过量表达NADH氧化酶加速光滑球拟酵母合成丙酮酸

Heterologous expression of H_2O-forming NADH oxidase in Torulopsis glabrata significantly enhance the pyruvate productivity of the host

【目的】进一步提高光滑球拟酵母(Torulopsis glabrata)发酵生产丙酮酸的生产强度。【方法】将来源于乳酸乳球菌(Lactococcus lactis)中编码形成水的NADH氧化酶noxE基因过量表达于丙酮酸工业生产菌株T.glabrata CCTCCM202019中,获得了一株NADH氧化酶活性为34.8U/mg蛋白的重组菌T.glabrata-PDnoxE。【结果】与出发菌株T.glabrata CCTCC M202019相比,细胞浓度、葡萄糖消耗速率和丙酮酸生产强度分别提高了168%、44.9%和12%,发酵进行到36h葡萄糖消耗完毕。补加50g/L葡萄糖继续发酵20h,则使丙酮酸浓度提高到67.2g/L。葡萄糖消耗速度和丙酮酸生产强度增加的原因在于形成水的NADH氧化酶过量表达,导致NADH和ATP含量分别降低了18.1%和15.8%,而NAD+增加了11.1%。【结论】增加细胞内NAD+含量能有效地提高酵母细胞葡萄糖的代谢速度及目标代谢产物的生产强度。

[Objective] The aim of this manuscript was to illuminate the effect of NADH oxidation pathway on the glycolytic rate and the pyruvate productivity. [Methods] The noxE gene encoding a waterorming NADH oxidase from Lactococcus lactis, was expressed in a pyruvate producing Torulopsis glabrata CCTCC M202019. A mutant strain T. glabrata-PDnoxE, with specific NADH oxidase activity of 34.8 U/mg protein, was obtained. [Results] During batch fermentation with 100 g/L glucose in the medium, the dry cell weight, the glucose consumption rate and pyruvate production rate were 168%, 44.9% and 12% higher than that of the parent strain, respectively. Only 2.5 g/L residual glucose was detected in the fermentation broth after 36 h culture, then 50 g/L glucose was supplemented to the culture broth and the concentration of pyruvate increased to 67.2 g/L. As the result of NADH oxidase overexpression, the intracellular NADH, NAD＋ and ATP concentrations of the mutant and the parent strain were deter- mined, the NADH and ATP content decreased 18.1% and 15.8% respectively, while the NAD＋ concentration increased 11.1%. [Conclusion] The increasing of intracellular NAD＋ concentration can efficiently enhance the rate of glucose consumption and the pyruvate production.