In biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research. In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1–30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica. We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

中文翻译：

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解脂耶氏酵母中的混合启动子工程策略：异戊醇生产作为一项测试研究

在生物细胞中，启动子通过 RNA 聚合酶的特异性结合来驱动基因表达。它们决定基因表达的起始位置、时间和水平。因此，合理微调启动子以调节靶基因的表达水平以优化代谢工程中的生物合成途径已成为最近的一个活跃研究领域。在这项研究中，我们系统地检测和表征了非常规酵母解脂耶氏酵母中的常见启动子元件，并构建了一个涵盖广泛启动子强度的人工混合启动子库。结果表明，杂交启动子强度可以通过启动子元件进行微调，即上游激活序列（UAS）、TATA盒和核心启动子。尤其，首次报道了酿酒酵母启动子的 UAS 在功能上转移到解脂耶氏酵母。随后，使用多功能平台化学异戊醇的生产作为测试研究，应用混合启动子库优化解脂耶氏酵母中的生物合成途径表达。通过表达关键途径基因 ScARO10 和启动子文库，异戊醇滴度比对照菌株解脂耶氏酵母 Po1g KU70Δ 提高了 1.1-30.3 倍。有趣的是，使用弱启动子 PUAS1B4-EXPm 表达 ScARO10 获得了最高滴度增加。这些结果表明，我们的混合启动子文库可以成为一个强大的工具包，用于鉴定在解脂耶氏酵母中表达代谢途径的最佳启动子。