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Applied and Environmental Microbiology, December 2005, p. 8873-8880, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8873-8880.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Directed Evolution of Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Enhancement of Penicillin G Expansion

Chia-Li Wei,¹ Yunn-Bor Yang,² Chan-Hui Deng,² Wen-Chi Liu,² Jyh-Shing Hsu,^1,2 Yu-Ching Lin,¹ Shwu-Huey Liaw,^1,3* and Ying-Chieh Tsai^1*

Institute of Biochemistry,¹ Faculty of Life Science, National Yang-Ming University, Taipei,³ Synmax Biochemical Co., Ltd., Hsinchu, Taiwan²

Received 25 April 2005/ Accepted 20 August 2005

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 k_cat/K_m 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 k_cat/K_m 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.

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* 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 for S.-H. Liaw: shliaw{at}ym.edu.tw. E-mail for Y.-C. Tsai: tsaiyc{at}ym.edu.tw.

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Applied and Environmental Microbiology, December 2005, p. 8873-8880, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8873-8880.2005
Copyright © 2005, 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]