Genetic engineering of Rhodococcus jostii RHA1 for lignin valorization strategies

Abstract

Lignin streams derived from biomass processing plants represent an untapped source of aromatic compounds that can be converted into high value molecules. The lignin-degrading bacterium Rhodococcus jostii RHA1 (hereinafter named RHA1) depolymerizes lignin using the peroxidase DypB into oxidized, lower molecular weight aromatic compounds, which are catabolized. Although the metabolism underlying the lignin fragments funneling to the Krebs cycle in RHA1 is still scantily understood, a recent study from Prof. Tim Bugg's group has shown that aryl C2 lignin fragments generated by RHA1 can be metabolized via 4-hydroxybenzoylformate/3-methoxy-4-hydroxybenzoylformate, which are converted to 4-hydroxybenzaldehyde/vanillin in a reaction catalyzed by 4-hydroxybenzoylformate decarboxylase (named RjDFBC). In this project, we intend to explore the metabolism of R. jostii RHA1 to develop mutant strains harboring strategic gene knockouts in catabolic aromatic pathways, aiming the accumulation of high-value molecules upon cultivation on lignin. Specifically, we aim to knockout the 4-hydroxybenzoylformate decarboxylase gene RjDFBC (RHA1_RS14520) in RHA1. We expect that this knockout strain will accumulate 3-methoxy-4-hydroxybenzoylformate/4-hydroxybenzoylformate when grown in minimal medium supplemented with lignin streams or lignocellulosic material, effectively valorizing these feedstocks in valuable products. 3-methoxy-4-hydroxybenzoylformate and 4-hydroxybenzoylformate are high-value aromatic compounds that could be converted to unusual amino acids 3-methoxy-4-hydroxyphenylglycine and 4-hydroxyphenylglycine, respectively, by transamination. These non-proteinogenic amino acids are building blocks for the biosynthesis of several clinically relevant products, such as semi-synthetic ²-lactamic and the vancomycin group of antibiotics. To obtain these knockout strains, we intend to use the novel CRISPR/Cas9 system under development by our group in Brazil, alongside traditional mutagenesis, phenotypic assays and analytical methods within the expertise of Prof. Tim Bugg's group at The University of Warwick, who is among the best researchers of bacterial degradation of lignin in the world. We expect that this work will not only provide strains and vectors for microbial lignin valorization into high-value molecules, but also contribute to the elucidation of lignin degradation and catabolic pathways in R. jostii RHA1. (AU)