Background

Lignocellulose is the most abundant renewable biomass resource on Earth, with approximately 20 billion tonnes regenerated globally each year. Heilongjiang Province produces about 90 million tonnes of crop straw annually, of which approximately 77 million tonnes are collectable, accounting for roughly one-tenth of the national total^[1].

Globally, the comprehensive utilization rate of lignocellulosic biomass remains high — yet this utilization is almost entirely concentrated in low-value outlets such as straw incorporation into fields (fertilization), animal feed, and direct combustion for heat and power, while industrial conversion projects offering high technological content and high added value remain extremely scarce^[2]. Therefore, how can lignin, the most recalcitrant aromatic component of this biomass, be utilized at a higher added value?

Lignin is composed of numerous benzene rings randomly interconnected through a variety of C–O–C ether bonds and carbon–carbon bonds, lacking any repeating unit that could be recognized in its entirety by a single enzyme system^[2-4].

Superimposed on this heterogeneous structure is the recalcitrance shaped over long-term plant evolution — highly cross-linked, hydrophobic, and chemically stable.

Although previous studies have already provided some solutions for the utilization of lignin from catalytic depolymerization to biological funneling, most biological strategies remain constrained by native catabolic logic. Diverse lignin-derived aromatics are typically funnelled into protocatechuate (PCA) or catechol, followed by aromatic ring cleavage and assimilation into central metabolism^[5-7]. Even state-of-the-art funneling systems, capable of converting diverse lignin-derived aromatics to PCA at up to 99.89%, remain challenged by substrate toxicity and metabolic flux imbalance^[8].

Inspired by established chemical strategies for lignin conversion, we developed a multienzyme funneling cascade to converge structurally diverse lignin-derived aromatic monomers into vanillic acid (VA)^[9-11], providing a unified entry point for downstream bioconversion.

However, VA has limited biological utilization routes. We therefore focused on chorismate (CHA), a central metabolic intermediate connected to diverse downstream products^[12]. Building on our laboratory’s previous establishment of an efficient 4-HB-to-CHA conversion, we designed a completely non-natural VA-to-4-HB-to-CHA pathway, creating a new biological route from lignin-derived VA to central metabolism and shifting the major bottleneck to the first step: demethoxylation of VA^[13,14].

[1]Paul, S. & Dutta, A. Challenges and opportunities of lignocellulosic biomass for anaerobic digestion. Resources, Conservation and Recycling. 130, 164-174 (2018). DOI: https://doi.org/10.1016/j.resconrec.2017.12.005
[2]Ragauskas, A. J., Beckham, G. T., Biddy, M. J., et al. Lignin valorization: Improving lignin processing in the biorefinery. Science 344, 1246843 (2014). DOI: https://doi.org/10.1126/science.12468433
[3]Zakzeski, J., Bruijnincx, P. C. A., Jongerius, A. L., et al. The catalytic valorization of lignin for the production of renewable chemicals. Chem. Rev. 110, 3552-3599 (2010). DOI: https://doi.org/10.1021/cr900354u
[4]Rinaldi, R., Jastrzebski, R., Clough, M. T., et al. Paving the way for lignin valorisation: Recent advances in bioengineering, biorefining and catalysis. Angew. Chem. Int. Ed. 55, 8164-8215 (2016). DOI: https://doi.org/10.1002/anie.201510351
[5]Linger, J. G., Vardon, D. R., Guarnieri, M. T., et al. Lignin valorization through integrated biological funneling and chemical catalysis. Proc. Natl. Acad. Sci. USA. 111, 12013-12018 (2014). DOI: https://doi.org/10.1073/pnas.1410657111
[6]Beckham, G. T., Johnson, C. W., Karp, E. M., et al. Opportunities and challenges in biological lignin valorization. Curr. Opin. Biotechnol. 42, 40-53 (2016). DOI: https://doi.org/10.1016/j.copbio.2016.02.030
[7]Johnson, C. W. & Beckham, G. T. Aromatic catabolic pathway selection for optimal production of Pyr and lactate from lignin. Metab. Eng. 28, 240-247 (2015). DOI: https://doi.org/10.1016/j.ymben.2015.01.005
[8]Xu, J., Zhang, J., Wang, X., et al. Engineering a robust and autonomous biological funneling for efficient valorization of lignin-related aromatics. Nat. Commun. 17, 7659 (2026). DOI: https://doi.org/10.1038/s41467-026-74424-y
[9]Guo, Y., Alvigini, L., Trajkovic, M., et al. Structure- and computational-aided engineering of an oxidase to produce isoeugenol from a lignin-derived compound. Nat. Commun. 13, 7195 (2022). DOI: https://doi.org/10.1038/s41467-022-34912-3
[10]De Simone, M., Alvigini, L., Alonso-Cotchico, L., et al. Rationally guided improvement of NOV1 dioxygenase for the conversion of lignin-derived isoeugenol to vanillin. Biochemistry. 62, 419-428 (2023). DOI: https://doi.org/10.1021/acs.biochem.2c00168
[11]Xu, L., Sethupathy, S., Liang, Z., et al. NAD⁺ regeneration-coupled enzymatic bioconversion of lignin-derived vanillin into VA: A cleaner production approach. Ind. Crops Prod. 222, 119921 (2024). DOI: https://doi.org/10.1016/j.indcrop.2024.119921
[12]Shende, V. V., Bauman, K. D. & Moore, B. S. The shikimate pathway: Gateway to metabolic diversity. Nat. Prod. Rep. 41, 604-648 (2024). DOI: https://doi.org/10.1039/D3NP00037K
[13]Abe, T., Masai, E., Miyauchi, K., et al. A tetrahydrofolate-dependent O-demethylase, LigM, is crucial for catabolism of vanillate and syringate in Sphingomonas paucimobilis SYK-6. J. Bacteriol. 187, 2030-2037 (2005). DOI: https://doi.org/10.1128/JB.187.6.2030-2037.2005
[14]Mallinson, S. J. B., Machovina, M. M., Silveira, R. L., et al. A promiscuous cytochrome P450 aromatic O-demethylase for lignin bioconversion. Nat. Commun. 9, 2487 (2018). DOI: https://doi.org/10.1038/s41467-018-04878-2