酿酒酵母W5单倍体的制备及其代谢水平上的变化

摘要/Abstract

摘要： 酿酒酵母（Saccharomyces cerevisiae）的无性繁殖会导致复杂且不稳定的基因组倍性变化，这一直是酵母遗传学研究及菌种改造的难题之一。研究表明，单倍体的制备是解决这种难题的重要途径。本研究旨在以二倍体酿酒酵母W5为出发菌株，通过单倍体构建2,3-丁二醇（2,3-butanediol, 2,3-BD）高产菌株。因此必须首先筛选最适产孢培养基，优化单倍体分离和制备的条件，获得单倍体细胞，利用MAT-PCR法和形态学观察鉴定单倍体，最后进行单倍体稳定性和代谢水平上的研究。结果表明，W5在改良SPM上30℃培养8 d，产孢率最高（35.57%）。在此条件下，将产孢菌悬液于3%蜗牛酶中处理2 h，孢子释放率高达47.06%；通过孢子分离及传代培养后共获得7株单倍体，PCR扩增后获得一株HO缺失的稳定传代单倍体菌株S. cerevisiae H14；以W5菌株为对照，摇瓶发酵数据表明H14菌株的葡萄糖利用率下降，乙醇产率降低21.67%；重要副产物乙酸、2,3-BD前体物质乙偶姻的产量分别提高了12.7%和57.14%，表明理想的单倍体制备成功。本研究为利用基因工程改造酿酒酵母，构建高产2,3-丁二醇的菌株提供技术保证。

关键词: 酿酒酵母W5, 单倍体制备, 单倍体验证, 代谢

Abstract: The complex and unstable genome ploidy variation of Saccharomyces cerevisiae, resulting from its asexual reproduction, have always been one of the challenges for genetics research and strains improvement of yeast. To resolve the problem, haploid strain must be prepared. In this paper, we aims at constructing an engineering S. cerevisiae with the capability of producing 2,3-butanediol (2,3-BD) by preparing haploid strain from S. cerevisiae W5, which is a diploid strain. Thus, the most appropriate sporulation medium must be selected firstly. Then, the haploid strain was obtained by exploring conditions of haploid separation and preparation which was then verified by MAT-PCR and morphology observation. At last, the stability and metabolism of the haploid strain were studied. The results showed that the highest sporulation rate (35.57%) of W5 was acquired when cultured on modified SPM medium at 30℃for 8 days. Under the condition, the sporulation suspension of W5 was treated in 3% snailase for 2 h and the release rate of the spores reached up to 47.06%, which was then separated and subcultured to screen the haploid strains. Finally, a stable haploid strain S. cerevisiae H14 with deletion of HO was obtained by MAT-PCR from 7 original haploid strains. Using the W5 as the control, the fermentation results demonstrated that the consumption of glucose decreased and the ethanol production reduced (21.67%). Meanwhile, the yield of important by-product acetic acid and acetoin that is the precursor of 2,3-BD increased by 12.7% and 57.14%, respectively, indicating the successfully preparation of the desired haploid strain. This paper will provide technical guarantee for S. cerevisiae modification to enhance 2,3-BD production by genetic engineering.

Key words: Saccharomyces cerevisiae, Haploid preparation, Haploid identification, Metabolism

中图分类号:

王长丽佟天奇刘文娟张云野宋刚葛菁萍. 酿酒酵母W5单倍体的制备及其代谢水平上的变化[J]. 食品科学, 0, (): 0-0.

jingping jingping GeGe. The haploid preparation of Saccharomyces cerevisiae W5 and its change of metabolism[J]. FOOD SCIENCE, 0, (): 0-0.

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