Extracellular production of biologically active deacetoxycephalosporin C synthase from Streptomyces clavuligerus in Pichia pastoris.
We have successfully expressed and observed secretion of the Streptomyces clavuligerus deacetoxycephalosporin C synthase (DAOCS) using the Pichia pastoris expression system. Two clones having multiple copies of the expression cassette were selected and used for protein-expression analysis. SDS-PAGE showed efficient expression and secretion of the bacterial recombinant DAOCS. The highest yield (120 microg/mL) was obtained when expression was induced with 2% methanol. Free and immobilized protein were assayed for biological activity and found to expand penicillin N (its natural substrate) and penicillin G to deacetoxycephalosporin C (DAOC) and deacetoxycephalosporin G (DAOG), respectively.
Adrio
J L
JL
Laboratorio de Bioquímica, Departamento de I+D, Antibióticos, S.A.U., Avda. Antibioticos, 59-61, 24009-León, Spain.
Velasco
J
J
Soler
G
G
Rodriguez-Saiz
M
M
Barredo
J L
JL
Moreno
M A
MA
D002511:Cephalosporins / Q000096:biosynthesis; D002999:Clone Cells; D003470:Culture Media / Q000032:analysis; D018274:Electroporation; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006863:Hydrogen-Ion Concentration; D019751:Intramolecular Transferases / Q000096:biosynthesis*; D000432:Methanol / Q000494:pharmacology*; D010400:Penicillin G / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism; D019897:Periplasm / Q000737:chemistry; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012997:Solvents / Q000494:pharmacology; D013302:Streptomyces / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors
D002511:Cephalosporins; D003470:Culture Media; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D011994:Recombinant Proteins; D012997:Solvents; C405006:deacetoxycephalosporin G; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase; C006414:penicillin N; D010400:Penicillin G; D000432:Methanol