The dacA gene of Bacillus stearothermophilus coding for D-alanine carboxypeptidase: cloning, structure and expression in Escherichia coli and Pichia pastoris.
The bacterial D-alanine carboxypeptidases (CPases) remove C-terminal D-alanyl residues from sugar-peptide cell wall precursors. The CPases have many characteristics in common with the high-M(r) penicillin-binding proteins (PBPs) whose inhibition by beta-lactam antibiotics is lethal. The CPases are attractive as model PBPs, because of their relatively lower M(r) and higher activity in vitro. We have cloned and sequenced the Bacillus stearothermophilus gene (dacA) coding for a membrane-bound CPase. The nucleotide (nt) sequence of the gene is homologous to that of the Escherichia coli and Bacillus subtilis dacA loci, which also code for membrane-bound CPases. E. coli host cells lysed when expression of B. stearothermophilus dacA was induced. The same coding sequence was expressed in the methylotrophic yeast, Pichia pastoris, using the alcohol oxidase-1 (AOX1) promoter. Over 100 micrograms/ml of CPase was efficiently secreted into the medium after induction by methanol, without adversely affecting this host. The yeast product is indistinguishable from the native enzyme in structure and activity. The ability to secrete large amounts of heterologous protein and the lack of endogenous peptidoglycan metabolism makes P. pastoris an attractive candidate for the production of PBPs.
Despreaux
C W
CW
Department of Molecular Genetics, Hoffmann-La Roche, Inc., Nutley, NJ 07110.
Manning
R F
RF
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000737:chemistry; D003001:Cloning, Molecular; D004926:Escherichia coli; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics*; D008961:Models, Structural; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010403:Penicillin Resistance; D010406:Penicillins / Q000378:metabolism; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D002352:Carrier Proteins; D005656:Fungal Proteins; D010406:Penicillins; D011993:Recombinant Fusion Proteins; D002267:Muramoylpentapeptide Carboxypeptidase