BACKGROUND The methanol-regulated AOX1 promoter (PAOX1) is the most widely used promoter in the production of recombinant proteins in the methylotrophic yeast Pichia pastoris. However, as the tight regulation and methanol dependence of PAOX1 restricts its application, it is necessary to develop a flexible induction system to avoid the problems of methanol without losing the advantages of PAOX1. The availability of synthetic biology tools enables researchers to reprogram the cellular behaviour of P. pastoris to achieve this goal. RESULTS The characteristics of PAOX1 are highly related to the expression profile of methanol expression regulator 1 (Mxr1). In this study, we applied a biologically inspired strategy to reprogram regulatory networks in P. pastoris. A reprogrammed P. pastoris was constructed by inserting a synthetic positive feedback circuit of Mxr1 driven by a weak AOX2 promoter (PAOX2). This novel approach enhanced PAOX1 efficiency by providing extra Mxr1 and generated switchable Mxr1 expression to allow PAOX1 to be induced under glycerol starvation or carbon-free conditions. Additionally, the inhibitory effect of glycerol on PAOX1 was retained because the synthetic circuit was not activated in response to glycerol. Using green fluorescent protein as a demonstration, this reprogrammed P. pastoris strain displayed stronger fluorescence intensity than non-reprogrammed cells under both methanol induction and glycerol starvation. Moreover, with single-chain variable fragment (scFv) as the model protein, increases in extracellular scFv productivity of 98 and 269% were observed in Mxr1-reprogrammed cells under methanol induction and glycerol starvation, respectively, compared to productivity in non-reprogrammed cells under methanol induction. CONCLUSIONS We successfully demonstrate that the synthetic positive feedback circuit of Mxr1 enhances recombinant protein production efficiency in P. pastoris and create a methanol-free induction system to eliminate the potential risks of methanol.

中文翻译：

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通过转录因子Mxr1的合成正反馈电路提高巴斯德毕赤酵母AOX1启动子的效率。

背景技术甲醇调节的AOX1启动子（PAOX1）是在甲基营养酵母巴斯德毕赤酵母中重组蛋白生产中使用最广泛的启动子。但是，由于PAOX1的严格调节和对甲醇的依赖性限制了其应用，因此有必要开发一种灵活的感应系统来避免甲醇问题而又不失去PAOX1的优势。合成生物学工具的可用性使研究人员能够重新编程巴斯德毕赤酵母的细胞行为，以实现这一目标。结果PAOX1的特性与甲醇表达调节剂1（Mxr1）的表达谱高度相关。在这项研究中，我们应用了生物学启发的策略来重新编程巴斯德毕赤酵母中的调控网络。重新编程的P。通过插入由弱AOX2启动子（PAOX2）驱动的Mxr1的合成正反馈电路来构建pastoris。这种新方法通过提供额外的Mxr1并生成可转换的Mxr1表达来增强PAOX1的效率，从而使PAOX1在甘油饥饿或无碳条件下被诱导。此外，保留了甘油对PAOX1的抑制作用，因为合成回路未响应于甘油而被激活。以绿色荧光蛋白为例，在甲醇诱导和甘油饥饿的情况下，这种重编程的巴斯德毕赤酵母菌株显示出比未重编程的细胞更强的荧光强度。此外，以单链可变片段（scFv）作为模型蛋白，与在甲醇诱导下非重编程细胞中的生产率相比，在甲醇诱导和甘油饥饿下的Mxr1重编程细胞中分别观察到了98％和269％的细胞外scFv生产率增加。结论我们成功地证明了Mxr1的合成正反馈电路提高了巴斯德毕赤酵母中重组蛋白的生产效率，并创建了不含甲醇的诱导系统，以消除甲醇的潜在风险。