Wu Li, Wang Mengzhu, Zha Genhan, Zhou Jungang, Yu Yao, Lu Hong
State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200438, China.
Shanghai Engineering Research Center of Industrial Microorganisms, Shanghai 200438, China.
MethodsX. 2020 Nov 12;7:101138. doi: 10.1016/j.mex.2020.101138. eCollection 2020.
Genome shuffling is a process to combine advantage traits by the recombination of the entire genome and it has been successfully applied in the laboratory evolution of various industrial microorganisms. However, genome shuffling has not been described in (KM), a promising yeast host for the expression of heterologous proteins. In this protocol, genome shuffling in KM is performed by sexual reproduction and is combined with high-throughput screening to obtain high-yielding strains. Notably, the screening of diploid clones risen from one mating mixture is carried out to improve the effectiveness of evolution. Mating-sporulation-mating cycles are repeated to obtain KM strain with ideal traits. •The method combines genome shuffling with high-throughput to achieve strains displaying high yielding of heterologous proteins.•This method can be applied to the genome shuffling of other species when only a few starting strains are available for sexual reproduction.
基因组改组是一种通过整个基因组重组来组合优势性状的过程，并且已成功应用于各种工业微生物的实验室进化。然而，在克鲁维酵母（KM）中尚未描述基因组改组，KM是一种用于表达异源蛋白的有前景的酵母宿主。在本方案中，KM中的基因组改组通过有性繁殖进行，并与高通量筛选相结合以获得高产菌株。值得注意的是，对从一个交配混合物中产生的二倍体克隆进行筛选，以提高进化的有效性。重复交配-孢子形成-交配循环以获得具有理想性状的KM菌株。•该方法将基因组改组与高通量相结合，以获得显示高产异源蛋白的菌株。•当只有少数起始菌株可用于有性繁殖时，该方法可应用于其他物种的基因组改组。
