降低光滑球拟酵母电子传递链活性加速丙酮酸合成  

The Decrease of The Activity of Electron Transfer Chain of Torulopsis glabrata Enhanced Pyruvate Productivity

作　　者：刘立明[1] 陈坚[1] 李华钟[2] 李寅[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 [2]江南大学生物工程学院,无锡214036

出　　处：《微生物学报》2004年第6期800-804,共5页Acta Microbiologica Sinica

基　　金：江苏省自然科学基金 (BK2 0 0 2 0 72 );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国家重点实验室开放课题 ;教育部留学回国人员科研启动基金~~

摘　　要：光滑球拟酵母CCTCCM2 0 2 0 19经溴化乙锭诱变 ,挑选假阳性呼吸缺陷型菌株共 4 0株。对其中 7株丙酮酸产量提高的突变株进行发酵性底物 (葡萄糖 )和非发酵性底物 (甘油、乙酸 )的利用能力测试 ,鉴定得到 3株呼吸缺陷型突变株RD 16、RD 17和RD 18。相对于出发菌株 ,呼吸缺陷型突变株生长速率下降 ,最终菌体浓度降低 2 1%～2 9% ,胞内ATP含量下降 15 %～ 2 1% ,但单位细胞耗葡萄糖能力和单位细胞产丙酮酸能力分别提高了 2 0 7%～30 7%和 30 7%～ 5 5 5 %。进一步研究发现 ,呼吸缺陷型突变株线粒体复合体Ⅰ、Ⅰ +Ⅲ、Ⅱ +Ⅲ和Ⅳ的活性分别下降了 34%～ 4 1%、38 6 %～ 5 2 6 %、2 1%～ 2 5 %、15 0 %～ 6 30 % ,表明线粒体电子传递链氧化NADH的功能受到抑制。为使酵解产生的NADH正常氧化 ,在RD 18菌株的对数生长期流加 2 1mmol L外源电子受体乙醛。发现细胞合成丙酮酸能力提高 2 1 6 % ,且葡萄糖消耗速度明显加快 ,发酵周期缩短 14h。Torulopsis glabrata CCTCC M202019 was mutated by ethidium bromide to screen for respiratory-deficient mutants. Seven mutants that produced pyruvate higher than that of the parent were subjected to the tests of the capability assimilating fermentable substrate (glucose) and non-fermentable substrates (glycerol and acetate) to characterize true respiratory-deficient mutants. Mutants RD-16, RD-17 and RD-18 were unable to assimilate acetate or glycerol and were therefore identified as respiratory-deficient mutants. Compared to the parent strain, the growth the intracellular ATP content of those mutants decreased by 21%～29% and 15%～21%, respectively, while the glucose consumption per cell and the pyruvate production per cell of those mutants were enhanced by 20.7%～30.7% and 30.7%～55.5%, respectively. Qualitative analysis of cytochromes involved in electron transfer chain showed that mutants RD-16 and RD-18 lacked both cytochrome aa3 and b, while mutant RD-17 lacked cytochrome b. Enzymes analysis indicated that the activities of ATPase, succinate-cytochrome c reductase (complex Ⅰ), complex Ⅰ+Ⅲ, complex Ⅱ+Ⅲ, and complex Ⅳ of those mutants decreased by 14.6%～22.2%, 34%～41%, 38.6%～52.6%, 21%～25%, and 150%～630%, respectively. However, increased glucose consumption per cell was not observed in those mutants, which might be due to that the NADH generated in glycolysis can not be completely oxidized via electron transfer chain. To avoid the accumulation of NADH, 2.1mmol/L acetaldehyde was added to the culture broth of mutant RD-17 at 26h of fermentation. Using this strategy, the amount of pyruvate produced increased by 21.6% while the fermentation time was shortened from 62h to 48h.

关 键 词：光滑球拟酵母 呼吸缺陷型 电子传递链 细胞色素 线粒体复合体 丙酮酸 

分 类 号：TQ920[轻工技术与工程—发酵工程]