Acetovanillone catabolism and the biocatalytic valorization of lignin

NAVAS, LAURA E.; DEXTER, GARA; BAJWA, HARBIR; LEVY-BOOTH, DAVID; LIU, JIE; GRIGG, JASON C.; NASSERI, SEYED A.; JANG, SOO-KYEONG; RENNECKAR, SCOTT; MOHN, WILLIAM W.; ELTIS, LINDSAY D.

Vancouver

Congreso; LIGNOBIOTECH SYMPOSIUM 2022; 2022

One potential biorefining strategy to extract more value from lignin is the sequentialchemo- and bio-catalytic conversion of biomass. The success of this strategy dependsin part on the efficient biocatalytic conversion of the aromatic compounds produced fromthe chemo-catalytic conversion of lignin. Bacterial catabolic pathways have tremendousbiocatalytic potential, due in part to their convergent organization and their amenabilityto engineering. However, our lack of knowledge of relevant enzymes and pathways haslimited its application. Here we describe a bacterial pathway responsible for thecatabolism of acetovanillone, a major component of several industrial softwood biomassstreams. To elucidate the pathway, we first performed transcriptomic analyses ofRhodococcus rhodochrous GD02, previously isolated for growth on acetovanillone, toreveal an eight-gene cluster up-regulated during growth on acetovanillone and 4-hydroxyacetophenone. The hydroxyphenylethanone (Hpe) pathway was predicted toproceed via phosphorylation and carboxylation before β-elimination yields vanillate fromacetovanillone (Figure 1). Consistent with this prediction, the purified carboxylase,HpeCBA, catalyzed the ATP-dependent carboxylation of 4-phospho-acetovanillone butnot acetovanillone. The carboxylase’s specificity for 4-phospho-acetophenone (kcat/KM= 34 ±2 mM-1 s-1) was approximately an order of magnitude higher than for 4-phospho-acetovanillone. HpeD catalyzed the efficient dephosphorylation of the carboxylatedproducts. The biocatalytic potential of the Hpe pathway was demonstrated by the abilityof GD02 to grow on a preparation of pine biomass subjected to oxidative catalyticfractionation (OCF). Notably, GD02 depleted the major monoaromatic products of OCF:acetovanillone, vanillin and vanillate. By identifying a pathway responsible for thedegradation of acetovanillone, this study facilitates the engineering of bacteria for use inupgrading lignin.