Abstract
Monolignol-derived dimers containing β–β linkages are synthesized by vascular plants and can be released during lignin depolymerization. In this work, we isolated a bacterium, Novosphingobium rhizosphaerae LY, that grows with the β–β lignan (+)-pinoresinol as a sole growth substrate. Sequence analysis suggested that this strain encodes a broad range of pathways for assimilation of aromatic monomers as well as one enzyme implicated in pinoresinol catabolism but lacks other known pathways for aromatic dimer catabolism. We constructed a genome-wide barcoded transposon library and identified genes required for pinoresinol catabolism. Using feeding studies, compound isolation, targeted synthesis, and analysis of purified enzymes, we elucidated the biochemical intermediates and reaction pathway involved in pinoresinol catabolism. We demonstrated that the first enzymatic reaction is the reductive cleavage of a furan ring in (±)-pinoresinol with retention of configuration to yield lariciresinol. We addition ally confirmed that the final pathway enzyme, PinU, is related to lignostilbene dioxy genases and oxidatively cleaves a diguaiacylbutadiene intermediate to yield vanillin and coniferaldehyde. Finally, based on the enzyme characterization, we demonstra ted that the strain can grow with a second β–β lignan, (–)-syringaresinol, as a sole growth substrate. In combination, these results demonstrate a new biocatalytic route for transforming a widely occurring group of plant phenylpropanoid natural products. IMPORTANCE Plants synthesize a variety of aromatic phenylpropanoid compounds containing β–β linkages, including lignin, a major structural polymeric component of the vascular plant cell wall, and lignans, biochemically related secondary metabolites with a wide range of bioactivities. Although microbial catabolic pathways have been described for dimeric phenylpropanoids featuring other interunit linkages, relatively little is known about pathways for catabolism of β–β-linked compounds. In this work, we isolated a Novosphingobium strain capable of degrading the β–β-linked lignan (+)-pinor esinol and elucidated the catabolic pathway. Understanding how bacteria catabolize β–β-linked compounds provides a basis for new biocatalytic transformations of lignans and oligolignols and has the potential to improve bacterial lignin valorization.
| Original language | English |
|---|---|
| Journal | mBio |
| Volume | 16 |
| Issue number | 11 |
| DOIs | |
| State | Published - Jan 2025 |
Funding
J.N.E., and J.K.M.) and metabolite analysis (R.J.G.) were supported by the Center for Bioenergy Innovation (CBI), U.S. Department of Energy, of Science, Biological and Environmental Research Program under Award Number ERKP886. Metabolite synthesis (F.L. and J.R.) was supported by the Great Lakes Bioenergy Research Center, a U.S. DOE Bioenergy Research Center supported by the of Biological and Environmental Research in the DOE of Science (DE-SC0018409). PinZ expression (M.A.C.), PinZ characterization (D.L.B.), and synthesis of substrates and products, as well as HPLC reversed phase and chiral column chromatography (S.G.A.M.), were partially supported by the USDA National Institute of Food and Agriculture, Hatch umbrella project WNP7003632. The funders played no role in the study design, article preparation, or decision to publish. This paper has been authored by UT-Battelle, LLC, under Contract No. DE AC05-00OR22725 with the U.S. Department of Energy (DOE). This work was primarily supported by the U.S. DOE, Office of Science, Office of Biological and Environmental Research, though an Early Career Award to J.K.M. Isolation of N. rhizosphaerae LY (G.N.P., J.N.E., and J.K.M.) and metabolite analysis (R.J.G.) were supported by the Center for Bioenergy Innovation (CBI), U.S. Department of Energy, Office of Science, Biological and Environmental Research Program under Award Number ERKP886. Metabolite synthesis (F.L. and J.R.) was supported by the Great Lakes Bioenergy Research Center, a U.S. DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science (DE-SC0018409). PinZ expression (M.A.C.), PinZ characterization (D.L.B.), and synthesis of substrates and products, as well as HPLC reversed phase and chiral column chromatography (S.G.A.M.), were partially suppor ted by the USDA National Institute of Food and Agriculture, Hatch umbrella project WNP7003632. The funders played no role in the study design, article preparation, or decision to publish. Environmental samples from Yellowstone National Park were collected under sampling permit YELL-2018-SCI-5741. M.N.A. performed the majority of experiments involving strain LY and wrote the original manuscript. F.L. synthesized standards for the identification of pathway intermediates. G.N.P. isolated strain LY. H.R.V. isolated DGBD. D.L.B. purified PinZ and performed related enzyme assays. M.A.C. cloned and expressed PinZ. S.G.A.M. performed lignan chemical synthesis and HPLC reversed-phase and chiral analyses. C.C.A. assisted with feeding assays for DGBD isolation. D.P.V. performed PinU expression, purification, and analysis. D.M.K. assisted with transposon library sequencing. L.H.H. assisted with genome sequencing. A.R.F. and B.C.S. assisted with DGPD isolation and characterized the isolated compound. L.D.E. assisted with DGPD isolation. R.J.G. analyzed metabolite production from wild-type and deletion strains of LY, as well as PinU reaction products. L.B.D. and N.G.L. supervised the analysis of all experiments using PinZ. J.R. supervised the synthesis of putative pathway intermediates. J.G.E. supervised the isolation of strain LY. J.K.M. conceived of the project, acquired funding, supervised the project, and wrote the original manuscript. F.L., D.P.V., D.M.K., L.H.H., A.R.F., B.C.S., R.J.G., L.B.D., N.G.L., and J.R. also contributed sections of the original manuscript and provided input on various manuscript drafts.
Keywords
- Novosphingobium
- lignin
- pinoresinol