PCR-mediated screening and cloning of a malate synthase genefrom Streptomyces griseus NCIMB 9001

Malate synthase is an essential metabolic enzyme of the glyoxylate bypass t hat makes possible the replenishment of carbon intermediates to cells grown on acetate. A polymerase chain reaction (PCR)-based molecular screening in vestigation of full-length malate synthase genes from Streptomyces spp. was initiated by our group. To this end, consensus primers were designed based on known streptomycete malate synthase sequences and successful amplificat ion was obtained for Streptomyces griseus, S. fimbriatus and S. lipmanii. T he putative full-length malate synthase gene from S. griseus was subsequent ly cloned, sequenced and expressed. Sequence analysis of this gene showed v ery high identity with other streptomycete malate synthase genes. Furthermo re, high malate synthase activity was detected after heterologous expressio n in Escherichia coli, thus demonstrating successfully the rapid cloning an d functional verification of a streptomycete malate synthase gene. Growth s tudies of S. griseus revealed that malate synthase activity was induced by the presence of acetate, which is a two-carbon source. Interestingly, the a ctivity peaked during late growth phase when the biomass was declining, sug gesting that the enzyme may have a late role in metabolism.