Overproduction and localization of components of the polyketide synthase of Streptomyces glaucescens involved in the production of the antibiotic tetracenomycin C.
Three proteins, including the beta-keto acyl synthase and the acyl carrier protein, involved in the synthesis of the polyketide antibiotic tetracenomycin C by Streptomyces glaucescens GLA.0 were produced in Escherichia coli by using the T7 RNA polymerase-dependent pT7-7 expression vector. Changing the N-terminal codon usage of two of the genes greatly increased the level of protein produced without affecting mRNA levels, suggesting improvements in translational efficiency. Western immunoblot analysis of cytoplasmic and membrane fractions of S. glaucescens with antibodies raised to synthetic oligopeptides corresponding to the two presumed components of the beta-keto acyl synthase indicated that both proteins were membrane bound; one appears to be proteolytically cleaved before or during association with the membrane. The beta-keto acyl synthase could be detected in stationary-phase cultures but not in rapidly growing cultures, correlating with the time of appearance of tetracenomycin C in the medium.
Department of Genetics, John Innes Institute, Norwich, England.
H C
HC
Gramajo
J
J
White
C R
CR
Hutchinson
M J
MJ
Bibb
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000235:genetics; D000213:Acyl Carrier Protein / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003062:Codon / Q000235:genetics; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D009279:Naphthacenes / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D010446:Peptide Fragments / Q000032:analysis / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D000213:Acyl Carrier Protein; D003062:Codon; D009097:Multienzyme Complexes; D009279:Naphthacenes; D010446:Peptide Fragments; C020938:tetracenomycin C; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase