Production Of 5-methylpyrazine-2-carboxylic Acid By Whole-cell Catalysis Of Recombinant Escherichia Coli

5-Methylpyrazine-2-carboxylic acid(MPCA)is an important pharmaceutical intermediate and is widely used in the production of hypoglycemic agents,lipid-lowering drugs and drugs for tuberculosis.The synthesis of MPCA by the traditional chemical synthesis,biological transformation by wild strain and enzymatic catalysis face many problems like environmental pollution,long production period and the use of toxic xylene,etc.Therefore,a whole-cell biocatalysis method for the environmentally friendly production of MPCA by recombinant Escherichia coli was established in this research.Firstly,to construct the MPCA synthesis pathway in E.coli,we cloned xyl M and xyl A encoding xylene monooxygenase(XMO),xyl B encoding benzyl alcohol dehydrogenase(BADH)and xyl C gene encoding benzaldehyde dehydrogenase(BZDH)from Pseudomonas putida ATCC 33015 to the expression vector p ET28 a.By optimizating the cell harvesting phase and balancing the expression of those enzymes,the recombinant E.coli MABC1 was obtained.Then whole-cell biocatalysis conditions were optimizited,the optimal conditions were determined as follows: OD600=100,p H8.0,temperature 20 oC,substrate 2,5-dimethylpyrazine(DMP)concentration 12 g?L-1,catalytic time 48 h.During this process,XMO was found to be the key enzyme limiting the increase of MPCA production.By optimizing the RBS sequences preceding xyl A,the gene encoding electron transfer protein of XMO,a positive strain E.coli ARBS3 was obtained.The MPCA titer was improved to 10.2 g?L-1 and the yield of MPCA on DMP reached 0.665 mol/mol.Secondly,intergrating the MPCA pathway into the genome and further fine-tuning the copy number ratio of xyl M and xyl A in the genome by using CRISPR/Cas9,a high-yield and plasmid-free strain E.coli MPCA8 was constructed.The MPCA titer of it reached 15.6 g·L-1 and the yield of MPCA on DMP reached 1.0 mol/mol.Meanwhile,the cell concentration dropped to OD600=75 and the catalytic time was shortened to 36 h.Finally,we used threading method to model the protein structure of hydroxylase playing a catalytic role in XMO.The software Discovery Studio was used to perform molecular docking and we obtained 5 key amino acids.After site-directed saturation mutagenesis,the mutant strain L151 F with enhanced catalytic activity was obtained and the catalytic activity was 67 mg·h-1·g DCW-1.Catalytic vertification of the mutant showed that the titer of MPCA reached 11.6 g·L-1,which was higher than that of plasmid-dependent E.coli ARBS3.