Presentation Information
[P02-268]Biological Funneling of Industrial Black Liquor into a Polymer Building Block Using Engineered Sphingobium lignivorans SYK-6
○Naofumi Kamimura1, Mina Endo1, Banri Nakamura1, Takuya Akiyama2, Ryo Kato1, Mitsuru Kawazoe1, Masaya Fujita1, Takuma Araki3, Yuzo Suzuki3, Tsuyoshi Michinobu4, Yuichiro Otsuka3, Masaya Nakamura3, Eiji Masai1 (1. Department of Materials Science and Bioengineering, Nagaoka University of Technology (Japan), 2. Graduate School of Agricultural and Life Sciences, The University of Tokyo (Japan), 3. Department of Forest Resource Chemistry, Forestry and Forest Products Research Institute (Japan), 4. Department of Materials Science and Engineering, Institute of Science Tokyo (Japan))
Keywords:
Lignin,Black liquor,Sphingobium lignivorans,2-Pyrone-4,6-dicarboxylic acid,Biological funneling
[Purpose] Lignin is the second most abundant biopolymer on Earth; however, its effective utilization remains a major challenge due to its heterogeneous structure. Biological funneling, a strategy that channels heterogeneous lignin-derived aromatic compounds into a single target molecule through microbial catabolism, has emerged as a promising approach for lignin biorefineries. 2-Pyrone-4,6-dicarboxylic acid (PDC) is a promising polymer building block producible from lignin-derived aromatics via microbial fermentation. Here, we engineered the catabolic pathways of Sphingobium lignivorans SYK-6, a bacterium capable of utilizing a broad range of lignin-derived monomers and dimers, to enable efficient production of PDC from industrial black liquor (BL) generated during wood pulping.
[Method] BL from softwood (Japanese cedar) and hardwood (birch) was prepared by oxygen–soda–anthraquinone pulping, and the aromatic compound composition of the BL was analyzed by HPLC–MS. Gene disruption was performed by homologous recombination. Growth assays and biochemical analyses were conducted to identify genes responsible for the metabolic branching of syringyl-type compounds. PDC production was evaluated using model substrates and BL.
[Results] In softwood BL, dominated by guaiacyl-type aromatics such as vanillin, the PDC hydrolase-deficient mutant (ΔligI) achieved a PDC yield of 135 mol% based on the identified compounds. Hardwood BL was dominated by syringyl-type compounds, such as syringaldehyde, which were partially metabolized via branched pathways bypassing PDC. We identified desY, a previously uncharacterized gene encoding a 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoic acid hydrolase in the branched 3-O-methylgallate catabolic pathway. The triple mutant (ΔligI ligM desY) produced PDC at yields of 91 mol% from syringic acid and 62 mol% from birch BL.
[Consideration] A yield exceeding 100% from softwood BL suggests that SYK-6 converts unidentified compounds, potentially including aromatic oligomers, into PDC. This finding highlights the broad catabolic capacity of SYK-6 and its potential advantages over conventional microbial production hosts such as Pseudomonas putida. Identification of desY enabled metabolic engineering to redirect the catabolic flux of syringyl-type aromatics toward PDC.
[Conclusion] SYK-6 catabolizes an exceptionally broad range of lignin-derived aromatics in BL and serves as a robust microbial platform for PDC production. Identification of desY and subsequent pathway engineering significantly improved PDC production from syringyl-derived aromatics. These findings highlight the potential of engineered SYK-6 for efficient PDC production from diverse lignin streams through biological funneling.
[FUNDING] This work was supported by NARO BRAIN (01014B), JST COI-NEXT (JPMJPF2104), and JST CREST (JPMJCR23L4), JSPS KAKENHI 24K01891 and 25H01196, and the JSPS J-PEAKS JPJS00420240017.
[Method] BL from softwood (Japanese cedar) and hardwood (birch) was prepared by oxygen–soda–anthraquinone pulping, and the aromatic compound composition of the BL was analyzed by HPLC–MS. Gene disruption was performed by homologous recombination. Growth assays and biochemical analyses were conducted to identify genes responsible for the metabolic branching of syringyl-type compounds. PDC production was evaluated using model substrates and BL.
[Results] In softwood BL, dominated by guaiacyl-type aromatics such as vanillin, the PDC hydrolase-deficient mutant (ΔligI) achieved a PDC yield of 135 mol% based on the identified compounds. Hardwood BL was dominated by syringyl-type compounds, such as syringaldehyde, which were partially metabolized via branched pathways bypassing PDC. We identified desY, a previously uncharacterized gene encoding a 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoic acid hydrolase in the branched 3-O-methylgallate catabolic pathway. The triple mutant (ΔligI ligM desY) produced PDC at yields of 91 mol% from syringic acid and 62 mol% from birch BL.
[Consideration] A yield exceeding 100% from softwood BL suggests that SYK-6 converts unidentified compounds, potentially including aromatic oligomers, into PDC. This finding highlights the broad catabolic capacity of SYK-6 and its potential advantages over conventional microbial production hosts such as Pseudomonas putida. Identification of desY enabled metabolic engineering to redirect the catabolic flux of syringyl-type aromatics toward PDC.
[Conclusion] SYK-6 catabolizes an exceptionally broad range of lignin-derived aromatics in BL and serves as a robust microbial platform for PDC production. Identification of desY and subsequent pathway engineering significantly improved PDC production from syringyl-derived aromatics. These findings highlight the potential of engineered SYK-6 for efficient PDC production from diverse lignin streams through biological funneling.
[FUNDING] This work was supported by NARO BRAIN (01014B), JST COI-NEXT (JPMJPF2104), and JST CREST (JPMJCR23L4), JSPS KAKENHI 24K01891 and 25H01196, and the JSPS J-PEAKS JPJS00420240017.
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