通过体外代谢途径构建确定乳酸合成关键酶

Determination of key enzymes for lactate synthesis through in vitro metabolic pathway construction

基于代谢控制分析理论提出了一种通过体外代谢途径构建和分析确定关键酶的方法并应用其确定乳酸高产菌株中的关键酶。首先获得高产菌株的粗酶液并测定葡萄糖到乳酸合成途径中各种蛋白的绝对浓度,进而通过向粗酶液中分别添加同等比例的各纯酶对途径进行扰动,并由扰动前后的途径通量变化计算出各酶的通量控制系数以确定关键酶。结果表明,该菌株乳酸合成途径中丙酮酸激酶和甘油醛-3-磷酸脱氢酶对途径通量影响最大,由此预测在该菌株基础上进一步过表达这两个酶对提高乳酸生成速率可能最为有效。

A method was presented for key enzyme determination through in vitro metabolic pathway analysis based on the metabolic control analysis theory and it was used to determine the key enzymes in lactate-producing strain. Firstly, crude enzyme extracts were obtained from the strain and the absolute protein concentrations for all enzymes in the pathway from glucose to pyruvate were measured. Then individual pure enzymes were added to the crude extract and the pathway flux changes were measured to calculate the flux control coefficients （FCCs） of the enzymes to determine the key enzymes. It was found that pyruvate kinase （PykA） and glyceraldehyde-3-phosphate dehydrogenase （GapA） had the largest effect on FCCs, and thus should be the gene overexpression targets for further improvement of lactate production.