Malate synthase from Streptomyces clavuligerus NRRL3585: cloning, molecular characterization and its control by acetate

Abstract

SUMMARY: Malate synthase is a key enzyme of the glyoxylate cycle, which is an anaplerotic pathway essential for growth on acetate as the sole carbon source. The aceB gene, encoding malate synthase from Streptomyces clavuligems NRRL 3585, was cloned using PCR and fully sequenced. The ORF obtained encodes 541 amino acids with a deduced M, of 6OOOO, consistent with the observed M, (62000-64000) of most malate synthase enzymes reported so far. The aceB gene has a high G+C content (71.5 molO/O), especially in the third codon position. A 50 bp region upstream of the malate synthase ORF was predicted to be a prokaryotic promoter region. The relationship between carbon source, antibiotic (cephalosporin) biosynthesis and malate synthase activity was investigated. Growth of S. clavuligerus on acetate as the major carbon sorce was delayed, compared to that on glycerol. Furthermore, high levels of malate synthase activity were associated with the presence of acetate in the growth medium. Growth on acetate also resulted in lower levels of cephalosporin production, compared to that on glycerol. The cloned 5. clavuligerus aceB gene was expressed in Escherichia coli BL2l (DE3).Transformants exhibited an approximately 713old increase in malate synthase activity, compared to the control, thereby demonstrating high-level expression of soluble and enzymically active malate synthase in the heterologous host.