An Engineered Synthetic Pathway for Discovering Nonnatural Nonribosomal Peptides in Escherichia coli

Abstract

Peptides that are synthesized independently of the ribosome in plants, fungi, and bacteria can have clinically relevant anticancer, antihemochromatosis, and antiviral activities, among many other. Despite their natural origin, discovering new natural products is challenging, and there is a need to expand the chemical diversity that is accessible. In this work, we created a novel, compressed synthetic pathway for the heterologous expression and diversification of nonribosomal peptides (NRPs) based on homologs of siderophore pathways from Escherichia coli and Vibrio cholerae. To enhance the likelihood of successful molecule production, we established a selective pressure via the iron-chelating properties of siderophores. By supplementing cells containing our synthetic pathway with different precursors that are incorporated into the pathway independently of NRP enzymes, we generated over 20 predesigned, novel, and structurally diverse NRPs. This engineering approach, where phylogenetically related genes from different organisms are integrated and supplemented with novel precursors, should enable heterologous expression and molecular diversification of NRPs.

Striving for sustainable biosynthesis: discovery, diversification, and production of antimicrobial drugs in Escherichia coli

“…New pathway cloning strategies facilitate fast or even automated cloning and refactoring of BGCs for experimental characterization [ 38 , 42 , 99 , 100 ]. Several approaches have been used to assemble libraries of natural, engineered, or even combinatorial pathway variants [ 101–103 ], yet they remain to be applied for the discovery of antimicrobial NPs. Lastly, for the bioactivity screening step, the field is moving towards medium- and high-throughput approaches that enable parallel screening of larger pathway libraries.…”

Repurposing Modular Polyketide Synthases and Non-ribosomal Peptide Synthetases for Novel Chemical Biosynthesis

“…In many cases, the screening step is the bottleneck, which requires rapid and accurate selection of the desired clone among the extensive libraries. Leveraging the biological activity of the product for screening was the traditional screening assay logic that involves an inhibition zone assay for antibacterial activity, colorimetric assay for pigment product, and growth assay for the auxotrophic strain of the product such as siderophore (Balouiri et al, 2016;Cleto and Lu, 2017;Wehrs et al, 2019). As these methods were limited to specific products and the small size of the libraries, the universal and high-throughput screening strategies such as yeast surface display combined with FACS (Zhang et al, 2013), Ppant ejection strategy detecting the intermediate of the rate-limiting step by protease (Meluzzi et al, 2008), and biosensor development for the PKS and NRPS product such as the macrolide biosensor MphR (Kasey et al, 2018), along with mass spectrometry, would be a more favorable method.…”

Reconstruction of Secondary Metabolic Pathway to Synthesize Novel Metabolite in Saccharopolyspora erythraea

“…Heterologous production of natural products has attracted more attention in terms of microbial technology and the discovery of new active compounds (Cleto and Lu, 2017;Huo et al, 2019). Not only can it produce more valuable compounds and higher yield in more suitable heterologous hosts (Horbal and Luzhetskyy, 2016;Tan et al, 2017), but it can also dig out new compounds through biosynthetic engineering and metabolic engineering (Zhang et al, 2016;Lopatniuk et al, 2017;Reynolds et al, 2017).…”