Directed Mutation Of Deacetowcephalosporin C Synthase-the Key Enzyme In Synthesis Of Cephalosporins Intermediate

Streptomyces clavuligerus deacetoxycephalosporin C synthase (ScDAOCS) wasmodified using site-directed mutagenesis, iterative combionatorial mutagenesis, andtargeted mutation to improve its ability to catalyze the ring expansion reaction ofpenicillin G. Ten residues (Y184, V245, S261, C37, T42, V51, S59, A61, Q126, andT213) not directly involved in substrate recognition were selected as site-directedmutagenesis targets，Q126M, T213V, S261M, S261A, and Y184A showed improvedactivity toward penicillin G, with1.45-to4.50-fold increment in the kcat/Km; Aniterative combinatorial mutagenesis (ICM) strategy was used to engineerdeacetoxycephalosporin C synthase of Streptomyces clavuligerus (scDAOCS) forimproved activity toward penicillin G. Seven mutational sites were repeatedlycombined onto a starter mutant (C155Y Y184H V275I C281Y) of scDAOCS. Elevenimproved combinatorial mutants were identified from20mutants in four rounds ofICM and six improved combinatorial mutants showed46.4-to87.2-fold increases inkcat/Km relative to that of the WT enzyme; Based on the crystal structure ofStreptomyces clavuligerus DAOCS,2-oxoglutarate, and penicillin G triple complex,targeted mutation was used to engineering scDAOCS in four area near the substratebinding pocket. Twenty eight improved mutants were identified by bioassay. Andthree improved mutants were were identified using specific activity.