Directed evolution of deacetoxycephalosporin C synthase from Streptomyces clavuligerus for improved conversion of penicillin G

• Main Authors: Chia-Li Wei, 魏佳俐
• Other Authors: Ying-Chieh Tsai
• Format: Others
• Language: en_US
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/80179153100229021639

博士 === 國立陽明大學 === 生物化學研究所 === 91 === Cephalosporins are antibiotic compounds used clinically for the treatment of bacterial infection. Three pharmaceutically important oral cephalosporins, including cephradine, cephalexin and cephadroxil, are derived from 7-aminodeacetoxycephalosporanic acid (7-ADCA) to which different side chains are added. The industrial process to synthesize 7-ADCA mainly involves a chemical ring expansion of penicillin G to phenylacetyl-7-ADCA, and followed an enzymatic side chain cleavage of phenylacetyl-7-ADCA. The chemical ring expansion is complex and expensive, and causes a significant negative environmental impact. Therefore, an enzymatic reaction is greatly desirable to replace such chemical reaction. The primary object of this study was to engineer Streptomyces clavuligerus deacetoxycephalosporin C synthase (DAOCS) using directed evolution in an effort to improve conversion of penicillin G for industrial production of 7-ADCA. A random library of DAOCS mutants was firstly created using hydroxylamine as a chemical mutagen and subjected to a developing 2-step screening procedure. After analysis of 5,500 clones, three mutants G79E, V275I, and C281Y were selected and showed 2- to 6-fold increases in kcat/Km values compared to the wild-type enzyme. Another PCR-based random mutagenesis of the enzyme followed by screening of 6,400 clones identified additional 10 mutations, M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, L277Q, and I305L with 2- to 6-fold increased kcat/Km ratio. These mutants together with mutations N304K and I305M, which were produced by Dr. Yunn-Bor Yang (Synmax Biochemical) using site-directed mutagenesis and had 11- and 14-fold increased kcat/Km ratio, were further combined using DNA shuffling. After analysis of 10,600 clones, three mutants with 41-, 51-, and 60-fold increased kcat/Km ratio were thus obtained. This study showed clearly that directed evolution could be used to optimize DAOCS-penicillin G reaction. Prior to this work, it is only an approximately 2-fold increase in penicillin G conversion activity were found from mutants with modifications on the C-terminal part. Therefore, our work not only provided powerful DAOCSs for industrial usage, but also revealed more residues that are significantly involved in the enzymatic catalysis.