用于改变酿酒酵母中基因表达的 tetO 工具包,ACS Synthetic Biology

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用于改变酿酒酵母中基因表达的 tetO 工具包
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2015-03-17 00:00:00 , DOI: 10.1021/sb500363y
Josh T Cuperus ₁ , Russell S Lo ₁ , Lucia Shumaker ₁ , Julia Proctor ₁ , Stanley Fields ₁

Affiliation

优化代谢途径的策略通常涉及构建大量菌株，每个菌株都包含调节途径基因表达的不同版本的序列。在这里，我们开发了在酿酒酵母中高效执行该过程的试剂和方法。我们鉴定了大肠杆菌tet 操纵子 (tetO) 序列的变体，这些变体以不同的亲和力结合 TetR-VP16 激活剂，因此导致不同的 TetR-VP16 激活剂驱动的表达。通过重组通路基因上游的这些变体，我们生成了独特的表达水平组合。在这里，我们构建了一个 tetO 工具包，其中包括 I-OnuI 归巢核酸内切酶以创建双链断裂，从而将同源重组增加 10⁵ ; 携带六个变体 tetO 序列的质粒，两侧是 I-OnuI 位点，解偶联转化和重组步骤；酿酒酵母优化的 TetR–VP16 激活剂；以及将构建体整合到酵母基因组中的载体。我们将酵母合成番茄红素所需的来自Erwinia herbicola的三个crt基因引入酿酒酵母基因组，并进行重组过程以产生具有调节这三个基因的 tetO 变体排列的细胞群。我们确定该人群中有 0.7% 的人制造可检测到的番茄红素，其中绝大多数在所有三个crt都经历了重组基因。我们估计每个目标位点的重组率为 ~20%，远高于其他研究中获得的重组率。将该工具包应用于医学或工业上重要的最终产品可以减少优化一组代谢基因表达所需的时间和劳动力。

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A tetO Toolkit To Alter Expression of Genes in Saccharomyces cerevisiae

Strategies to optimize a metabolic pathway often involve building a large collection of strains, each containing different versions of sequences that regulate the expression of pathway genes. Here, we develop reagents and methods to carry out this process at high efficiency in the yeast Saccharomyces cerevisiae. We identify variants of the Escherichia coli tet operator (tetO) sequence that bind a TetR–VP16 activator with differential affinity and therefore result in different TetR–VP16 activator-driven expression. By recombining these variants upstream of the genes of a pathway, we generate unique combinations of expression levels. Here, we built a tetO toolkit, which includes the I-OnuI homing endonuclease to create double-strand breaks, which increases homologous recombination by 10⁵; a plasmid carrying six variant tetO sequences flanked by I-OnuI sites, uncoupling transformation and recombination steps; an S. cerevisiae-optimized TetR–VP16 activator; and a vector to integrate constructs into the yeast genome. We introduce into the S. cerevisiae genome the three crt genes from Erwinia herbicola required for yeast to synthesize lycopene and carry out the recombination process to produce a population of cells with permutations of tetO variants regulating the three genes. We identify 0.7% of this population as making detectable lycopene, of which the vast majority have undergone recombination at all three crt genes. We estimate a rate of ∼20% recombination per targeted site, much higher than that obtained in other studies. Application of this toolkit to medically or industrially important end products could reduce the time and labor required to optimize the expression of a set of metabolic genes.

更新日期：2015-03-17

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