Journal
ENZYME AND MICROBIAL TECHNOLOGY
Volume 134, Issue -, Pages -Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2019.109488
Keywords
Whole-cell biocatalyst; Xylene monooxygenase; 2, 5-dimethylpyrazine; 5-methylpyrazine-2-carboxylic acid; Escherichia coli BL21 (DE3)
Categories
Funding
- National Natural Science Foundation of China [31622001, 31671845, 31600068]
- Natural Science Foundation of Jiangsu Province [BK20160176]
- Fundamental Research Funds for the Central Universities [JUSRP51713B]
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5-Methylpyrazine-2-carboxylic acid (MPCA) is an important pharmaceutical intermediate and is used in the production of hypoglycemic agents and lipid-lowering drugs. This work aimed to develop a whole-cell biocatalytic process for the efficient synthesis of MPCA from 2, 5-dimethylpyrazine (DMP). Firstly, a whole-cell biocatalyst Escherichia coli strain was constructed by plasmid-based expression of xylene monooxygenase (XMO), benzyl alcohol dehydrogenase (BADH), and benzaldehyde dehydrogenase (BZDH) from Pseudomonas putida ATCC 33015, resulting in MPCA titer of 5.0 g/L. Then, the reaction conditions were optimized and the MPCA titer was further increased to 9.1 g/L. Next, the Ribosome Binding Site (RBS) Calculator v2.0 was used to predict and compare the translation initiation rates of the RBS sequences preceding xylM and xylA genes, encoding the two subunits of XMO. By optimizing the RBS sequences preceding xylA, the MPCA titer was increased to 10.2 g/L and the yield of MPCA on DMP reached 0.665 mol/mol. Finally, to achieve plasmid-free production of MPCA, we integrated the genes encoding for XMO, BADH and BZDH in the genome by using CRISPR/Cas9 and further fine-tuned the copy number ratios of xylM and xylA in the genome, improving the MPCA titer to 15.6 g/L and the yield of MPCA on DMP to 1.0 mol/mol. This work developed a high-yield and plasmid-free biocatalysis process for the environmentally friendly production of MPCA with 100% substrate conversion, and paved the way for the commercial production of MPCA in the future.
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