Malate synthase from Streptomyces clavuligerus NRRL3585: cloning, molecular characterization and its control by acetate.
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 clavuligerus NRRL 3585, was cloned using PCR and fully sequenced. The ORF obtained encodes 541 amino acids with a deduced Mr of 60000, consistent with the observed Mr (62000-64000) of most malate synthase enzymes reported so far. The aceB gene has a high G+C content (71.5 mol%), 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 source 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 S. clavuligerus aceB gene was expressed in Escherichia coli BL21(DE3). Transformants exhibited an approximately 71-fold 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.
Chan
Maurice
M
Department of Microbiology, National University of Singapore,10 Kent Ridge Crescent, Singapore 1 19260.
Sim
Tiow-Suan
TS
Department of Microbiology, National University of Singapore,10 Kent Ridge Crescent, Singapore 1 19260.
D000085:Acetates / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology; D005798:Genes, Bacterial; D008292:Malate Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D000085:Acetates; D003470:Culture Media; D011994:Recombinant Proteins; D008292:Malate Synthase