Increase of Clavulanic acid production by using recombinant Streptomyces clavuligerus strain including claR gene

Abstract

Introduction : Clavulanic acid is a major β-lactam antibiotic which is produced by Streptomyces clavuligerus. Clavulanic acid is used in combination of strong but sensitive to β-lactamase antibiotics. The claR gene has an important role in regulation of clavulanic acid production and is needed for the expression of the genes in final step of clavulanic acid biosynthesis. Materials and methods: The recombinant construct pMTclaR which contains claR gene is obtained from Isfahan University and plasmid extraction was done from Streptomyces lividans for next steps. The Streptomyces clavuligerus protoplast was prepared and transformation was done by using polyethylene glycol. Transformation was confirmed by plasmid extraction and PCR. Finally, bioassay method was used to survey the effect of extra copy of claR on clavulanic acid production . Results : The typical chalky white colony of Streptomyces clavuligerus was seen on GYME plates containing thiostrepton antibiotic. Plasmid extraction was initially carried out. Furthermore, PCR reaction was done by claR specific primers and the 1334 bp band which was belonging to claR was detected. Finally, the bioassay was done and the diameters of zone of inhibition in control and sample were compared. The results of the bioassay show that amplification of the claR gene in multicopy plasmids resulted in a 2.5 fold increase in clavulanic acid production . Discussion and conclusion : In this study the 3.3 fold increase in clavulanic acid production was obtained by using an expression vector containing claR. According to the clinical use of clavulanic acid, production of bacterial strains which are able to produce high level of antibiotic can help significantly in customization of antibiotic production.

Keywords

• Streptomyces clavuligerus
• Clavulanic acid
• Bioassay
• pMTclaR plasmid
• ClaR gene