Construction And Optimization Of Lignin Monomer Demethoxyl Engineering Bacteria

Lignin is the main component of lignocellulosic biomass,and it is the most abundant renewable aromatic compound in nature.However,most of the lignocellulosic residues are dumped and burned at will,which not only wastes resources,but also pollutes the environment.Demethylation and demethoxylation of lignin monomer are effective strategies for lignin valorization.In this paper,the research of lignin high-quality was carried out from two aspects:the construction of engineered bacteria to degrade lignin and the optimization of methoxyl removing enzyme.First of all,we selected five kinds of demethoxygenase from ligninoids,which were VDH from Pseudomonas putida KT2440?Van AB from Amycolatopsis mediterranei U32?PEE from Arabidopsis thaliana?lig M from Sphingobium sp.(strain NBRC 103272/SYK-6?Van A-Van B from P.putida and Rhodococcus.A lignin demethoxy reaction path was constructed in Saccharomyces cerevisiae,and lig M with the highest efficiency was screened out through the reaction activity.Then the degradation efficiency of lig M under different conditions was studied.Copy number,inoculation amount,temperature,p H and different substrates were investigated respectively.The best fermentation results were obtained by using vanillic acid with substrate concentration of 400mg/L,OD₆₀₀=0.2,temperature of 30?,p H6 and on a single copy plasmid,the highest protocatechuic acid yield was 5.77 mg/L.Rhodococcus-derived lignin monomeric demethoxyenzyme Van A-Van B is two genes that catalyze the cleavage of methoxy groups in lignin monomers.Through protein fusion by using two different linker,the highest yield of protocatechuic acid obtained by converting substrate vanillic acid is 5.04 mg/L,which is 1.8 times of the original.Using error-prone PCR to carry out multiple rounds of directed evolution on lig M,the optimized mutant screened can convert substrate vanillic acid to protocatechuic acid with the highest yield of 22.00 mg/L,which is 3.5 times that of lig M.Four mutants D268G,Y273H,V454A and W464R were obtained by sequencing,and their structures were verified and analyzed.It is speculated that the decrease of amino acid negative charge and the increase of alkalinity at the four sites increased the yield.A high-throughput screening method was established by color reaction of vanillic acid,protocatechuic acid and ferric chloride ions.This study can be used for reference in the biodegradation of lignin monomers and lignin,the utilization of biomass energy and the development of renewable energy.