Yu Haibo, Xu Jianzhong, Liu Liming, Zhang Weiguo
The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2021 Jan 25;37(1):228-241. doi: 10.13345/j.cjb.200270.
2,5-dimethylpyrazine (2,5-DMP) is of important economic value in food industry and pharmaceutical industry, and is now commonly produced by chemical synthesis. In this study, a recombinant Escherichia coli high-efficiently converting L-threonine to 2,5-DMP was constructed by combination of metabolic engineering and cofactor engineering. To do this, the effect of different threonine dehydrogenase (TDH) on 2,5-DMP production was investigated, and the results indicate that overexpression of EcTDH in E. coli BL21(DE3) was beneficial to construct a 2,5-DMP producer with highest 2,5-DMP production. The recombinant strain E. coli pRSFDuet-tdh(Ec) produced (438.3±23.7) mg/L of 2,5-DMP. Furthermore, the expression mode of NADH oxidase (NoxE) from Lactococcus cremoris was optimized, and fusion expression of EcTDH and LcNoxE led to balance the intracellular NADH/NAD⁺ level and to maintain the high survival rate of cells, thus further increasing 2,5-DMP production. Finally, the accumulation of by-products was significantly decreased because of disruption of shunt metabolic pathway, thereby increasing 2,5-DMP production and the conversion ratio of L-threonine. Combination of these genetic modifications resulted in an engineered E. coli Δkbl ΔtynA ΔtdcB ΔilvA pRSFDuet-tdhEcnoxELc-PsstT (EcΔkΔAΔBΔA/TDH(Ec)NoxE(Lc)-PSstT) capable of producing (1 095.7±81.3) mg/L 2,5-DMP with conversion ratio of L-threonine of 76% and a yield of 2,5-DMP of 28.8% in 50 mL transformation system with 5 g/L L-threonine at 37 °C and 200 r/min for 24 h. Therefore, this study provides a recombinant E. coli with high-efficiently catalyzing L-threonine to biosynthesize 2,5-DMP, which can be potentially used in biosynthesis of 2,5-DMP in industry.
2,5-二甲基吡嗪（2,5-DMP）在食品工业和制药工业中具有重要的经济价值，目前通常通过化学合成法生产。在本研究中，通过代谢工程和辅因子工程相结合的方法构建了一种能高效将L-苏氨酸转化为2,5-DMP的重组大肠杆菌。为此，研究了不同的苏氨酸脱氢酶（TDH）对2,5-DMP产量的影响，结果表明在大肠杆菌BL21(DE3)中过表达EcTDH有利于构建2,5-DMP产量最高的生产菌株。重组菌株大肠杆菌pRSFDuet-tdh(Ec)产生了(438.3±23.7)mg/L的2,5-DMP。此外，对来自乳酸乳球菌的NADH氧化酶（NoxE）的表达模式进行了优化，EcTDH和LcNoxE的融合表达使细胞内NADH/NAD⁺水平达到平衡，并维持了细胞的高存活率，从而进一步提高了2,5-DMP的产量。最后，由于旁路代谢途径的中断，副产物的积累显著减少，从而提高了2,5-DMP的产量和L-苏氨酸的转化率。这些基因改造的组合产生了一种工程大肠杆菌ΔkblΔtynAΔtdcBΔilvA pRSFDuet-tdhEcnoxELc-PsstT（EcΔkΔAΔBΔA/TDH(Ec)NoxE(Lc)-PSstT），在50 mL转化体系中，以5 g/L L-苏氨酸为底物，于37℃、200 r/min培养24 h时，该菌株能够产生(1 095.7±81.3)mg/L的2,5-DMP，L-苏氨酸的转化率为76%，2,5-DMP的产率为28.8%。因此，本研究提供了一种能高效催化L-苏氨酸生物合成2,5-DMP的重组大肠杆菌，其在工业上具有潜在的2,5-DMP生物合成应用价值。
