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Applied and Environmental Microbiology, April 2003, p. 2306-2312, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2306-2312.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Engineering Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Optimal Ring Expansion Activity toward Penicillin G

Chia-Li Wei,1 Yunn-Bor Yang,2 Wen-Ching Wang,3 Wen-Chi Liu,2 Jyh-Shing Hsu,1,2 and Ying-Chieh Tsai1*

Institute of Biochemistry, National Yang-Ming University, Taipei,1 Synmax Biochemical Co., Ltd., Hsinchu,2 Department of Life Sciences, National Tsing-Hua University, Hsinchu, Taiwan3

Received 16 September 2002/ Accepted 15 January 2003

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 kcat/Km 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 kcat/Km 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 kcat/Km 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.

* Corresponding author. Mailing address: Institute of Biochemistry, National Yang-Ming University, 155, Sec. 2, Li-Nong St., Pei-Tou, Taipei 11221, Taiwan. Phone: (886) 2-2826-7125. Fax: (886) 2-2826-4843. E-mail: tsaiyc{at}ym.edu.tw.

Applied and Environmental Microbiology, April 2003, p. 2306-2312, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2306-2312.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Goo, K. S., Chua, C. S., Sim, T.-S. (2008). Relevant Double Mutations in Bioengineered Streptomyces clavuligerus Deacetoxycephalosporin C Synthase Result in Higher Binding Specificities Which Improve Penicillin Bioconversion. Appl. Environ. Microbiol. 74: 1167-1175 [Abstract] [Full Text]  
  • Wei, C.-L., Yang, Y.-B., Deng, C.-H., Liu, W.-C., Hsu, J.-S., Lin, Y.-C., Liaw, S.-H., Tsai, Y.-C. (2005). Directed Evolution of Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Enhancement of Penicillin G Expansion. Appl. Environ. Microbiol. 71: 8873-8880 [Abstract] [Full Text]  
  • Hsu, J.-S., Yang, Y.-B., Deng, C.-H., Wei, C.-L., Liaw, S.-H., Tsai, Y.-C. (2004). Family Shuffling of Expandase Genes To Enhance Substrate Specificity for Penicillin G. Appl. Environ. Microbiol. 70: 6257-6263 [Abstract] [Full Text]  
  • Chin, H. S., Goo, K. S., Sim, T. S. (2004). A Complete Library of Amino Acid Alterations at N304 in Streptomyces clavuligerus Deacetoxycephalosporin C Synthase Elucidates the Basis for Enhanced Penicillin Analogue Conversion. Appl. Environ. Microbiol. 70: 607-609 [Abstract] [Full Text]  


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