College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China.
Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, 100029, China.
Folia Microbiol (Praha). 2023 Apr;68(2):207-217. doi: 10.1007/s12223-022-01006-y. Epub 2022 Oct 6.
During yeast dough fermentation, such as the high-sucrose bread-making process, the yeast cells are subjected to considerable osmotic stress, resulting in poor outcomes. Invertase is important for catalyzing the irreversible hydrolysis of sucrose to free glucose and fructose, and decreasing the catalytic activity of the invertase may reduce the glucose osmotic stress on the yeast. In this study, we performed structural design and site-directed mutagenesis (SDM) on the Saccharomyces cerevisiae invertase (ScInV) in an Escherichia coli expression system to study the catalytic activity of ScInV mutants in vitro. In addition, we generated the same mutation sites in the yeast endogenous genome and tested their invertase activity in yeast and dough fermentation ability. Our results indicated that appropriately reduced invertase activity of yeast ScInV can enhance dough fermentation activity under high-sucrose conditions by 52%. Our systems have greatly accelerated the engineering of yeast endogenous enzymes both in vitro and in yeast, and shed light on future metabolic engineering of yeast.
在酵母面团发酵过程中，例如高蔗糖面包制作过程中，酵母细胞会受到相当大的渗透胁迫，导致结果不佳。转化酶对于催化蔗糖不可逆水解为游离葡萄糖和果糖非常重要，降低转化酶的催化活性可能会降低酵母的葡萄糖渗透胁迫。在这项研究中，我们在大肠杆菌表达系统中对酿酒酵母转化酶（ScInV）进行了结构设计和定点突变（SDM），以研究 ScInV 突变体在体外的催化活性。此外，我们在酵母内源基因组中生成了相同的突变位点，并在酵母和面团发酵中测试了它们的转化酶活性。我们的结果表明，适当降低酵母 ScInV 的转化酶活性可以提高高蔗糖条件下面团发酵活性 52%。我们的系统大大加快了酵母内源酶的体外和酵母工程改造，为未来的酵母代谢工程提供了思路。
