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Published on: January 26, 2018
A 2-pyrone-4,6-dicarboxylic acid biosensor for lignin valorization based on a newly identified transcriptional
Kenta Tanatani1, Hyo Suzuki1, Masaya Fujita1
1Department of Materials Science and Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, 940-2188, Japan.
Background:
Lignin is the most abundant renewable aromatic resource on Earth, yet its efficient conversion into value-added chemicals through biological funneling remains a key challenge. Sphingobium lignivorans SYK-6 exhibits broad metabolic capacity for lignin-derived aromatic compounds, ranging from monomers to dimers, making it a suitable host for biological funneling. Production systems for the polymer building block 2-pyrone-4,6-dicarboxylic acid (PDC) from lignin-derived aromatic compounds have been constructed using SYK-6. In this study, we developed a PDC-responsive biosensor to facilitate high-throughput screening of improved PDC-producing strains.
Results:
PDC-assimilating bacteria were isolated from environmental samples, and their genomes were analyzed to obtain candidate PDC-responsive transcriptional regulatory systems. Among these, the LysR-type transcriptional regulator PdcR and its target promoter PPDC from Cupriavidus sp. 8B were identified and characterized as a PDC-responsive system, in which PdcR is suggested to act as both a repressor and an activator. A reporter plasmid pCup1 carrying these components was introduced into a PDC hydrolase gene-deficient strain (ΔligI) of SYK-6 to construct the PDC biosensor ΔligI(pCup1). The biosensor was specific for PDC, with no response observed for its upstream metabolites. When vanillic acid or protocatechuic acid was supplied as a substrate, concentration-dependent fluorescence responses were observed over a range of 0.1-5 mM, demonstrating that PDC production from lignin-derived aromatic compounds can be monitored. Fluorescence increased synchronously with PDC accumulation, suggesting real-time monitoring of PDC production. Furthermore, the biosensor responded to PDC produced from aromatic compounds in softwood black liquor extract.
Conclusions:
ΔligI(pCup1) enables real-time, specific detection of PDC produced from lignin-derived aromatic compounds, including those in black liquor extract derived from lignocellulosic biomass, demonstrating applicability to screening for lignin valorization. Integration of this biosensor with diverse strain engineering approaches, including random mutagenesis, adaptive laboratory evolution, and metagenomics-based enzyme discovery, would provide a platform for efficient selection of SYK-6 strains with enhanced PDC production capability.
