Yoshihiro Katayama

Genetic and Biochemical Investigations on Bacterial Catabolic Pathways for Lignin-Derived Aromatic Compounds

Lignins are the most abundant aromatic compounds in nature, and their decomposition is essential to the terrestrial carbon cycle. White rot fungi secreting phenol oxidases are assumed to be involved in the initial degradation of native lignin, whereas bacteria play a main role in the mineralization of lignin-derived low-molecular-weight compounds in soil. There are a number of reports on the degradation pathways for lignin-derived aromatic compounds, but their catabolism has not been enzymatically or genetically characterized. Sphingomonas paucimobilis SYK-6 is one of the best-characterized lignin-degrading bacteria. It can grow on a wide variety of lignin-related biaryls and monoaryls, including β-aryl ether, biphenyl, diarylpropane, and phenylpropane. These compounds are degraded via the protocatechuate (PCA) 4,5-cleavage pathway or multiple 3-O-methylgallate (3MGA) catabolic pathways. In this review, the enzyme systems for β-aryl ether and biphenyl degradation, O demethylation linked with one carbon metabolism, the PCA 4,5-cleavage pathway, and the multiple 3MGA catabolic pathways in SYK-6 are outlined.

Efficient production of 2-pyrone 4,6-dicarboxylic acid as a novel polymer-based material from protocatechuate by microbial function

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 respectively encode protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase, into a broad host range plasmid vector, pKT230MC. The resulting plasmid, pDVABC, was introduced into the PpY1100 strain of Pseudomonas putida, and we found that PDC could be stably produced from protocatechuate 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 100xa0mM glucose and determined that PDC was stably produced from protocatechuate to yield 10xa0g/L or more.

Enzymatic properties of terephthalate 1,2-dioxygenase of Comamonas sp. strain E6.

The tphA1 II and tphA2 II A3 II genes of Comamonas sp. E6 perhaps code for the terephthalate (TPA) 1,2-dioxygenase (TPADO). To characterize E6 TPADO, these genes were expressed in a His-tagged form in Escherichia coli, and the recombinant proteins were purified. TPADO activity was reconstituted from TphA1II and TphA2IIA3II, indicating that TPADO consists of a reductase (TphA1II) and a terminal oxygenase component (TphA2II and TphA3II). TphA1II contains FAD, and the presence of a plant-type [2Fe-2S] cluster was suggested. These results indicate that TPADO is a class IB aromatic ring-hydroxylating dioxygenase. NADH and NADPH were effective as electron donors for TphA1II, but NADPH appeared to be the physiological electron donor, based on the kinetic parameters. TPADO showed activity only toward TPA, and Fe2+ was required for it. The Km values for TPA and the V max were determined to be 72±6 μM and 9.87±0.06 U/mg respectively.