Mining And Engineering Of Oxidoreductases For One-step Synthesis Of 2,3,5-trimethyl Hydroquinone From 2,3,6-trimethyl Phenol

As one of the largest vitamin products in the market,vitamin E has wide applications in food,medicine,feed,and cosmetics industries.2,3,5-trimethyl hydroquinone?TMHQ?is an important precursor of synthetic vitamin E.The main methods of TMHQ synthesis are TMP oxidation-reduction method and isophorone method.Chemical routes have common problems such as long reaction steps,harsh reaction conditions,and environmental pollution caused by the use of catalysts and solvents.In contrast,biocatalysis is an green alternative with mild reaction conditions.To this end,an oxidoreductase library was constructed in this study,from which some biocatalysts capable of catalyzing TMP to TMHQ within one step were screened out,and their catalytic activity were improved by optimization of reaction conditions and directed evolution.The project started wirg gene mining of oxidoreductases by searching the National Center for Biotechnology Information databank,followed by cloning of oxidoreductase genes from different species and recombinant expression in E.coli BL21?DE3?.Expression of these enzymes was optimized by adjusting the inducing time and inducing remperature as well as co-expression with a molecular chaperone,resulting in soluble expression of six recombinant proteins.In this enzyme library,4 proteins catalyzed the conversion of TMP to TMHQ using NADH as hydrogen donor,namely,HapC and UbiH-K from E.coli,yjiB from Bacillus subtilis and P450_BM3 from Bacillus megatherium,among which P450_BM3 showed the highest activity.In order to improve the catalytic activity of P450_BM3 towards TMP,the reaction system was optimized in seven aspects including coenzyme specificity,temperature,pH,substrate concentration,cosolvent and its concentration,and metal ions.The results showed that P450_BM3 could use both NADH and NADPH as cofactor at a substrate concentration of 0.5 mM,the optimum reaction temperature was 25 ?,the optimum pH was 7.0,5%acetone could be used as a cosolvent,and Fe2+ played a role in this catalysis.The TMHQ yield in the P450_BM3-catalyzed reaction increased from 0.4%to 3.19%?by nearly 7 times?after optimization of reaction conditions.Finally,protein engineering of P450_BM3 was performed by directed evolution to improve its catalytic activity towards TMP.Taking into consideration the modification experience of P450_BM3 towards unnatural substrate,a mutant library was constructed by site-saturation mutagenesis.In combination with the MTT/PMS-based color developing high-throughout screening method,two positive mutants L181Q and I259P were obtained,with 71.9%and 57.5%activity improvement,respectively.Determination of kinetic parameters showed that the Km value of both mutants decreased as compared to the wild type,indicating improved affinity to the substrate,and the total catalytic activity?kcat/Km?of the mutants was higher than the wild type.The results of molecular docking indicated that L181Q mutant was conducive to the formation of hydrogen bonds between the key amino acids to stabilize catalytic center and promote the reaction,whereas I259P mutant drew the substrate closer to the Fe atom by influencing the interaction between the substrate and the amino acid residues in the substrate channel.The results support the feasibility of substituting chemical synthesis of drug intermediates with biocatalytic synthesis.The screening and engineering of oxidoreductase for one-step synthesis of 2,3,5-trimethyl hydroquinone from 2,3,6-trimethyl phenol would lay a foundation for TMHQ biosynthesis.