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J. Biochem, 2004, Vol. 135, No. 3 305-317
© 2004 The Japanese Biochemical Society

BIOTECHNOLOGY

Molecular Breeding of 2,3-Dihydroxybiphenyl 1,2-Dioxygenase for Enhanced Resistance to 3-Chlorocatechol

Kouhei Ohnishi*,§,1, Akiko Okuta,1, Jiansong Ju2, Tohru Hamada{ddagger},1, Haruo Misono3 and Shigeaki Harayama++,1

1 Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001; 2 Department of Applied Bioresource Science, The United Graduate School of Agricultural Sciences, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566; and 3 Department of Bioresources Science, Faculty of Agriculture, Kochi University, 200 Monobe, Nankoku, Kochi 783-8502

3-Chlorobiphenyl is known to be mineralized by biphenyl-utilizing bacteria to 3-chlorobenzoate, which is further metabolized to 3-chlorocatechol. An extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHB12O; EC 1.13.11.39), which is encoded by the bphC gene, catalyzes the third step of the upper pathway of 3-chlorobiphenyl degradation. In this study, two full-length bphCs and nine partial fragments of bphCs fused to the 3' end of bphC in Pseudomonas pseudoalcaligenes KF707 were cloned from different biphenyl-utilizing soil bacteria and expressed in Escherichia coli. The enzyme activities of the expressed DHB12Os were inhibited to varying degrees by 3-chlorocatechol, and the E. coli cells overexpressing DHB12O could not grow or grew very slowly in the presence of 3-chlorocatechol. These sensitivities of enzyme activity and cell growth to 3-chlorocatechol were well correlated, and this phenomenon was employed in screening chimeric BphCs formed by family shuffling of bphC genes isolated from Comamonas testosteroni KF704 and C. testosteroni KF712. The resultant DHB12Os were more resistant by a factor of two to 3-chlorocatechol than one of the best parents, KF707 DHB12O.

* To whom correspondence should be addressed. Research Institute of Molecular Genetics, Kochi University. Tel: +81-88-864-5213, Fax: +81-88-864-5109, E-mail: kouheio{at}rimg.kochi-u.ac.jp

§ Present address: Research Institute of Molecular Genetics, Kochi University, 200 Monobe, Nankoku, Kochi 783-8502

Present address: Japan Biological Information Research Center, JBIC, 2-41-6 Aomi, Kouto-ku, Tokyo 135-0064

{ddagger} Present address: Intellectual Property Division, Gifu R & D Center, Amano Enzyme Inc, 4-179-35 Sue-cho, Kakamigahara, Gifu 509-0108

++ Present address: NITE Biological Resource Center, National Institute of Technology and Evaluation, 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818


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