New England Biolabs, Ipswich, Massachusetts, USA.
Genetics & Experimental Bioinformatics, Institute of Biology III, University of Freiburg, Freiburg, Germany.
Appl Environ Microbiol. 2019 Jul 1;85(14). doi: 10.1128/AEM.00542-19. Print 2019 Jul 15.
The yeast has been a successful host for the production of heterologous proteins for over 30 years. Currently, the galactose-/lactose-inducible and glucose-repressible promoter (P ) is the most widely used promoter to drive recombinant protein expression in However, P is not fully repressed in the presence of glucose and significant protein expression still occurs. Thus, P is not suitable in processes where tight regulation of heterologous gene expression is required. In this study, we devised a novel promoter system that is both strong and tightly controllable. We first tested several different endogenous promoters for their ability to express recombinant proteins. A novel hybrid promoter (termed P) was created by combining segments of two promoters, namely, the strong constitutive P promoter and the carbon source-sensitive P promoter. We demonstrate that P is tightly repressed in the presence of glucose or glycerol and becomes derepressed upon depletion of these compounds by the growing cells. We further illustrate the utility of P-controlled protein expression in shake flask and high-cell-density bioreactor cultivation strategies. The P hybrid promoter is a strong derepressible promoter for use in autoinduction of one-step fermentation processes for the production of heterologous proteins in The yeast is an important host for the expression of recombinant proteins at both laboratory and industrial scales. However, the system lacks a tightly regulated promoter that permits controlled expression of heterologous proteins. In this study, we report the engineering of a highly regulated strong hybrid promoter (termed P) for use in P is tightly repressed by glucose or glycerol in the medium but strongly promotes gene expression once the carbon source has been consumed by the cells. This feature permits heterologous protein expression to be "autoinduced" at any scale without the addition of a gratuitous inducer molecule or changing feed solutions.
酵母作为生产异源蛋白的宿主已有 30 多年的历史。目前，半乳糖/乳糖诱导和葡萄糖抑制的启动子（P）是最广泛用于驱动重组蛋白在酵母中表达的启动子。然而，在葡萄糖存在的情况下，P 没有完全被抑制，仍然会发生显著的蛋白表达。因此，P 不适用于需要严格调控异源基因表达的过程。在本研究中，我们设计了一种新型的强且可严格调控的启动子系统。我们首先测试了几种不同的内源性启动子，以评估它们表达重组蛋白的能力。通过组合两个启动子，即强组成型启动子 P 和碳源敏感启动子 P 的片段，我们创建了一个新的杂交启动子（称为 P）。我们证明，在葡萄糖或甘油存在的情况下，P 被严格抑制，而当这些化合物被生长细胞耗尽时，P 被去抑制。我们进一步说明了 P 控制的蛋白表达在摇瓶和高细胞密度生物反应器培养策略中的应用。P 杂交启动子是一种强可诱导的启动子，可用于一步发酵过程，用于生产酵母中的异源蛋白。酵母是在实验室和工业规模表达重组蛋白的重要宿主。然而，该系统缺乏一种可严格调控的启动子，以允许对异源蛋白进行可控表达。在本研究中，我们报告了一种高度调控的强杂交启动子（称为 P）的工程设计，用于酵母。P 在培养基中被葡萄糖或甘油强烈抑制，但一旦细胞消耗了碳源，就会强烈促进基因表达。这个特性允许在任何规模下“自动诱导”异源蛋白表达，而无需添加额外的诱导分子或改变补料溶液。
