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Sphingobium sp. strain SYK-6 expresses the best characterized catabolic systems for lignin-derived aromatic compounds. However, the uptake systems for these aromatics remain unknown. In this study, we identified and characterized the protocatechuate (PCA) transporter gene SLG_12880 (pcaK) in SYK-6. Sequence comparisons revealed that PcaK belongs to the aromatic acid/H + symporter family of major facilitator superfamily transporters. Further, pcaK was highly conserved among Sphingomonadales possessing catabolic genes for vanillate and PCA. The growth of an SYK-6 pcaK mutant was significantly delayed on PCA medium and PCA uptake rate was only 8% of wild type. In addition, vanillate uptake rate was 78% of wild type, although the pcaK mutant grew as well as the wild type on vanillate. When pcaK was expressed in Sphingobium japonicum UT26S, the transformant acquired the capacity to uptake both PCA and vanillate. These results indicate that pcaK is responsible for the major proportion of PCA uptake and a minor fraction of vanillate uptake in SYK-6. The productivity of 2-pyrone-4,6-dicarboxylate (PDC), a building block of functional polymers, was evaluated using a PDC hydrolase SYK-6 mutant harboring a pcaK plasmid. The mutant exhibited 1.27-fold greater PCA conversion and 1.24-fold greater PDC production compared to the control strain, suggesting that enhanced expression of transporter genes for lignin-derived aromatics can be used to increase the production of value-added metabolites.

Pinoresinol reductase and pinoresinol/lariciresinol reductase play important roles in an early step of lignan biosynthesis in plants. The activities of both enzymes have also been detected in bacteria. In this study, pinZ, which was first isolated as a gene for bacterial pinoresinol reductase, was constitutively expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Higher reductive activity toward pinoresinol was detected in the resultant transgenic plants but not in wild-type plant. Principal component analysis of data from untargeted metabolome analyses of stem, root, and leaf extracts of the wild-type and two independent transgenic lines indicate that pinZ expression caused dynamic metabolic changes in stems, but not in roots and leaves. The metabolome data also suggest that expression of pinZ influenced the metabolisms of lignan and glucosinolates but not so much of neolignans such as guaiacylglycerol-8-O-4'-feruloyl ethers. In-depth quantitative analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) indicated that amounts of pinoresinol and its glucoside form were markedly reduced in the transgenic plant, whereas the amounts of glucoside form of secoisolariciresinol in transgenic roots, leaves, and stems increased. The detected levels of lariciresinol in the transgenic plant following β-glucosidase treatment also tended to be higher than those in the wild-type plant. Our findings indicate that overexpression of pinZ induces change in lignan compositions and has a major effect not only on lignan biosynthesis but also on biosynthesis of other primary and secondary metabolites.