Tominaga Masahiro, Shima Yoko, Nozaki Kenta, Ito Yoichiro, Someda Masataka, Shoya Yuji, Hashii Noritaka, Obata Chihiro, Matsumoto-Kitano Miho, Suematsu Kohei, Matsukawa Tadashi, Hosoya Keita, Hashiba Noriko, Kondo Akihiko, Ishii Jun
Engineering Biology Research Center, Kobe University, Kobe, Japan.
Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.
Nat Commun. 2024 Dec 19;15(1):10653. doi: 10.1038/s41467-024-54865-z.
Inducible promoters are essential for precise control of target gene expression in synthetic biological systems. However, engineering eukaryotic promoters is often more challenging than engineering prokaryotic promoters due to their greater mechanistic complexity. In this study, we describe a simple and reliable approach for constructing strongly inducible synthetic promoters with minimum leakiness in yeasts. The results indicate that the leakiness of yeast-inducible synthetic promoters is primarily the result of cryptic transcriptional activation of heterologous sequences that may be avoided by appropriate insulation and operator mutagenesis. Our promoter design approach has successfully generated robust, inducible promoters that achieve a > 10-fold induction in reporter gene expression. The utility of these promoters is demonstrated by using them to produce various biologics with titers up to 2 g/L, including antigens designed to raise specific antibodies against a SARS-CoV-2 omicron variant through chicken immunization.
可诱导启动子对于在合成生物学系统中精确控制靶基因表达至关重要。然而，由于真核启动子的机制更为复杂，对其进行工程改造往往比原核启动子更具挑战性。在本研究中，我们描述了一种简单可靠的方法，用于在酵母中构建渗漏最小的强诱导型合成启动子。结果表明，酵母诱导型合成启动子的渗漏主要是异源序列隐蔽转录激活的结果，通过适当的绝缘和操纵子诱变可以避免这种情况。我们的启动子设计方法成功地产生了强大的、可诱导的启动子，使报告基因表达实现了>10倍的诱导。通过使用这些启动子生产各种效价高达2 g/L的生物制品，证明了这些启动子的实用性，包括通过鸡免疫设计用于产生针对SARS-CoV-2奥密克戎变体的特异性抗体的抗原。
