Deciphering the metabolic distribution of vanillin in Rhodococcus opacus during lignin valorization

Vanillin bioconversion is important for the biological lignin valorization. In this study, the obscure vanillin metabolic distribution in Rhodoccous opacus PD630 was deciphered by combining the strategies of intermediate detection, putative gene prediction, and target gene verification. The results suggest that approximately 10% (mol/mol) of consumed vanillin is converted to vanillic acid for further metabolism, and a large amount is converted to dead-end vanillyl alcohol in R. opacus PD630. Subsequently, five vanillin reductases were identified in R. opacus PD630, among which Pd630_LPD03722 product exhibited the greatest activity. With the detected metabolic distributions of vanillin, the conversion of vanillin to muconic acid was facilitated by deleting domestic vanillin reductase genes and introducing vanillin dehydrogenase from Sphingobium sp. SYK-6. Ultimately, the muconic acid yield from vanillin increased to 97.83% (mol/mol) from the initial 10% (mol/mol). Moreover, this study demonstrated the existence of vanillin reductases in Escherichia coli, Bacillus subtilis, and Corynebacterium glutamicum.