Extracellular production of biologically active deacetoxycephalosporin C synthase from Streptomyces clavuligerus in Pichia pastoris

Biotechnol Bioeng. 2001 Nov 20;75(4):485-91. doi: 10.1002/bit.10028.

Abstract

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.

MeSH terms

  • Cephalosporins / biosynthesis
  • Clone Cells
  • Culture Media / analysis
  • Electroporation
  • Gene Expression
  • Gene Transfer Techniques
  • Hydrogen-Ion Concentration
  • Intramolecular Transferases / biosynthesis*
  • Methanol / pharmacology*
  • Penicillin G / metabolism
  • Penicillin-Binding Proteins*
  • Penicillins / metabolism
  • Periplasm / chemistry
  • Pichia / genetics*
  • Pichia / metabolism
  • Plasmids
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Solvents / pharmacology
  • Streptomyces / metabolism*
  • Substrate Specificity
  • Time Factors

Substances

  • Cephalosporins
  • Culture Media
  • Penicillin-Binding Proteins
  • Penicillins
  • Recombinant Proteins
  • Solvents
  • deacetoxycephalosporin G
  • Intramolecular Transferases
  • deacetoxycephalosporin C synthetase
  • penicillin N
  • Penicillin G
  • Methanol