光滑球拟酵母新霉素抗性株加速葡萄糖代谢  

Torulopsis glabrata neomycin-resistant mutant abolishes pyruvate production with enhancement of glucose consumption rate

作　　者：刘立明[1] 李华钟[1] 李寅[1] 陈坚[1] 

机构地区：[1]江南大学工业生物技术教育部重点实验室,无锡214036

出　　处：《微生物学报》2005年第4期617-620,共4页Acta Microbiologica Sinica

基　　金：江苏省高等学校研究生创新计划;华东理工大学生物反应器工程国家重点实验室开放课题;教育部高等学校博士点建设专项资金(20040294003)~~

摘　　要：为进一步提高光滑球拟酵母发酵生产丙酮酸的生产强度,在能量代谢分析的基础上提出了降低ATP合成酶活性、但不影响NADH氧化的育种策略。通过亚硝基胍诱变,获得一株新霉素抗性突变株N07,该菌株F1ATPase活性降低65%、丙酮酸产量高于48gL且单位细胞消耗葡萄糖能力提高38%。添加双环己基碳二亚胺(DCCD)、叠氮钠(NaN3)、新霉素显著降低出发株F1ATPase活性但不影响突变株F1ATPase活性。突变菌株胞内ATP含量下降23.7%导致生长速率和最终菌体浓度(为出发菌株的76%)均低于出发菌株,但葡萄糖消耗速度和丙酮酸生产速度分别提高34%和42.9%,发酵周期缩短12h。进一步研究发现,突变株糖酵解途径中关键酶磷酸果糖激酶、丙酮酸激酶和磷酸甘油醛激酶的活性提高了63.7%、28.8%和14.4%,电子传递链关键酶活性提高10%。结果表明降低真核微生物F1ATPase活性有效地提高了糖酵解关键酶活性而加速葡萄糖代谢。To further increase the rate of glucose consumption by multi-vitamin auxotrophic yeast Torulopsis glabrata. A neomycin-resistant mutant N07, with the activity of F-1-ATPase decreased roughly 35% but glucose consumed per cell was increased 38% than that of parent strain, was breed based on analysis of energy metabolic pathway. The typical inhibitors of F-1F-0-ATPase, DCCD, NaN3 and neomycin, depressed the F-1-ATPase activity of parental strain but no effect on that of mutant strain. Strain N07 was cultured in a pyruvate fermentation medium containing 100g/L of glucose using flask. It was found that the rate of glucose consumption and pyruvate production were higher by 34% and 42.9% in the mutant than in the parent, respectively. However, the rate and yield of growth (about 24%) of the mutant was lower than that of the parent. The content of intracellular ATP of the mutant also decreased 23.7% than that of the parent. The activities of key enzymes in glycolytic pathway and electron transfer chain of the mutant and the parent were determined. Enzymatic analysis revealed that, compared with the parent strain CCTCC M202019.The activities of key enzymes, phosphofructokinase, pyruvate kinase, glyceraldyde-3-phosphate dehydrogenase of the mutant N07 increased 63.7%,28.8% and 14.4%, respectively, all the key enzymes of electron transfer chain in the mutant N07 also increased roughly 10%.

关 键 词：光滑球拟酵母 丙酮酸生产 糖酵解 F1-ATPase 新霉素抗性 

分 类 号：TQ920[轻工技术与工程—发酵工程]