Biochemical and genetic investigations for the production of β-lactam metabolites from Streptomyces clavuligerus ATCC27064 and Streptomyces pratensis ATCC33331

Abstract

Streptomyces are recognized for their ability to produce a wide range of antimicrobial agents. In this thesis, two of the Streptomyces bacteria, Streptomyces clavuligerus and Streptomyces pratensis were the subject of my investigations. Streptomyces clavuligerus has the ability to produce a diverse set of β-lactams compounds; the β-lactamase inhibitor clavulanic acid (CA), the 5S clavams, and cephamycin C (Ceph-C). I first investigated the molecular features of two genes, cpe (orf12) and orf14, from the CA biosynthetic gene cluster (BGC) of S. clavuligerus and their roles in CA and 5S clavam biosynthesis. The two genes are essential for the production of CA since deletion of any of them abolishes CA biosynthesis. The two genes are thought to be involved in converting clavaminic acid into clavaldehyde during the late steps of CA biosynthesis. Different protein variants of CPE and ORF14 were prepared by site-directed mutagenesis and used to investigate their effects on CA and 5S clavam production. In addition, the regulatory impact of cpe on the transcription level of CA and 5S clavam biosynthetic genes was tested in cpe deleted and overexpressed mutants compared to the wild-type strain. Next, a comparative genomic study was conducted for CA and CA-like BGCs between the CA producer (S. clavuligerus) and non-producers (Streptomyces pratensis, Saccharomonospora. viridis, and Streptomyces sp. M41). One of the main differences is a large gene, nocE, resides within the CA-like BGCs of the non-producers species. Deletion and overexpression of nocE in S. clavuligerus were achieved, and their effects on physiology, general metabolism, and specialized metabolism were investigated. In the third part of the study, genomic analysis for BGCs was conducted for the S. pratensis ATCC33331 genome, which predicted 27 BGCs. None of the predicted products has been previously reported to be produced by this species. Therefore, I followed the one strain many compounds (OSMAC) approach to investigate the ability of S. pratensis to produce specialized metabolites. Our results revealed the production of a bioactive substance (SB) that has antimicrobial activity against Klebsiella pneumoniae. Biochemical and genetic procedures were performed to discover the identity of this substance.