Graduate School of Environmental, Life, Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
Faculty of Environmental, Life, Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
FEMS Yeast Res. 2024 Jan 9;24. doi: 10.1093/femsyr/foae032.
Promoters for artificial control of gene expression are central tools in genetic engineering. In the budding yeast Saccharomyces cerevisiae, a variety of constitutive and controllable promoters with different strengths have been constructed using endogenous gene promoters, synthetic transcription factors and their binding sequences, and artificial sequences. However, there have been no attempts to construct the highest strength promoter in yeast cells. In this study, by incrementally increasing the binding sequences of the synthetic transcription factor Z3EV, we were able to construct a promoter (P36) with ~1.4 times the strength of the TDH3 promoter. This is stronger than any previously reported promoter. Although the P36 promoter exhibits some leakage in the absence of induction, the expression induction by estradiol is maintained. When combined with a multicopy plasmid, it can express up to ~50% of total protein as a heterologous protein. This promoter system can be used to gain knowledge about the cell physiology resulting from the ultimate overexpression of excess proteins and is expected to be a useful tool for heterologous protein expression in yeast.
启动子是人工控制基因表达的重要工具，在遗传工程中被广泛应用。在 budding 酵母 Saccharomyces cerevisiae 中，人们利用内源性基因启动子、合成转录因子及其结合序列和人工序列，构建了多种组成型和可控性的启动子，它们的强度各有不同。然而，目前还没有人尝试在酵母细胞中构建最强的启动子。在这项研究中，我们通过逐步增加合成转录因子 Z3EV 的结合序列，成功构建了一个强度约为 TDH3 启动子 1.4 倍的启动子（P36）。这比以前报道的任何启动子都要强。尽管 P36 启动子在没有诱导物的情况下存在一定的泄漏，但雌二醇的表达诱导仍然能够维持。当与多拷贝质粒结合使用时，它可以表达高达约 50%的总蛋白作为异源蛋白。这个启动子系统可以用来研究由于过度表达多余蛋白质而导致的细胞生理学，有望成为酵母中异源蛋白表达的有用工具。
