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

Moore, Marcus A. (2015) Manipulation of Streptomyces clavuligerus for the purification of δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase and the mobilization of plasmid DNA. Masters thesis, Memorial University of Newfoundland.

[English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (4MB)

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.

Actions (login required)

View Item