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食品与发酵工业  2020, Vol. 46 Issue (8): 7-13    DOI: 10.13995/j.cnki.11-1802/ts.023147
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
酿酒酵母pdc基因缺陷菌株的构建及其丙酮酸发酵特性
李亿1, 秦艳1, 申乃坤2, 朱婧1, 梁戈1, 王青艳1*
1 (广西科学院,国家非粮生物质能源工程技术研究中心,非粮生物质酶解国家重点实验室,广西生物炼制重点实验室,广西生物质工程技术研究中心, 广西 南宁, 530007)
2 (广西民族大学 海洋科学与生物技术学院,广西微生物与植物资源利用重点实验室, 广西 南宁, 530008)
Construction of pdc-deficient Saccharomyces cerevisiae and its pyruvic acid fermentation characteristics
LI Yi1, QIN Yan1, SHEN Naikun2, ZHU Jing1, LIANG Ge1, WANG Qingyan1*
1 (National Engineering Research Center for Non-Food Bio-refinery, State Key Laboratory of Non-Food Biomass and Enzyme Technology, Guangxi Key Laboratory of Bio-refinery, Guangxi Biomass Engineering Technology Research Center, Guangxi Academy of Sciences, 98 Daling Road, Nanning 530007, China)
2 (School of Marine Sciences and Biotechnology, Guangxi Key Laboratory of Utilization of Microbial and Botanical Resources, Guangxi University for nationalities, Nanning 530008, China)
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摘要 丙酮酸是一种重要的有机酸,在聚合物、化妆品、食品添加剂、医药等领域具有广泛应用。酿酒酵母被认为是丙酮酸生产的潜在最适微生物,但其糖酵解过程产生的丙酮酸会在胞质内的丙酮酸脱羧酶催化作用下降解为CO2和乙醛,造成碳代谢流的损失。为将更多的碳代谢流引向丙酮酸合成,该研究通过敲除丙酮酸脱羧酶的3个结构基因(pdc1pdc5pdc6),显著地促进了丙酮酸积累,但也造成突变菌株XY-156生长缓慢。与进化之前相比,采用适应性进化策略驯化得到的菌株XY-156A,在细胞生长、葡萄糖消耗以及丙酮酸积累方面均获得显著提高;进一步利用2 L发酵罐进行批次补料发酵试验,发酵76 h丙酮酸产量可达105 g/L,生产强度为1.38 g/(L·h),得率为0.5 g/g葡萄糖。结果表明,采用失活丙酮酸脱羧酶与适应性定向进化相结合的策略改造酿酒酵母,能够实现其高效积累丙酮酸,可为生物基丙酮酸工业化生产奠定坚实的基础。
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李亿
秦艳
申乃坤
朱婧
梁戈
王青艳
关键词:  酿酒酵母  基因敲除  丙酮酸脱羧酶  适应性进化  丙酮酸发酵    
Abstract: Pyruvic acid is an important organic acid with wide applications in polymer, cosmetic, food additive, pharmaceutical and other fields. Saccharomyces cerevisiae is considered as the best candidate microorganism for pyruvic acid production. However, pyruvic acid produced during glycolysis could be catalyzed by pyruvate decarboxylase in the cytoplasm to degrade into CO2 and acetaldehyde, resulting in the loss of carbon metabolic flux. In order to redirect more carbon metabolic flux to pyruvic acid synthesis, the deletions of the three structural genes of pyruvate decarboxylase (pdc1, pdc5 and pdc6) were performed, which significantly promoted pyruvic acid accumulation. Nevertheless, poor growth of the mutant strain XY-156 was also observed. Compared with the non-evolved strain XY-156, the evolved strain XY-156A obtained by adaptive evolution showed significant improvement in cell growth, glucose consumption and pyruvate accumulation. Fed-batch fermentation was further performed in a 2 L stirred bioreactor, and up to 105 g/L pyruvic acid was obtained within 76 h, with a productivity of 1.38 g/(L·h) and a yield of 0.5 g/g glucose. The results showed that the combination of inactivating pyruvate decarboxylase with adaptive evolution could achieve efficient pyruvic acid accumulation in S. cerevisiae, and could lay a solid foundation for the industrial production of biological pyruvic acid.
Key words:  Saccharomyces cerevisiae    gene knockout    pyruvate decarboxylase    adaptive evolution    pyruvate fermentation
收稿日期:  2019-12-18                出版日期:  2020-04-25      发布日期:  2020-05-20      期的出版日期:  2020-04-25
基金资助: 国家自然科学基金项目(31660022);广西科技计划项目(2018-15-Z03-1202;桂科AB19110041);广西科学院基本科研业务费资助项目(2017YJJ23013);广西自然科学基金青年科学基金项目(2019GXNSFBA245026)
作者简介:  硕士,助理研究员(王青艳研究员为通讯作者,E-mail:qingyanw@126.com)
引用本文:    
李亿,秦艳,申乃坤,等. 酿酒酵母pdc基因缺陷菌株的构建及其丙酮酸发酵特性[J]. 食品与发酵工业, 2020, 46(8): 7-13.
LI Yi,QIN Yan,SHEN Naikun,et al. Construction of pdc-deficient Saccharomyces cerevisiae and its pyruvic acid fermentation characteristics[J]. Food and Fermentation Industries, 2020, 46(8): 7-13.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.023147  或          http://sf1970.cnif.cn/CN/Y2020/V46/I8/7
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