Clavam metabolite production in streptomyces: investigations into clavulanic acid and clavamycin biosynthesis

Abstract

Streptomyces are renowned for their ability to produce various bioactive compounds, such as antibiotics, antifungals, anti-tumor agents, and more. The clavam group of compounds include the clinically important ?-lactamase inhibitor clavulanic acid (CA) and the subgroup of antibiotics called the 5S clavams, both of which are produced by the organism Streptomyces clavuligerus. CA is industrially produced by fermentation of S. clavuligerus, during which a cluster of 16 genes that encode proteins and enzymes necessary for CA production are activated. The later steps in the CA biosynthetic pathway are not well understood, and the function of many of the gene products in the CA biosynthetic gene cluster (BGC) are unknown. Orf12, also known as cpe (for cephalosporin esterase), is one of these genes of unknown function, and it has been shown in previous studies to be essential for CA production. It has been recently demonstrated that when cpe is overexpressed in S. clavuligerus, there is production of a 5S clavam in conditions that does not normally permit its production. In this thesis, I investigated these findings and attempted to uncover the role of cpe in CA biosynthesis by making the equivalent cpe knockout and overexpression strains in Streptomyces katsurahamanus, another CA producer. Additionally, I performed untargeted metabolomic analysis to assess the effects of cpe disruption and overexpression on global metabolite production. Finally, I conducted co-culture experiments of the S. clavuligerus cpe knockout strain with various other CA non-producer knockout strains. I report that cpe overexpression does induce production of 2-hydroxymethylclavam (2-HMC) through the activation of genes in the 5S clavam pathway and thus propose that CPE has a secondary role in pathway signaling, contributing to the tight regulation of antibiotic production in S. clavuligerus.