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Applied and Environmental Microbiology, August 2009, p. 5195-5201, Vol. 75, No. 16
0099-2240/09/$08.00+0     doi:10.1128/AEM.00880-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of Three Alcohol Dehydrogenase Genes Involved in the Stereospecific Catabolism of Arylglycerol-β-Aryl Ether by Sphingobium sp. Strain SYK-6 ^,

Yusuke Sato,^1, Hideki Moriuchi,^1, Shojiro Hishiyama,² Yuichiro Otsuka,² Kenji Oshima,¹ Daisuke Kasai,¹ Masaya Nakamura,² Seiji Ohara,² Yoshihiro Katayama,³ Masao Fukuda,¹ and Eiji Masai^1*

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188,¹ Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687,² Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan³

Received 18 April 2009/ Accepted 11 June 2009

Degradation of arylglycerol-β-aryl ether is the most important process in bacterial lignin catabolism. Sphingobium sp. strain SYK-6 degrades guaiacylglycerol-β-guaiacyl ether (GGE) to -(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV), and then the ether linkage of MPHPV is cleaved to generate -glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol. We have characterized three enantioselective glutathione S-transferase genes, including two genes that are involved in the ether cleavage of two enantiomers of MPHPV and one gene that is involved in the elimination of glutathione from a GS-HPV enantiomer. However, the first step in the degradation of four different GGE stereoisomers has not been characterized. In this study, three alcohol dehydrogenase genes, ligL, ligN, and ligO, which conferred GGE transformation activity in Escherichia coli, were isolated from SYK-6 and characterized, in addition to the previously cloned ligD gene. The levels of amino acid sequence identity of the four GGE dehydrogenases, which belong to the short-chain dehydrogenase/reductase family, ranged from 32% to 39%. Each gene was expressed in E. coli, and the stereospecificities of the gene products with the four GGE stereoisomers were determined by using chiral high-performance liquid chromatography with recently synthesized authentic enantiopure GGE stereoisomers. LigD and LigO converted (R,βS)-GGE and (R,βR)-GGE into (βS)-MPHPV and (βR)-MPHPV, respectively, while LigL and LigN transformed (S,βR)-GGE and (S,βS)-GGE to (βR)-MPHPV and (βS)-MPHPV, respectively. Disruption of the genes indicated that ligD is essential for the degradation of (R,βS)-GGE and (R,βR)-GGE and that both ligL and ligN contribute to the degradation of the two other GGE stereoisomers.

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* Corresponding author. Mailing address: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan. Phone and fax: 81-258-47-9428. E-mail: emasai{at}vos.nagaokaut.ac.jp

Published ahead of print on 19 June 2009.

Supplemental material for this article may be found at http://aem.asm.org/.

Y.S. and H.M. equally contributed to this work.

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Applied and Environmental Microbiology, August 2009, p. 5195-5201, Vol. 75, No. 16
0099-2240/09/$08.00+0     doi:10.1128/AEM.00880-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.