Directed evolution of Streptomyces clavuligerus deacetoxycephalosporin C synthase for enhancement of penicillin G expansion.
The deacetoxycephalosporin C synthase from Streptomyces clavuligerus was directly modified for enhancement of penicillin G expansion into phenylacetyl-7-aminodeacetoxycephalosporanic acid, an important intermediate in the industrial manufacture of cephalosporin antibiotics. Nine new mutants, mutants M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, and L277Q with 1.4- to 5.7-fold increases in the kcat/Km ratio, were obtained by screening 6,364 clones after error-prone PCR-based random mutagenesis. Subsequently, DNA shuffling was carried out to screen possible combinations of substitutions, including previous point mutations. One quaternary mutant, the C155Y/Y184H/V275I/C281Y mutant, which had a kcat/Km ratio that was 41-fold higher was found after 10,572 clones were assayed. The distinct mutants obtained using different mutagenesis methods demonstrated the complementarity of the techniques. Interestingly, most of the mutated residues that result in enhanced activities are located within or near the unique small barrel subdomain, suggesting that manipulation of this subdomain may be a constructive strategy for improvement of penicillin expansion. Several mutations had very distinct effects on expansion of penicillins N and G, perhaps due to different penicillin-interacting modes within the enzyme. Thus, the present study provided not only promising enzymes for cephalosporin biosynthesis but also a large number of mutants, which provided new insights into the structure-function relationship of the protein that should lead to further rational engineering.
Wei
Chia-Li
CL
Institute of Biochemistry, National Yang-Ming University, 155, Sec. 2, Li-Nong St., Pei-Tou, Taipei 11221, Taiwan.
Yang
Yunn-Bor
YB
Deng
Chan-Hui
CH
Liu
Wen-Chi
WC
Hsu
Jyh-Shing
JS
Lin
Yu-Ching
YC
Liaw
Shwu-Huey
SH
Tsai
Ying-Chieh
YC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019020:Directed Molecular Evolution / Q000379:methods*; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010400:Penicillin G / Q000378:metabolism*; D046915:Penicillin-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010406:Penicillins / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*
D046915:Penicillin-Binding Proteins; D010406:Penicillins; D011994:Recombinant Proteins; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase; D010400:Penicillin G