Engineering Streptomyces clavuligerus deacetoxycephalosporin C synthase for optimal ring expansion activity toward penicillin G.

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  • Author(s): Wei CL;Wei CL; Yang YB; Wang WC; Liu WC; Hsu JS; Tsai YC
  • Source:
    Applied and environmental microbiology [Appl Environ Microbiol] 2003 Apr; Vol. 69 (4), pp. 2306-12.
  • Publication Type:
    Evaluation Study; Journal Article; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: American Society for Microbiology Country of Publication: United States NLM ID: 7605801 Publication Model: Print Cited Medium: Print ISSN: 0099-2240 (Print) Linking ISSN: 00992240 NLM ISO Abbreviation: Appl Environ Microbiol Subsets: MEDLINE
    • Publication Information:
      Original Publication: Washington, American Society for Microbiology.
    • Subject Terms:
      Cephalosporins/*chemistry ; Cephalosporins/*metabolism ; Penicillin G/*metabolism ; Streptomyces/*enzymology ; Streptomyces/*genetics; Amino Acid Substitution ; Chromatography, High Pressure Liquid ; Genetic Engineering/methods ; Industrial Microbiology ; Models, Molecular ; Mutagenesis, Site-Directed ; Penicillin G/chemistry
    • Abstract:
      The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k(cat)/K(m) ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k(cat)/K(m) values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k(cat)/K(m) ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction.
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    • Accession Number:
      0 (Cephalosporins)
      26924-74-3 (deacetoxycephalosporin C)
      Q42T66VG0C (Penicillin G)
    • Publication Date:
      Date Created: 20030405 Date Completed: 20030624 Latest Revision: 20210526
    • Publication Date:
      20240829
    • Accession Number:
      PMC154807
    • Accession Number:
      10.1128/AEM.69.4.2306-2312.2003
    • Accession Number:
      12676714