Understanding Thioamitide Biosynthesis Using Pathway Engineering and Untargeted Metabolomics

Abstract

Thiostreptamide S4 is a thioamitide, a family of promising antitumour ribosomally synthesised and post-translationally modified peptides (RiPPs). The thioamitides are one of the most structurally complex RiPP families, yet very few thioamitide biosynthetic steps have been elucidated, even though the gene clusters of multiple thioamitides have been identified. We hypothesised that engineering the thiostreptamide S4 gene cluster in a heterologous host could provide insights into its biosynthesis when coupled with untargeted metabolomics and targeted mutations of the precursor peptide. Modified gene clusters were constructed, and in-depth metabolomics enabled a detailed understanding of the biosynthetic pathway, including the identification of an effector-like protein critical for amino acid dehydration. We use this biosynthetic understanding to bioinformatically identify new widespread families of RiPP biosynthetic gene clusters, paving the way for future RiPP discovery and engineering.

Total In Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway

“…a) The order of modifications has been proposed based on untargeted metabolomic studies. The role of phosphotransferase (TsaE)and cysteine protease (TsaK) is not fully understood 2. b) The formation of AviMeCys is carried out by the concerted action of flavin-dependent oxidoreductase (SpaF) and lanthipeptide synthetase (SpaKC) located outside the gene cluster.…”

Genomic Comparisons and Phenotypic Diversity of Dickeya zeae Strains Causing Bacterial Soft Rot of Banana in China

“…Strains from banana showed significant diversity in this antibiosis phenotype because of different core components in their biosynthetic gene cluster. MS_2018 had genes similar to those encoding RiPP-like thiostreptamide ( Eyles et al, 2020 ); MS2 and MS_2014 contained one more specific NRP protein ( C1O30_RS05075 , HJ580_05040 ) with a domain similar to that of Streptoalloteichus tallysomycin biosynthesis genes; no such genes or proteins existed in MS1. This differentiation was according to their different abilities to inhibit bacterial growth.…”