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

作　　者：于海波 徐建中[1] 刘立明[1,2] 张伟国[1] Haibo Yu;Jianzhong Xu;Liming Liu;Weiguo Zhang(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)

机构地区：[1]江南大学,生物工程学院,工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学,食品科学与技术国家重点实验室,江苏无锡214122

出　　处：《生物工程学报》2021年第1期228-241,共14页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.31601459)资助

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,LcNox E)并优化其表达方式发现通过融合表达EcTDH和Lc Nox E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-PSst T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(1095.7±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。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 p RSFDuet-tdh Ec produced(438.3±23.7)mg/L of 2,5-DMP.Furthermore,the expression mode of NADH oxidase(Nox E)from Lactococcus cremoris was optimized,and fusion expression of EcTDH and Lc Nox E 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.coliΔkblΔtynAΔtdcBΔilvA pRSFDuet-tdh EcnoxELc-Psst T(EcΔkΔAΔBΔA/TDHEc Nox ELc-PSst T)capable of producing(1095.7±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℃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.

关 键 词：2 5-二甲基吡嗪 L-苏氨酸 全细胞催化 EcTDH-LcNoxE融合蛋白 L-苏氨酸转运蛋白SstT 辅因子工程 

分 类 号：TQ25[化学工程—有机化工]