J Wood Sci (2002) 48:434-439 9 The Japan Wood Research Society 2002 Tomonori Sonoki 9Yuichiro Otsuka 9Seiichiro Ikeda Eiji Masai 9Shinya Kajita 9 Yoshihiro Katayama Tetrahydrofolate-dependent vanillate and syringate O-demethylation links tightly to one-carbon metabolic pathway associated with amino acid synthesis and DNA methylation in the lignin metabolism of Sphingomonaspaucimobilis SYK-6 Received: September 25, 2001 / Accepted: November 30, 2001 Abstract Sphingomonas paucimobilis SYK-6 strain can de- grade various lignin-related compounds. In the lignin meta- bolic pathway of this bacterium, vanillate and syringate are demethylated by the tetrahydrofolate (THF)-dependent O-demethylation system, which requires the enzymatic function of LigH. Upstream of the ligH gene is the 5,10- methylene-THF reductase gene. Its gene product was es- sential for one-carbon metabolism involved in the amino acid synthesis and DNA methylation in all organisms. When the metFgene was inactivated in the genome of SYK- 6, the resultant mutant, DLmetF, could not grow on vanil- late and syringate as a sole carbon source. Furthermore, DLmetF showed significant accumulation of methyl-THF as a result of vanillate and syringate O-demethylation. We report here that THF-dependent vanillate and syringate O- demethylation links tightly to the one-carbon metabolic pathway that is associated with amino acid synthesis and DNA methylation, and the methyl group is the sole one- carbon source in S. paucimobilis SYK-6. Key words Lignin biodegradation 9 Tetrahydrofolate 9 One-carbon metabolism Introduction Lignin is the most abundant aromatic compound in the biosphere, and its degradation is a significant step in the global carbon cycle. Sphingomonas paucimobilis SYK-6, a bacterium that can grow on 5,5'-dehydrodivanillic acid T. Sonoki ([]) 9 Y. Otsuka - S. Ikeda. S. Kajita. Y. Katayama Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan Tel. +81-42-388-7364; Fax +81-42-388-7364 e-mail: tomonori.sonoki@kyowa.co.jp E. Masai Department of Bioengineering, Nagaoka University of Engineering, Nigata 940-2188, Japan (2,2'-dihydroxy-3,3'-dimethoxy-5,5'-dicarboxybiphenyl) (DDVA) as a sole carbon source, was isolated from pulp- bleaching wastewater in Japan. This bacterium can also grow on several dimeric model compounds of lignin such as fi-aryl ether, phenylcoumarane, pinoresinol, and diarylpro- pane. The metabolic pathway of DDVA and other dimeric model compounds of lignin in this bacterium was previously reported. 1'2 The ability of S. paucirnobilis SYK-6 to grow on several low-molecular-weight lignin compounds as a sole source of carbon and energy indicates that SYK-6 strain produces unique and specific enzymes, such as O- demethylases, fi-etherases, and ring fission dioxygenases. We have identified several genes and characterized the function of these genes involved in this pathwayY 4 In the lignin metabolic pathway of S. paucimobilis SYK-6, the methylether cleavage (O-demethylation) step is essential for the substrate of enzymes such as LigZ 4 and LigAB.9'~~ S. paucimobilis SYK-6 has O-demethylation systems for three substrates [5,5'-dehydrodivanillic acid DDVA, syringate, vanillate] in the metabolic pathway (Fig. 1). DDVA-specific O-demethylation is an oxygenative system catalyzed by the enzyme encoded by the ligX gene. 12 On the other hand, vanillate- and syringate-specific O-demethylation is a tetrahydrofolate (THF)-dependent system required the enzyme function encoded by the ligH gene. 3 The role of THF in the biologic steps is the one-carbon (C1) carrier for amino acid (e.g., methionine, serine) synthesis, DNA methylation, acetyl-coenzyme A (CoA) synthesis, and nucleic acid synthesis. Generally, the THF- dependent C1 metabolic pathway consists of four enzymes: formyl-THF synthetase (EC 6.3.4.3); 5,10-methylene-THF dehydrogenase (EC 1.5.1.5); 5,10.methenyl-THF cyclohydrolase (EC 3.5.4.9); and 5,10-methylene-THF re- ductase (MetF; EC 1.5.1.20) t5 17 (see Fig. 6). In the present study, we revealed the role of THF- dependent O-demethylation of vanillate and syringate in lignin metabolism of S. paucimobilis SYK-6. In the lignin- degrading bacterium S. paucimobilis SYK-6, THF- dependent O-demethylation is tightly linked to the C1 metabolic pathway, especially MetF. The methyl group in the chemical structure of lignin is essential for C1 anabolic