Manipulation of Streptomyces clavuligerus for the purification of δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase and the mobilization of plasmid DNA

Abstract

Streptomyces clavuligerus is a filamentous bacterium renowned for its production of medicinally relevant natural products such as cephamycin C and clavulanic acid. Genome sequencing of this organism has revealed a treasure trove of cryptic/silent pathways, including a large linear plasmid with numerous secondary metabolite (SM) gene clusters. This research sought to transfer the S. clavuligerus megaplasmid (pSCL4) to a SM overproducing “super” strain, Streptomyces coelicolor (M1154), for the purpose of activating putative cryptic gene clusters encoded by it. Furthermore, synthetic constructs were designed for the overexpression and purification of mega-enzymes involved in producing important antibiotics in Streptomyces spp. The nonribosomal peptide synthetase, δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine Synthetase (ACVS), is a core enzyme in the biosynthesis of bacterial cephalosporins and penicillins. ACVS was fused to a 8 His-tag and expressed using the constitutive ermEp* promoter and was purified using nickel affinity chromatography. Reported here is the first targeted isolation of ACVS and the tools developed in this study can be applied to a variety of enzymes and SM gene clusters.