Deciphering Vanillin Metabolic Distributions In Rhodococcus Opacus And The Strain’s Capability Of Co-Utilization Lignin-Derived Aromatics

Lignocellulosic biomass is the most abundant renewable organic resource in nature.Biofuels and biological products made from lignocellulosic biomass are actively regarded as substitutes for fossil resource based fuels,chemicals,and other products.In current lignocellulosic refinery routes,cellulose and hemicellulose have been converted to value-added products,such as bioethanol and biolipids.However,due to the complex structure of lignin,most lignin is left after the conversion of cellulose and hemicellulose,and usually burned to provide heat and power for refinery plants.Considering the high proportion of 15-30wt% lignin in lignocellulosic biomass,efficient lignin utilization is beneficial to improving the economic feasibility of lignocellulose biorefinery.Vanillin,as a representative compound of G-type lignin monomers,its bioconversion is important for biological lignin valorization.In this study,the obscure vanillin metabolic distributions in Rhodoccous opacus PD630 were 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 compound 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 unveiled metabolic distributions of vanillin,the conversion of vanillin to muconic acid was facilitated by deleting domestic vanillin reductase genes and introducing heterogenous vanillin dehydrogenase in engineered strain R.opacus PD630-MA6.Resultly,the muconic acid yield from vanillin increased to 97.83%(mol/mol)from the initial 10%(mol/mol).During lignin decomposition process,a variety of aromatic monomers and oligomers with different structures are produced.As well known,when cultivated with two or more carbon sources,many microorganisms have a sequence of assimilating these carbon sources.In this study,lignin-derived aromatic monomers were combined in pairs and used as carbon sources to cultivate R.opacus PD630 to investigate the utilization preference of R.opacus PD630 for these lignin-derived compounds.Results show that R.opacus PD630 is capable of efficiently utilizing a variety of lignin-derived aromatic monomers at the same time.But when catechol was used as one of the carbon sources,the carbon inhibition of catechol on the other carbon sources appeared in varying degrees.Further results showed that the presence of catechol weakened the promoter strength of feruloyl-Co A synthetase gene by 51%,which may be the reason for the carbon inhibition of catechol on ferulic acid.To sum up,this study clarified the metabolic nodes of vanillin in R.opacus PD630,and preliminarily analyzed the substrate preference of R.opacus PD630 in presence of multiple lignin-derived aromatic compounds.These results laid a foundation for further designing rational metabolic engineering strategy and promoting the conversion of lignin to multiple products by R.opacus PD630,as well as engineering R.opacus PD630 to a more efficient cell chassis for biotechnology application.