Liuyan Gu,Haibo Yuan,Xueqin Lv,Guangsheng Li,Rigang Cong,Jianghua Li,Guocheng Du,Long Liu
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.
Li Ran,Yongjun Wu,Jing Jin,Lincheng Zhang,Cen Li,Shuoqiu Tong,Feng Wen,ZhongHui Mou
The globalization of food culture has driven the growth of the flavor market, with Bacillus subtilis- fermented soybean products widely used as traditional Asian condiments due to their distinctive soy sauce-like aroma. However, the molecular mechanism underlying aroma formation remains incompletely understood. Building on previous systematic multi-omics studies, this study identifies a key role for l -threonine catabolism in regulating pyrazine biosynthesis. Using homologous recombination, we performed targeted knockouts of the tdh and kbl genes to generate single mutants (BJ3-2Δ tdh and BJ3-2Δ kbl ) and a double mutant (BJ3-2Δ tdh - kbl ). Headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS), along with sensory evaluation, revealed that the tdh knockout drastically reduced 2,5-dimethylpyrazine (2,5-DMP) levels by 96.79%. The double knockout achieved a 97.73% reduction and completely eliminated 3-ethyl-2,5-dimethylpyrazine (EDMP). Interestingly, the kbl knockout alone enhanced 2,5-DMP production by 65.34%, and the odor activity value of EDMP increased to 10.50. Exogenous supplementation experiments confirmed that 2,5-DMP is a critical contributor to nutty, sweet, and buttery aromas, while also intensifying smoky undertones. Overall, this study elucidates the regulatory function of l -threonine catabolism in pyrazine biosynthesis, linking microbial metabolism to food flavor and offering a metabolic engineering strategy for targeted aroma modulation in fermented foods. Practical Applications This research provides valuable insights into the mechanism underlying the formation of soy sauce-like aroma, laying the foundation for optimizing 2,5-dimethylpyrazine (2,5-DMP) concentrations to fine-tune aroma profiles. These findings may support the environmentally friendly production of high-quality soy sauce-like aroma foods, potentially reducing reliance on artificial additives.
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