Appl Microbiol Biotechnol (2006) 71: 608–614 DOI 10.1007/s00253-005-0203-7 BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING Yuichiro Otsuka . Masaya Nakamura . Kiyotaka Shigehara . Kosuke Sugimura . Eiji Masai . Seiji Ohara . Yoshihiro Katayama Efficient production of 2-pyrone 4,6-dicarboxylic acid as a novel polymer-based material from protocatechuate by microbial function Received: 11 June 2005 / Revised: 18 September 2005 / Accepted: 23 September 2005 / Published online: 2 December 2005 # Springer-Verlag 2005 Abstract Sphingomonas paucimobilis SYK-6, which can degrade various low molecular weight compounds derived from plant polyphenols such as lignin, lignan, and tannin, metabolizes these substances via 2-pyrone-4,6-dicarboxylic acid (PDC). We focused on this metabolic intermediate as a potential raw material for novel, bio-based polymers. We cloned the ligAB and ligC genes of SYK-6, which re- spectively encode protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydro- genase, into a broad host range plasmid vector, pKT 230MC. The resulting plasmid, pDVABC, was introduced into the PpY1100 strain of Pseudomonas putida, and we found that PDC could be stably produced from proto- catechuate and accumulated. In addition, we examined the efficiency of production of PDC from protocatechuate on a 5-L scale in a Luria–Bertani medium containing 100 mM glucose and determined that PDC was stably produced from protocatechuate to yield 10 g/L or more. Introduction Lignin is the most abundant natural aromatic biomass. In trees, high levels of lignin are synthesized in wood and account for 15–36% of the dry weight of wood. However, because lignin forms a highly complicated three-dimen- sional network in which the monolignols synthesized in plant cells are randomly polymerized, an advanced system for the utilization of lignin as biomass has not been established. In addition, the chemical treatment system of lignin, such as aqueous alkaline oxidation, can decompose high molecular lignin to low molecular compounds, and the various low molecular compounds were produced. However, only few parts of these compounds are utilized for aroma chemicals, etc., and almost these compounds are burned or discarded (Parke et al. 2000). On the other hand, various soil microorganisms in nature degrade these various aromatic substances via complicated metabolic pathways and utilize it as an energy source. Sphingomonas paucimobilis SYK-6 that was isolated from pulping waste liquor is able to grow on various dimeric lignin com- pounds, including β-aryl ether, biphenyl, and diarylpropane, as sole carbon and energy sources (Masai et al. 1999), and we have characterized the enzymes and genes in- volved in β-aryl ether cleavage (Masai et al. 1991; 1993) and biphenyl degradation (Peng et al. 1998; 1999) by this bacterium. The unique, specific degradation enzymes for various lignin derivatives in SYK-6 would be suitable tools for the conversion of lignin to useful intermediate metabolites. The metabolic pathways of degradation of low molec- ular lignin compounds by SYK-6, which have been identified in previous studies, are shown in Fig. 1. By these metabolic pathways, various low molecular weight lignin compounds are all converted within SYK-6 cells into such compounds as vanillin, vanillate, and syringate. After conversion into the metabolic intermediate 2-pyrone-4, 6-dicarboxylic acid (PDC) via the protocatechuate 4,5- cleavage pathway, they are then completely degraded (Nishikawa et al. 1998; Noda et al. 1990; Hara et al. 2000; Sonoki et al. 2000). Therefore, in SYK-6 cells, various low molecular weight lignin compounds converge for transfor- mation into PDC and are subsequently degraded. We focused here on the study of PDC, a substance with a unique structure containing a pyran ring with two carbox- ylic acid groups. Artificial organic synthesis of this structure for mass production is desirable but has not yet been achieved. Also, because PDC has two carboxylic acid groups, the conversion of these groups into derivatives Y. Otsuka . M. Nakamura . S. Ohara Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan K. Shigehara . K. Sugimura . Y. Katayama (*) Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, 184-8588 Tokyo, Japan e-mail: kyamam@cc.tuat.ac.jp Tel.: +81-42-3887364 Fax: +81-42-3887364 E. Masai Nagaoka University of Technology, Nagaoka, Niigata, Japan