strategies for strain improvement.

Fig. 4

Schematic illustration of fermentation process control based on the extracellular and intracellular levels. A. Control of the fermentation process at the extracellular level. The fermentation process is affected by changes in various extracellular factors, including substrate and process parameters (e.g. DO), as well as cell growth (e.g. biomass). B. Fermentation control system. C. The physiological state of cells can be monitored and regulated by controlling the fermentation process at the intracellular level, which includes controlling the cell morphology and cell metabolism, as well as gene expression.