异源表达NADH选择性氧化酶提高光滑球拟酵母酵解速率及丙酮酸生产强度  

Significantly increase of glycolytic flux and pyruvate productivity in Torulopsis glabrata by heterologous expression of NADH alternative oxidase

作　　者：秦义[1] 董志姚[1] 周景文[1] 刘立明[1] 陈坚[1] 

机构地区：[1]江南大学,食品科学与技术国家重点实验室,无锡214122

出　　处：《微生物学报》2009年第11期1483-1488,共6页Acta Microbiologica Sinica

基　　金：国家杰出青年基金(20625619);国家自然科学基金(20706025);国家“973项目”(2007CB7l4303);全国优秀博士学位论文作者专项资金(200962)~~

摘　　要：【目的】进一步提高光滑球拟酵母(Torulopsis glabrata)葡萄糖代谢速率及丙酮酸生产强度。【方法】将源于荚膜胞浆菌(Histoplasma capsulatum)的编码选择性氧化酶的AOX1基因过量表达于T.glabrata中,获得了一株线粒体内NADH氧化途径发生改变且胞内总NADH氧化酶活性提高1.8倍的重组菌株AOX。【结果】与出发菌株CON比较,细胞浓度以及发酵周期降低了20.3%和10.7%,而平均比葡萄糖消耗速率和丙酮酸合成速率分别提高了34.7%和54.1%。其原因在于NADH选择性氧化酶过量表达,导致胞内NADH/NAD+比率和ATP含量分别降低了74.7%和52.9%,而磷酸果糖激酶和丙酮酸激酶活性分别提高了185.0%和28.1%。【结论】构建线粒体内非产能的NADH氧化途径,加速NADH氧化的同时降低ATP合成,能有效地提高酵母细胞糖酵解代谢速度及目标代谢产物的生产强度。[Objective] This study aimed at increasing the glycolytic flux and pyruvate productivity in Torulopsis glabrata with glucose as carbon source. [ Methods ] For this target, we introduced a mitochondrial located alternative oxidase encoded by Histoplasma capsulatum AOX1 gene into T. glabrata, and a mutant strain named as AOX with total NADH oxidase activity was 1.8-fold higher than that of the parent stain, was achieved. [ Results] The heterologous expression of NADH alternative oxidase resulted in decrease of the dry cell weight and fermentation time by 20.3% and 10.7%, but the specific rate of glucose consumption and pyruvate production increased 34.7 % and 54.1% higher, respectively. The reasons for high glycolytic flux were the intracellular NADH/NAD ＋ ratio and ATP concentration decreased 74.7 % and 52.9 % respectively, the specific activity of phosphofructokinase, pyruvate kinase increased 185.0% and 28.1%. [ Conclusion]Introduction of a novel NADH oxidation pathway by alternative oxidase can efficiently increase the rate of glucose consumption and the target metabolite productivity.

关 键 词：光滑球拟酵母 NADH选择性氧化酶 NADH 糖酵解速率 

分 类 号：TQ921.7[轻工技术与工程—发酵工程]