Family shuffling of expandase genes to enhance substrate specificity for penicillin G.
Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the k(cat)/K(m) ratio compared to the S. clavuligerus expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds.
Hsu
Jyh-Shing
JS
Institute of Biochemistry, National Yang-Ming University, 155 Li-Nong St., Sec. 2, Pei-Tou, Taipei 11221, Taiwan.
Yang
Yunn-Bor
YB
Deng
Chan-Hui
CH
Wei
Chia-Li
CL
Liaw
Shwu-Huey
SH
Tsai
Ying-Chieh
YC
D039903:Actinobacteria / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002678:Chimera / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D057927:Hydrophobic and Hydrophilic Interactions; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010400:Penicillin G / Q000378:metabolism*; D046915:Penicillin-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D013379:Substrate Specificity
D046915:Penicillin-Binding Proteins; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase; D010400:Penicillin G