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 > 10³-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.

诱导型启动子对于精确控制合成生物系统中的靶基因表达至关重要。然而，由于它们的机制复杂性更高，工程化真核启动子通常比工程化原核启动子更具挑战性。在这项研究中，我们描述了一种简单可靠的方法来构建强诱导性合成启动子，在酵母中具有最小的泄漏。结果表明，酵母诱导的合成启动子的泄漏主要是异源序列的隐蔽转录激活的结果，这可以通过适当的绝缘和操纵子诱变来避免。我们的启动子设计方法已成功生成稳健的诱导型启动子，可在报告基因表达中实现 > 10³ 倍诱导。通过使用这些启动子生产滴度高达 2 g/L 的各种生物制剂来证明这些启动子的效用，包括旨在通过鸡免疫产生针对 SARS-CoV-2 omicron 变体的特异性抗体的抗原。