The construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) was attempted by engineering a strong methanol-inducible DAS1 promoter using Citrobacter braakii phytase production as a model case. Constitutive expression of KpTRM1, formerly PRM1-a positive transcription regulator for methanol-utilization (MUT) genes of K. phaffii,was demonstrated to produce phytase without addition of methanol, especially when a DAS1 promoter was used but not an AOX1 promoter. Another positive regulator, Mxr1p, did not have the same effect on the DAS1 promoter, while it was more effective than KpTrmp1 on the AOX1 promoter. Removing a potential upstream repression sequence (URS) and multiplying UAS1DAS1 in the DAS1 promoter significantly enhanced the yield of C. braakii phytase with methanol-feeding, which surpassed the native AOX1 promoter by 80%. However, multiplying UAS1DAS1 did not affect the yield of methanol-free expression by constitutive KpTrm1p. Another important region to enhance the effect of KpTrm1p under a methanol-free condition was identified in the DAS1 promoter, and was termed ESPDAS1. Nevertheless, methanol-free phytase production using an engineered DAS1 promoter outperformed phytase production with the GAP promoter by 25%. Difference in regulation by known transcription factors on the AOX1 promoter and the DAS1 promoter was also illustrated.

中文翻译：

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设计 Komagataella phaffii（毕赤酵母）的表达系统：开发无甲醇表达系统的尝试。

通过使用 Citrobacter braakii 植酸酶生产作为模型案例设计强甲醇诱导型 DAS1 启动子，尝试构建 Komagataella phaffii（毕赤酵母）的无甲醇表达系统。KpTRM1 的组成型表达，以前是 PRM1 - 一种用于 K. phaffii 甲醇利用 (MUT) 基因的正转录调节因子，被证明可以在不添加甲醇的情况下产生植酸酶，特别是当使用 DAS1 启动子而不是 AOX1 启动子时。另一个正调节因子 Mxr1p 对 DAS1 启动子没有相同的影响，但它对 AOX1 启动子的作用比 KpTrmp1 更有效。去除潜在的上游抑制序列 (URS) 并在 DAS1 启动子中繁殖 UAS1DAS1 显着提高了甲醇喂养的 C. braakii 植酸酶的产量，超过了天然 AOX1 启动子 80%。然而，增加 UAS1DAS1 不会影响组成型 KpTrm1p 的无甲醇表达的产量。在 DAS1 启动子中发现了在无甲醇条件下增强 KpTrm1p 作用的另一个重要区域，称为 ESPDAS1。尽管如此，使用工程化 DAS1 启动子的无甲醇植酸酶生产比使用 GAP 启动子生产的植酸酶高 25%。还说明了已知转录因子对 AOX1 启动子和 DAS1 启动子的调控差异。使用工程化 DAS1 启动子的无甲醇植酸酶生产比使用 GAP 启动子的植酸酶生产高 25%。还说明了已知转录因子对 AOX1 启动子和 DAS1 启动子的调控差异。使用工程化 DAS1 启动子的无甲醇植酸酶生产比使用 GAP 启动子的植酸酶生产高 25%。还说明了已知转录因子对 AOX1 启动子和 DAS1 启动子的调控差异。