GALACTOSE AS A GRATUITOUS INDUCED OF GAL GENE-EXPRESSION IN YEASTS GROWING ON GLUCOSE
GALACTOSE AS A GRATUITOUS INDUCED OF GAL GENE-EXPRESSION IN YEASTS GROWING ON GLUCOSE
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DOI：
10.1016/0378-1119(89)90403-4
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发表时间：
1989-11-15
期刊：
影响因子：
3.5
通讯作者：
SCLAFANI, RA
中科院分区：
文献类型：
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作者：
HOVLAND, P;FLICK, J;SCLAFANI, RA
酿酒酵母的高表达和严格调控的GAL基因的启动子对于在该生物体中表达蛋白质是有用的。然而，有两个问题使它们的使用变得复杂。首先，由于在葡萄糖上生长会导致对GAL表达的长期抑制，细胞在非发酵碳源上生长后最快被诱导，而非发酵碳源通常支持较差的生长。其次，由于Gal基因的诱导剂(半乳糖)也是碳源，在Gal+菌株的生长过程中，诱导剂的水平不断下降，这可能导致Gal表达减少。为了解决第一个问题，我们使用了携带REG1-501突变的菌株，这种突变消除了GAL表达的葡萄糖抑制。该基因已被证明位于第四染色体的右臂，远端但与TRP1基因紧密连锁。我们证明了在这些REG1菌株中，通过在葡萄糖培养基中添加半乳糖，可以有效和快速地诱导GAL启动子的表达。低至0.02%的半乳糖水平可用于在该菌株中从GAL启动子获得1500倍的基因表达诱导。为了解决第二个问题，我们使用了一个胆汁突变体，它缺乏催化半乳糖利用的第一步的酶。我们发现，在这些突变体中，GAL启动子的高水平表达是迅速实现的，对这些突变体来说，半乳糖是一个无偿的诱导剂。为了在固定化的、未分裂的细胞的连续发酵中产生蛋白质，我们还证明了在用酵母肽激素α-因子阻断的REG1-501细胞中有效地产生了两种蛋白质，细胞内细菌β-半乳糖苷酶和分泌的小鼠淀粉酶。
The promoters of the highly expressed and stringently regulated GAL genes of Saccharomyces cerevisiae, are useful for expressing proteins in this organism. However, two problems complicate their use. First, because growth on glucose causes prolonged repression of GAL expression, cells are most rapidly induced after growth on nonfermentable carbon sources, conditions which usually support poor growth. Second, because the inducer of the GAL genes (galactose) also serves as a carbon source, the level of inducer is continually diminishing during growth of a Gal+ strain, which may lead to reduced GAL expression. To solve the first problem, we have employed strains that carry the reg1-501 mutation, which eliminates glucose repression of GAL expression. This gene has been shown to be located on the right arm of chromosome IV, distal but tightly linked to the TRP1 gene. We demonstrate that expression from GAL promoters is efficiently and rapidly induced in these reg1 strains by the addition of galactose to a culture growing in glucose medium. Levels of galactose as low as 0.02% can be used to obtain a 1500-fold induction of gene expression from GAL promoters in this strain. To surmount the second problem, we have used a gall mutant, deficient in the enzyme that catalyzes the first step of galactose utilization. We show that high levels of expression from GAL promoters are achieved rapidly in these mutants, for which galactose is a gratuitous inducer. With the goal of producing proteins during continuous fermentation of immobilized, nondividing cells, we also show that two proteins, intracellular bacterial .beta.-galactosidase and secreted mouse amylase, are produced efficiently in reg1-501 cells arrested with the yeast peptide hormone, .alpha.-factor.