重组大肠杆菌全细胞催化L-苏氨酸合成2,5-二甲基吡嗪

Biosynthesis of 2,5-dimethylpyrazine from L-threonine by whole-cell biocatalyst of recombinant Escherichia coli

摘要:

2,5-二甲基吡嗪 (2,5-dimethylpyrazine，2,5-DMP) 在食品香料与医药方面具有重要的经济价值，工业上普遍采用环境不友好且反应条件苛刻的化学合成法来生产。文中结合代谢工程和辅因子工程策略设计高效催化L-苏氨酸合成2,5-DMP的全细胞催化剂，实现微生物转化法合成2,5-DMP。本研究首先分析了不同微生物来源的苏氨酸脱氢酶 (Threonine dehydrogenase，TDH) 对2,5-DMP合成的影响，发现来源于大肠杆菌Escherichia coli中EcTDH具有最佳的催化能力，2,5-DMP产量达到 (438.3±23.7) mg/L。随后结合辅因子工程，通过引入乳脂链球菌Lactococcus cremoris中NADH氧化酶 (NADH oxidase，LcNoxE) 并优化其表达方式发现通过融合表达EcTDH和LcNoxE可平衡胞内NADH/NAD+水平，维持较高细胞存活率，进一步提高2,5-DMP产量。最后，通过阻断合成2,5-DMP的支路代谢途径，可以显著减少副产物积累，增加2,5-DMP产量，同时提高L-苏氨酸转化率。最终获得的重组菌EcΔkΔAΔBΔA/TDHEcNoxELc-PSstT在含有5 g/L L-苏氨酸的转化体系中于37 ℃、200 r/min孵化24 h，可积累 (1 095.7±81.3) mg/L的2,5-DMP，L-苏氨酸转化率达到76%，产物得率为0.288 g/(g L-苏氨酸)。因此，文中构建的重组菌可以实现高效催化L-苏氨酸合成2,5-DMP，具有一定的工业应用潜力。

Abstract:

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-tdhEc 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/TDHEcNoxELc-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.