酿酒酵母W5单倍体的制备及其代谢水平分析

摘要

为获得遗传稳定的单倍体酵母菌株,本实验以二倍体酿酒酵母Saccharomyces cerevisiae W5为出发菌株,优化单倍体制备和分离的条件,获得单倍体细胞;利用随机孢子分析法和MAT-聚合酶链式反应法对单倍体菌株进行分离和鉴定,通过连续传代培养的方式验证单倍体菌株的稳定性,并从代谢水平分析单倍体菌株的发酵性能.研究表明:S.cerevisiae W5在改良SPM培养基中30℃条件下培养8 d,产孢率最高为(35.57±0.82)%.经30 mg/mL蜗牛酶液处理2 h后,菌悬液的孢子释放率达到(47.06±0.23)%.本实验共分离出7 株单倍体菌株,其中菌株S.cerevisiae H14(MAT-a)遗传性能最稳定.当以葡萄糖为底物,30 ℃、150 r/min发酵30 h,乙醇质量浓度达到最大,为(40.46±1.06)g/L,较S.cerevisiae W5发酵24 h乙醇最大质量浓度(51.65±2.39)g/L下降了21.67%,乙酸、乙偶姻的产量分别提高了12.7%和57.14%,获得理想的单倍体菌株.本实验为S.cerevisiae单倍体的制备提供了一定研究经验,也丰富了酵母遗传学研究的理论基础.%The experimental conditions to obtain a genetically stable haploid strain from the Saccharomyces cerevisiae diploid strain W5 were optimized in this study. Seven haploid strains were isolated and identified by random spore analysis and MAT-PCR.Then, the stability of the haploid strains was evaluated by continuous passage culture. Ultimately, the fermentation performance of two selected haploid strains was analyzed. The results showed that the highest sporulation rate of the strain W5 of (35.57 ± 0.82)% was acquired when it was cultured on a modified SPM medium at 30 ℃ for 8 days. Spore release rate was(47.06 ± 0.23)% after 2 h treatment of the suspension with 3% snailase.S.cerevisiae H14(MAT-a)was the most genetically stable among seven haploid strains. Strain H14 gave the highest ethanol production of (40.46 ± 1.06) g/L when cultured at 30 ℃ with glucose as a substrate for 30 h with agitation at 150 r/min, which was 21.67% lower than that of W5 at 24 h, whereas the yields of acetic acid and acetoin were increased by 12.7% and 57.14%, respectively. This research can provide useful information for researchers to prepare haploid S.cerevisiae and also enrich the fundamental theory of yeast genetics.