In the present study, the adaptive evolution of a metabolically engineered Saccharomyces cerevisiae strain in the presence of an enzyme inhibitor terbinafine for enhanced squalene accumulation via serial transfer leads to the development of robust strains. After adaptation for nearly 1500 h, a strain with higher squalene production efficiency was identified at a specific growth rate of 0.28 h⁻¹ with a final squalene titer of 193 mg/L, which is 16.5-fold higher than the BY4741 and 3-fold higher over the metabolically engineered SK22 strain. Whole-genome sequencing comparison between the reference strain and the evolved variant SK23 has led to the identification of 462 single-nucleotide variants (SNVs) between both strains, with 102 SNVs affecting metabolism-related genes. It was also established that F420I mutation of ERG1 in S. cerevisiae improves squalene synthesis. Further, the effect of increased squalene on lipid droplet and neutral lipid pattern in the evolved mutant strains was investigated by fluorescent techniques proving that the neutral lipid content and clustering of lipid droplets increase with an increase in squalene.

中文翻译：

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用于角鲨烯生产改进的工程酵母的适应性进化及其全基因组分析

在本研究中，代谢工程酿酒酵母菌株在酶抑制剂特比萘芬存在下的适应性进化，通过连续转移增强角鲨烯积累，导致产生稳健菌株。经过近 1500 h 的适应，在 0.28 h ^-1的特定生长速率下鉴定出具有较高角鲨烯生产效率的菌株最终角鲨烯滴度为 193 mg/L，比 BY4741 高 16.5 倍，比代谢工程 SK22 菌株高 3 倍。参考菌株和进化变体 SK23 之间的全基因组测序比较导致在两种菌株之间鉴定出 462 个单核苷酸变体 (SNV)，其中 102 个 SNV 影响代谢相关基因。会议还确定了的F420I突变ERG1在酿酒酵母提高角鲨烯合成。此外，通过荧光技术研究了增加的角鲨烯对进化的突变菌株中脂滴和中性脂质模式的影响，证明中性脂质含量和脂滴的聚集随着角鲨烯的增加而增加。