Methanol independent induction in Pichia pastoris by simple derepressed overexpression of single transcription factors
Methanol independent induction in Pichia pastoris by simple derepressed overexpression of single transcription factors
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DOI：
10.1002/bit.26529
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发表时间：
2018-04-01
影响因子：
3.8
通讯作者：
Glieder, Anton
中科院分区：
文献类型：
--
作者：
Vogl, Thomas;Sturmberger, Lukas;Glieder, Anton
碳源调控的启动子是控制基因表达的标准工具。获得对启动子的天然调节的控制对于代谢工程和合成生物学应用是重要的。在常用的蛋白质生产宿主Komagataella phaffii（巴斯德毕赤酵母）中，使用甲醇诱导型启动子，因为它们的严格调控和异常强度。然而，用有毒和易燃的甲醇诱导可能是相当大的安全风险，并且不能应用于许多现有的发酵装置。在这里，我们研究了最常用的醇氧化酶1启动子（P-AOX 1）的基础上，这是紧密抑制在抑制碳源的存在下，并强烈诱导甲醇的新的监管电路。我们比较了不同的过表达策略，为假定的碳源依赖性监管机构确定的同源性搜索相关的酵母和以前发表的文献，以现有的甲醇依赖性表达菌株转化为无甲醇系统。虽然组成型过表达仅显示出边缘或有害的影响，但去阻遏表达（当阻遏碳源耗尽时激活）显示三种转录因子（TF）单独适合于在巴斯德毕赤酵母中强烈激活P-AOX 1，而不依赖于任何特定工程化的宿主菌株。转录组分析表明，Mxr 1，Mit 1和Prm 1调节部分重叠和独特的基因组。单一TF的去阻遏过表达足以将现有的基于P-AOX 1的表达菌株改造成葡萄糖/甘油调节的无甲醇系统。考虑到碳源调节启动子的广泛适用性、在大规模生物反应器中控制碳源进料速率的简单性和低成本，与巴斯德毕赤酵母中类似的方法也可用于其他生物体。
Carbon source regulated promoters are well-studied standard tools for controlling gene expression. Acquiring control over the natural regulation of promoters is important for metabolic engineering and synthetic biology applications. In the commonly used protein production host Komagataella phaffii (Pichia pastoris), methanol-inducible promoters are used because of their tight regulation and exceptional strength. Yet, induction with toxic and flammable methanol can be a considerable safety risk and cannot be applied in many existing fermentation plants. Here we studied new regulatory circuits based on the most frequently used alcohol oxidase 1 promoter (P-AOX1), which is tightly repressed in presence of repressing carbon sources and strongly induced by methanol. We compared different overexpression strategies for putative carbon source dependent regulators identified by a homology search in related yeasts and previously published literature in order to convert existing methanol dependent expression strains into methanol free systems. While constitutive overexpression showed only marginal or detrimental effects, derepressed expression (activated when the repressing carbon source is depleted) showed that three transcription factors (TFs) are single handedly suitable to strongly activate P-AOX1 in P. pastoris without relying on any specifically engineered host strains. Transcriptome analyses demonstrated that Mxr1, Mit1, and Prm1 regulate partly overlapping and unique sets of genes. Derepressed overexpression of a single TF was sufficient to retrofit existing P-AOX1 based expression strains into glucose/glycerol regulated, methanol-free systems. Given the wide applicability of carbon source regulated promoters, the simplicity and low cost of controlling carbon source feed rates in large scale bioreactors, similar approaches as in P. pastoris may also be useful in other organisms.