克雷伯氏菌1,3-丙二醇合成途径甘油脱水酶同工酶的研究

1,3-propanediol (1,3-PD) is one of the most important commodity chemical. Klebsiella pneumoniae metabolizes glycerol to synthesize 1,3–PD with good performance. So, K. pneumoniae is an advantage strain for industrialization of 1,3–PD. While glycerol dehydratase limits the reaction rate of synthesizing 1,3-PD. It had been confirmed that increasing the expression of glycerol dehydratase failed to accelerate glycerol dehydration reaction. The activity of glycerol dehydration reaction is not only affected by the amount of glycerol dehydratase, but also by some other factors. In the genome of K. pneumoniae, It’s found that encoding genes for glycerol dehydratase, glycerol dehydratase reactivation factor, diol dehydratase, and diol dehydratase reactivation factor, and these enzymes and factors combined carry out the reaction of glycerol dehydration. However, the effects of each enzyme on the activity of glycerol dehydration or on the production of 1,3-PD are not known. To study the relationship between the enzymes and the rate of glycerol dehydration reaction and the production for 1,3-PD, Firstly, the influence of glycerol dehydratase and diol dehydratase on synthesizing 1,3-PD or on metabolic flux will be investigated, secondly, the reactivation mechanisms and the enzyme kinetics of activated factors and enzyme kinetics of two dehydratases will be determined, lastly, the correlation between the yields with combinations of different reactivation factors and dehydratase were examined. According the experiments above, this study would remove the limit of glycerol dehydration reaction on producing 1,3-PD and lay the foundation for improving 1,3-PD production by K. pneumoniae.

1,3-丙二醇（1,3-PD）是一种重要的大宗化学品。克雷伯氏菌代谢甘油合成1,3-PD性能较好，是一株有优势的产业化菌株。甘油脱水酶是1,3-PD合成途径的限速酶，增加甘油脱水酶的表达不能解除限制。甘油脱水反应不仅受甘油脱水酶表达量的影响，还受其他因素的影响。克雷伯氏菌同时编码甘油脱水酶、甘油脱水酶激活因子以及同功酶二醇脱水酶、二醇脱水酶激活因子等催化甘油脱水反应的组成单元。各个组成单元对甘油脱水反应活性的调控机制以及调控克雷伯氏菌合成1,3-PD性能的变化规律均未见报道。针对以上问题，本项目考察甘油脱水酶和二醇脱水酶对克雷伯氏菌1,3-PD合成性能和代谢流分配的影响；明确激活因子和脱水酶的激活效应以及催化动力学性质；探明激活因子和脱水酶组合表达调控克雷伯氏菌1,3-PD合成性能的变化规律。研究成果为解除甘油脱水反应的限制以及提高克雷伯氏菌合成1,3-PD的性能提供理论指导。

克雷伯氏菌是一株性能优良的代谢甘油合成1,3-丙二醇的菌株。其甘油脱水反应由编码的甘油脱水酶、甘油脱水酶激活因子以及同功酶二醇脱水酶、二醇脱水酶激活因子等组成单元共同催化。而各个组成单元对甘油脱水反应活性的调控机制以及调控克雷伯氏菌合成1,3-PD性能的变化规律均未见报道。.针对以上问题，本项目获得以下结论：.1、甘油脱水酶和丙二醇脱水酶均能催化甘油脱水反应，但是失活其中一个酶后对碳源在氧化支路和还原支路之间的分配有影响。突变株K. pneumoniae ΔpduCDE合成1,3-丙二醇的水平提高，而乳酸和乙醇等副产物的产量减少，表明甘油脱水酶（DhaB）有利于碳源流向还原支路；突变株K. pneumoniae ΔdhaB合成1,3-丙二醇的产量减少，而乳酸和乙醇的产量增多，表明丙二醇脱水酶（PduCDE）促使更多碳源流向氧化支路。.2、甘油脱水酶和丙二醇脱水酶在催化脱水反应过程都需要活化因子恢复酶活性。甘油脱水酶活化因子的大亚基由orfZ编码，丙二醇脱水酶活化因子的大亚基由pduG编码。为了考察活化因子与脱水酶的相互关系以及对菌体代谢合成1,3-丙二醇的影响，针对脱水酶编码基因dhaB/pduCDE和活化因子编码基因orfZ/pduG进行组合敲除。根据工程菌合成1,3-丙二醇的性能，发现orfZ编码的活化因子具有专一性，只能够活化甘油脱水酶；而pduG编码的活化因子对甘油脱水酶和丙二醇脱水酶都有活化作用。此外，OrfZ/DhaB比PduG/PduCDE具有较高的甘油脱水反应催化活性，在K. pneumoniae代谢合成1,3-丙二醇的途径中起主要作用。.3、K. pneumoniae ΔpduCDE在合成1,3-丙二醇方面展现出优良的性能。在该菌株基础上构建工程菌K. pneumoniae ΔpduCDE∆ldhA∆adhE合成1,3-丙二醇的性能进一步提高，发酵32 h，1,3-丙二醇的产量达到70 g/L，比初始菌株提高了27.3%。