Wu Li, Lyu Yilin, Wu Pingping, Luo Tongyu, Zeng Junyuan, Shi Tianfang, Zhou Jungang, Yu Yao, Lu Hong
State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China.
Shanghai Engineering Research Center of Industrial Microorganisms, Shanghai, China.
Front Bioeng Biotechnol. 2022 Jan 11;9:799756. doi: 10.3389/fbioe.2021.799756. eCollection 2021.
is the fastest-growing eukaryote and a promising host for producing bioethanol and heterologous proteins. To perform a laboratory evolution of thermal tolerance in , diploid, triploid and tetraploid strains were constructed, respectively Considering the genetic diversity caused by genetic recombination in meiosis, we established an iterative cycle of "diploid/polyploid - meiosis - selection of spores at high temperature" to screen thermotolerant strains. Results showed that the evolution of thermal tolerance in diploid strain was more efficient than that in triploid and tetraploid strains. The thermal tolerance of the progenies of diploid and triploid strains after a two-round screen was significantly improved than that after a one-round screen, while the thermal tolerance of the progenies after the one-round screen was better than that of the initial strain. After a two-round screen, the maximum tolerable temperature of Dip2-8, a progeny of diploid strain, was 3°C higher than that of the original strain. Whole-genome sequencing revealed nonsense mutations of and in the thermotolerant progenies. Deletion of either or in the original strain improved thermotolerance and two deletions displayed additive effects, suggesting and negatively regulated the thermotolerance of in parallel pathways. Therefore, the iterative cycle of "meiosis - spore screening" developed in this study provides an efficient way to perform the laboratory evolution of heat resistance in yeast.
是生长最快的真核生物，也是生产生物乙醇和异源蛋白的有前途的宿主。为了在中进行耐热性的实验室进化，分别构建了二倍体、三倍体和四倍体菌株。考虑到减数分裂中基因重组引起的遗传多样性，我们建立了一个“二倍体/多倍体 - 减数分裂 - 在高温下选择孢子”的迭代循环来筛选耐热菌株。结果表明，二倍体菌株的耐热性进化比三倍体和四倍体菌株更有效。经过两轮筛选后，二倍体和三倍体菌株后代的耐热性比一轮筛选后显著提高，而一轮筛选后后代的耐热性比初始菌株更好。经过两轮筛选后，二倍体菌株后代Dip2-8的最高耐受温度比原始菌株高3°C。全基因组测序揭示了耐热后代中 和 的无义突变。在原始菌株中删除 或 均可提高耐热性，且两个缺失显示出累加效应，表明 和 在平行途径中对 的耐热性起负调控作用。因此，本研究中开发的“减数分裂 - 孢子筛选”迭代循环为在酵母中进行耐热性的实验室进化提供了一种有效的方法。
