Metabolic pathways for lignin-related aromatic compounds elucidated in a Xanthomonas phytopathogen

Convergent microbial biocatalysis has emerged as a promising approach for the conversion of lignin side-streams into value-added chemicals in recent decades. However, the current knowledge of metabolic pathways directing the bioconversion of lignin-related aromatics is still limited to a few microbial species and unavailable for some of these compounds. Thus, the aim of this study was to identify the genes involved in the bioconversion of aromatic compounds in Xanthomonas citri subsp. citri 306 (X. citri 306), a bacterium belonging to a compelling yet untapped genus for studies on lignin-related aromatics metabolism. For this purpose, we used an integrative approach including genome data mining, RNA-seq, enzymology and gene knockout studies. The RNA-seq analysis revealed a total of 278 to 1464 differentially expressed genes (DEGs) in the aromatic-containing conditions compared to the control XVM2m-glucose, evidencing the importance of these compounds in modulating various physiological processes of X. citri 306 beyond the pathways related to their metabolism. Moreover, this work revealed the operon molRKAB, which plays a role in the first catabolic steps of the three main monolignols (p-coumaryl, coniferyl and sinapyl alcohols), besides showing all the enzymatic steps funneling them up to the tricarboxylic acid cycle. Additionally, the study uncovered aryl aldehyde reductases and efflux strategies that likely function to protect the pathogen from aromatics toxicity. Together, these findings enhance the current understanding of Xanthomonas metabolism and transcriptional responses to lignin-related aromatic compounds, shedding light on the diverse metabolic pathways available to enable the engineering of microbial chassis dedicated to lignin valorization. Overall design: To investigate the transcriptional profile of X. citri 306 in response to lignin-related compounds, samples of cells grown on XVM2m minimal medium supplemented with six lignin-related aromatic compounds, three complex mixtures of lignin-derived compounds, and glucose (reference) were collected in four biological replicates. Total RNA was extracted and used to generate RNA-Seq libraries. Differential expression analysis was carried out by pairwise comparisons between X. citri 306 grown in XVM2m supplemented with lignin-related aromatics and XVM2m(G) containing glucose (reference medium).