Elucidation of a bacterial pathway for catabolism of the β–β-linked dilignol pinoresinol

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, <i>Novosphingobium rhizosphaerae</i> 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 additionally confirmed that the final pathway enzyme, PinU, is related to lignostilbene dioxygenases and oxidatively cleaves a diguaiacylbutadiene intermediate to yield vanillin and coniferaldehyde. Finally, based on the enzyme characterization, we demonstrated 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.IMPORTANCEPlants 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 <i>Novosphingobium</i> strain capable of degrading the β-β-linked lignan (+)-pinoresinol 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.