大肠杆菌异丁醇合成途径中还原力调控的机制研究

NADPH is the reducing equivalent required for isobutanol production, while the common reducing equivalent under anaerobic glucose fermentation condition of Escherichia coli is NADH. There was a cofactor imbalance problem for anaerobic isobutanol synthesis, which decreased isobutanol yield. In this project, in order to increase NADPH supply, transhydrogenase (pntAB) and NAD kinase (yfjB) genes will be modulated in combination. On the other hand, glycolysis will be replaced by Entner-Doudoroff (ED) pathway followed by combinatorial modulation of key genes of ED pathway. The relationship between key enzyme activities, reducing equivalent concentration and isobutanol yield will further be investigated. The aim of this project is to identify 1) mechanisms of how reducing equivalent is regulated by transhydrogenase and NAD kinase; 2) mechanisms of how to engineer an efficient ED pathway and 3) mechanisms of how reducing equivalent is regulated by glucose utilization pathways, which will be useful for improving production of target compounds using NADPH as reducing equivalent within their synthetic pathways. The other aim of this project is to obtain robust strains for efficient isobutanol production with yield more than 95% of theoretical maximum, which can be used in future for industrial production of isobutanol.

异丁醇合成途径需要NADPH作为还原力，而大肠杆菌在葡萄糖厌氧发酵过程中产生的还原力是NADH形式，这就存在还原力的供给类型与需求类型之间的不平衡问题，从而降低了异丁醇合成的转化率。本项目拟通过组合激活转氢酶和NAD激酶，以及使用Entner-Doudoroff（ED）途径替代糖酵解途径并对其关键酶进行组合改造，提高细胞的NADPH供给能力。在此基础上，研究关键酶的活性、胞内各种类型还原力的浓度以及异丁醇转化率之间的相互关系。本研究的目标是解析出转氢酶和NAD激酶对胞内还原力调控的机制、ED途径高效转运的分子机理以及葡萄糖代谢途径对胞内还原力的调控机制，为将来优化以NADPH为辅因子的产品合成途径奠定理论基础。同时还将构建出高效生产异丁醇的工程菌，厌氧发酵转化率达到理论最大值的95%，为异丁醇生物制造的工业化应用建立菌种基础。

异丁醇合成途径需要NADPH作为还原力，而大肠杆菌在葡萄糖厌氧发酵过程中产生的还原力是NADH形式，这就存在还原力的供给类型与需求类型之间的不平衡问题。本项目设计了三种新策略用于解决异丁醇合成途径中还原力不平衡问题。（1）通过联产异丁醇和乙醇，避免了NADH的积累；另外激活磷酸戊糖途径提高NADPH供给，调减醇脱氢酶活性降低乙醇产量。该策略将异丁醇厌氧转化率提高了12%，达0.74 mol/mol；另外，总的糖醇转化率提高了23%，达到0.81 mol/mol。（2）激活ED途径替代天然的糖酵解途径，每消耗1个葡萄糖产生1个NADH和1个NADPH，使还原力平衡。该策略将异丁醇厌氧转化率提高了34.8%，达0.89 mol/mol；并且发现EDD和EDA是ED途径中的关键限速酶。（3）使用NADH依赖型醇酮酸还原异构酶KARI替代天然的NADPH依赖型IlvC，减少异丁醇合成途径对NADPH的依赖，并激活转氢酶和NAD激酶增加胞内NADPH的供给。该策略将异丁醇厌氧转化率提高了47%，达0.97 mol/mol，是三种策略中效果最好的。此外，通过固相萃取和亲水作用色谱-质谱相结合建立了胞内辅因子测定的方法，并使用该方法测定了转氢酶和NAD激酶调控过程中关键菌株胞内辅因子的浓度。结果发现，厌氧条件下异丁醇转化率越高的菌株中NADPH的浓度也越高，证明了NADPH供给的提高是厌氧条件下提高异丁醇产量的关键。本研究的实施一方面为提高胞内NADPH供给提供了理论基础和遗传改造方案，另一方面也为异丁醇生物制造的工业化应用提供了具有潜力的高效生产异丁醇的工程菌株。