Streptomyces clavuligerus is a gram-positive filamentous bacterium well known for its ability to produce an array of &amp / #61538 / -lactam compounds (secondary metabolites) including cephamycin C, clavulanic acid and other structurally related clavams. Of these, cephamycin C is a second generation cephalosporin antibiotic having great medical significance. Biosynthesis of the &amp / #946 / -lactam nucleus begins with the non-ribosomal condensation of L-&amp / #945 / -aminoadipic acid (&amp / #945 / -AAA), L-cysteine and L-valine to form the tripeptide &amp / #945 / -aminoadipiyl-cysteinyl-valine (ACV). In Streptomyces clavuligerus, &amp / #945 / -aminoadipic acid (&amp / #945 / -AAA) is a catabolic product of L-lysine produced from the lysine branch of the aspartate pathway and its biosynthesis represents a key secondary metabolic regulatory step in carbon flow to &amp / #946 / -lactam synthesis through this core pathway. The ask (aspartokinase)-asd (aspartate semialdehyde dehydrogenase) gene cluster which encodes for the first key enzymes of aspartate pathway has already been cloned from S. clavuligerus, characterized and heterologously expressed for the first time in our laboratory. Amplification of ask-asd cluster or ask gene alone in a multi-copy Streptomyces plasmid vector and determination of the effects of multiple copies on cephamycin C biosynthesis were the goals of the present study. For this purpose, three different strategies were employed. Of these, two strategies involving the use of vector pIJ702 did not work because of the instability of resulting recombinant plasmids. In the third and last strategy, we used another multicopy Streptomyces vector, pIJ486, which we showed in this study to be very stable for the same purpose. Meanwhile, an efficient protoplast transformation protocol was developed in our laboratory. Ask gene was cloned into this vector and S. clavuligerus protoplasts were efficiently transformed with the recombinant plasmid (pTB486) using the newly-developed protocol. After stable recombinants were obtained, the effects of the multiple copies of ask gene on cephamycin C biosynthesis were determined. There was a profound reduction in the rate and extent of growth of Ask overproducers, as experienced by testing two independent ask-multicopy recombinants. Although one such recombinant strain (designated S. clavuligerus TB 3585) had a 5.5 fold increased level of Ask activity as compared to the parental strain, it displayed only a 1.1 fold increase in specific production of cephamycin C.

Aspartokinase is the first enzyme of the aspartate family amino acids biosynthetic pathway. Cephamycin C is a &amp / #946 / -lactam antibiotic produced as a secondary metabolite via the enzymatic reactions in the lysine branch of this pathway in Streptomyces clavuligerus. The aspartokinase activity of S. clavuligerus is under concerted feedback inhibition by two of the end product amino acids, lysine plus threonine. It is also known that carbon flow through the lysine branch of the aspartate pathway is rate limiting step in the formation of cephamycin C. Therefore, genetic alterations in the regulatory regions of the aspartokinase enzyme are expected to lead to an increased cephamycin C production. The aim of this study was to obtain S. clavuligerus mutants that possess aspartokinase enzyme insensitive to feedback inhibition by lysine and threonine, identification of the mutation(s) accounting for the resistance being the ultimate goal. For this aim, chemical mutagenesis was employed to increase random mutation rate and a population of lysine anti-metabolite resistant S. clavuligerus mutants that can grow in the presence of S-(2-aminoethyl)-L-cysteine was obtained. The mutants were screened for their aspartokinase insensitivity via enzyme assays and one mutant exhibiting the highest level of deregulation was assessed for its cephamycin C production. The results revealed a 2-fold increase in specific production of the antibiotic. As a member of &amp / #946 / -lactam class antibiotics, cephamycin C has an importance in medicine. Therefore, the mutant strain obtained might be a candidate for industrial production of the compound.