Increasing sulphite formation in Saccharomyces cerevisiae by overexpression of MET14 and SSU1
Increasing sulphite formation in Saccharomyces cerevisiae by overexpression of MET14 and SSU1
复制标题
作者：
Donalies, UEB;Stahl, U
在硫酸盐向硫化物的同化还原过程中，酿酒酵母产生亚硫酸盐作为中间产物。MET3、MET14和MET16这三个基因对这种减少至关重要。我们研究了这些基因在高、中、低亚硫酸盐形成的酿酒酵母菌株中的转录水平。MET14-和MET16-mRNA水平随亚硫酸盐产量的变化而变化，而MET3-mRNA水平在几乎所有菌株中都很弱。我们还分析了MET14和MET16过表达对亚硫酸盐形成的影响。用含有MET14和MET16或同时含有MET14和MET16的高拷贝质粒转化亚硫酸盐产量低的两株菌株。这两个基因的过度表达导致亚硫酸盐形成2到3倍。此外，编码亚硫酸盐还原酶亚基的MET10失活也导致亚硫酸盐形成明显增加；然而，细胞变成蛋氨酸营养不良。SSU1是一种编码亚硫酸盐泵的基因，其过表达会使亚硫酸盐的积累轻微增加，而SSU1和MET14的过表达会使亚硫酸盐的形成增加10倍。此外，亚硫酸盐的形成强烈依赖于生长条件，例如，与在最小培养基中生长相比，在麦汁中生长的酵母转化体产生的亚硫酸盐量要高得多。在限氧条件下，葡萄糖的添加也可以增加过表达MET14和/或SSU1菌株的亚硫酸盐形成，而在好氧条件下，葡萄糖的添加没有显著的影响。版权所有:John Wiley Sons, Ltd。
Saccharomyces cerevisiae produces sulphite as an intermediate product during the assimilatory reduction of sulphate to sulphide. Three genes, MET3, MET14 and MET16, are essential for this reduction. We investigated the level of transcription of these genes in strains of S. cerevisiae with high, medium and low sulphite formation. The level of MET14- and MET16-mRNA varied with sulphite production, whereas the level of MET3-mRNA was very weak in almost all strains. We also analysed the effect of overexpression of MET14 and MET16 on sulphite formation. Two strains with low sulphite production were transformed with high-copy plasmids containing either or both MET14 and MET16. The overexpression of these two genes leads to a two- to three-fold sulphite formation. In addition, inactivation of MET10, encoding a subunit of the sulphite reductase, also leads to a distinct increase in sulphite formation; however, the cells became methionine auxotroph. The overexpression of SSU1, a gene encoding a putative sulphite pump, yields a slight increase in sulphite accumulation, whereas overexpression of SSU1, together with MET14, increases sulphite formation up to 10-fold. Furthermore, sulphite formation strongly depends on growth conditions, e.g. yeast transformants growing in wort produce much higher amounts of sulphite when compared to growth in minimal media. The addition of glucose can also increase the sulphite formation in strains overexpressing MET14 and/or SSU1 under oxygen-limiting conditions, while the addition of glucose has no significant effect under aerobic conditions. Copyright (C) 2002 John Wiley Sons, Ltd.