Metabolic Pathway Design And Process Intensification For Synthesizing Muconic Acid And Gallic Acid From Lignin Based On Rhodococcus Opacus PD630

As the most abundant renewable resource in nature,lignocellulosic biomass is mainly composed of cellulose,hemicellulose and lignin.The valorization of lignocellulosic biomass is regarded as a promising way to solve energy shortage and environmental pollution.In current biorefinery concept,cellulose and hemicellulose have been converted to value-added products,such as bioethanol and biolipid.However,most lignin generated during biorefinery process is un-utilized due to its complex and heterogeneity structure,leading to a serious obstacle for lignocellulosic biomass upgrading.To search an effective way for lignin valorization,serial studies including Rhodococcus opacus cell chassis development,lignin-based product innovation and bioconversion process intensification,were conducted in this study.(1)Development of Rhodococcus opacus PD630 cell chassis for valorizing lignin to muconic acidR.opacus PD630,which has efficient assimilation ability for lignin-derived aromatic compounds,is an ideal cell chassis for lignin valorization.However.the insufficient genome modification toolkit for this strain limits its application.To development an efficient genome modification method for R.opacus PD630,a markerless gene deletion/insertion system was constructed based on a mutant phenylalanyl-t RNA synthase gene as a counter-selection marker.With the developed gene deletion/insertion system,the muconate cycloisomerase gene and putative catechol 2,3-dioxygenase gene were deleted from R.opacus PD630 genome,and the generated R.opacus PD630-MA2 was successfully constructed for cis,cis-muconate(CCMA)production from lignin-derived aromatics using catechol and its upper metabolites.Further,to widen the substrate spectrum,an heterogenous protocatechuate decarboxylation system was introduced into the protocatechuate degradation way blocked mutant to bridge the conversion of protocatechuate and coumponds in its upper pathways to CCMA production.Results indicated the constructed strain R.opacus PD630-MA6 can assimilate at least 13 types of lignin-derived aromatics for CCMA production.Ultimately,1.63 g/L CCMA was produced using lignin from corn stover as substrate by a fed-batch fermentation process.(2)Integration of different bio-catalyzation modules to convert lignin components into gallic acidConsidering the high contents of aromatic conpounds in lignin,the aromatic compound gallate was set as a target product for lignin valorization.A novel extradiol dioxygenase which can ring-opening gallate was identified and deleted from R.opacus PD630 to block the gallate degradation pathway.Then,a rationally designed hydroxylase system was introduced into a gallate biodegradation pathway-blocked R.opacus PD630 mutant so that gallate could be accumulated from protocatechuate.Based on this strain,two H-type lignin components were successful catalyzed to gallate.Further,G-type lignin components were catalyzed to gallate with the native Rieske nonheme iron monooxygenase based O-demethylation system exits in R.opacus PD630.Furthermore,a heterologous O-demethylation system was employed,leading to some S-type lignin components being catalyzed to gallate.Finally,an aryl side-chain oxidase was engaged to broaden the substrate spectrum for gallate production.An efficient biocatalyst R.opacus PD630-GA4 was developed to selectively produce gallate from lignin components by integrating three main reactions: hydroxylation,Odemethylation,and aryl side-chain oxidation.(3)Investigate on gallic acid production from lignin by R.opacus PD630-GA4To search an effective way for gallate production from lignin,several types of lignin were applied for gallate production using R.opacus PD630-GA4,including alkali lignin,alkali lignin treated with laccase,AFEX lignin,alkaline pretreated lignin,and base-depolymerized AFEX lignin.As a result,the yields of gallate from lignin were respectively 0.132 g/g,0.053 g/g,0.054 g/g,0.407 g/g,and 0.630 g/g,indicating the gallate yield was significantly improved with base-catalyzed depolymerization.The results also suggested that a suitable chemo-bio hybrid method is beneficial to lignin biological conversion.Further analysis of alkaline pretreated lignin and basedepolymerized AFEX lignin samples before and after bio-catalyzation showed that the gallate produced from lignin was mainly attribute to low-molecular-lignin,indicating the significance for biological conversion of low-molecular-lignin.(4)Construction of bacterial co-culture system to enhance lignin bio-conversionIn addition to aromatic monomers,the conversion of low-molecular-lignin,such as lignin dimers,is also vital for lignin valorization.To improve the utilization of lowmolecular-lignin,two co-culture systems were developed by combining a lignin dimer depolyerizing bacterium Sphingomonas sp.SYK-6.The protocatechuate and gallate degradation pathway in Sphingomonas sp.SYK-6 were firstly blocked,generating SYK6-△lig AB and SYK6-△lig AB△des Z△des B-kan.Then,the MA6+SYK6-△lig AB and GA4+SYK6-△ lig AB △ des Z △ des B-kan co-culture systems were respectively constructed based on Sphingomonas sp.SYK-6 mutants and R.opacus PD630 derivates.Consequently,CCMA and gallate were generated respectively from diverse lignin dimers by the constructed co-culture systems.Furthermore,the application of alkali lignin as the substrate for CCMA production also demonstrated the effectiveness of the co-culture system.These results suggested that the co-culture systems can enhance the conversion of lignin dimers to produce value-added products,providing a new reference for lignin valorization by using bacterial consortia.