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Identification and validation of acetovanillone catabolism in Rhodococcus rhodochrous GD02 Dexter, Gara

Abstract

Bacteria play a major role in the mineralization of phenolic lignin depolymerization products and possess a multitude of pathways for the catabolism of these compounds, many of which have yet to be determined. Acetovanillone, a key component in various industrial lignin streams, including black liquor from the Kraft pulping process and oxidative catalytic fractionation of softwood biomass, has been a focus of our research. Herein, I describe a pathway for the catabolism of acetovanillone in bacteria. I enriched bacteria from compost capable of growth on acetovanillone as the sole carbon source. Two Rhodococcus rhodochrous strains, GD01 and GD02, were isolated and their genome sequences were analyzed for genes encoding potential aromatic catabolic pathways. The strains have an ANI of 99.1% and, in accordance with their highly similar genomes, utilize the same profile of aromatic substrates. However, GD02 grew on higher concentrations of acetovanillone and vanillin. A high-quality closed genome assembly of GD02 was generated by combining short and long-read sequence data, revealing a circular 5.58-Mb circular chromosome, a 721-kb linear plasmid, and a 165-kb circular plasmid. To gain insight into GD02’s ability to grow on acetovanillone, I performed transcriptomic experiments with cells grown on acetovanillone, 4-hydroxyacetophenone, acetophenone, and citrate as a control. The analyses revealed an eight-gene cluster up-regulated during growth on acetovanillone (>150-fold) and 4-hydroxyacetophenone (>500-fold). Bioinformatic analyses predicted that this hydroxyphenylethanone (Hpe) pathway is unusual in that it begins by phosphorylation and carboxylation, before β-elimination yields vanillate from acetovanillone or 4-hydroxybenzoate from 4-hydroxyacetophenone. This study also identified a pathway for acetophenone catabolism, which was first proposed in 1975, by identifying the genes and proteins responsible in GD02. The first step of the Hpe pathway was validated by expressing and characterizing the kinase, HpeHI, which had the highest apparent specificity (k꜀ₐₜ/KM = 2.2 ± 0.1 µM⁻¹ s⁻¹) for 4-hydroxyacetophenone. This enzyme belongs to a relatively unexplored group of kinases and is homologous to enzymes involved in diverse biological processes like gluconeogenesis and antibiotic resistance. Overall, this work expands our understanding of natural lignin degradation, uncovered new enzymes, and facilitates the design of bacterial strains to biocatalytically upgrade lignin.

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Attribution-NonCommercial-NoDerivatives 4.0 International