Improvement of galactose uptake in Saccharomyces cerevisiae through overexpression of phosphoglucomutase:: Example of transcript analysis as a tool in inverse metabolic engineering
Improvement of galactose uptake in Saccharomyces cerevisiae through overexpression of phosphoglucomutase:: Example of transcript analysis as a tool in inverse metabolic engineering
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DOI：
10.1128/aem.71.11.6465-6472.2005
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发表时间：
2005-11-01
影响因子：
4.4
通讯作者：
Nielsen, J
中科院分区：
文献类型：
--
作者：
Bro, C;Knudsen, S;Nielsen, J
通过全基因组转录本分析的参考菌株和两个重组酿酒酵母菌株具有不同的速率的半乳糖摄取，我们获得的信息的GAL基因调控网络的代谢工程的全球转录响应。其中一个重组菌株过表达编码转录激活因子Gal 4的基因，而在另一个菌株中，缺失了编码GAL系统负调节因子Gal 80、Gal 6和Mig 1的基因。尽管三种菌株的半乳糖摄取速率显著不同，但我们令人惊讶地没有发现编码催化途径第一步的酶的基因的表达有任何显著变化（即，编码Gal 2、Gal 1、Gal 7和Gal 10的基因）。然而，我们发现，PGM 2，编码的主要同工酶的磷酸葡萄糖变位酶，略有上调，在两个重组菌株具有较高的半乳糖摄取率。这表明，PGM 2在增加通过Leloir途径的通量方面是过表达的靶标，并且通过PGM 2的过表达，与参考菌株相比，半乳糖摄取率可以增加70%。基于我们的研究结果，我们得出结论，葡萄糖磷酸变位酶在控制通过Leloir途径的通量中起着关键作用，这可能是由于葡萄糖-1-磷酸向葡萄糖-6-磷酸的转化增加。该结论得到糖磷酸盐测量的支持，其显示在过表达PGM 2的菌株构建体中存在增加浓度的葡萄糖-6-磷酸、半乳糖-6-磷酸和果糖-6-磷酸。
Through genome-wide transcript analysis of a reference strain and two recombinant Saccharomyces cerevisiae strains with different rates of galactose uptake, we obtained information about the global transcriptional response to metabolic engineering of the GAL gene regulatory network. One of the recombinant strains overexpressed the gene encoding the transcriptional activator Gal4, and in the other strain the genes encoding Gal80, Gal6, and Mig1, which are negative regulators of the GAL system, were deleted. Even though the galactose uptake rates were significantly different in the three strains, we surprisingly did not find any significant changes in the expression of the genes encoding the enzymes catalyzing the first steps of the pathway (i.e., the genes encoding Gal2, Gal1, Gal7, and Gal10). We did, however, find that PGM2, encoding the major isoenzyme of phosphoglucomutase, was slightly up-regulated in the two recombinant strains with higher galactose uptake rates. This indicated that PGM2 is a target for overexpression in terms of increasing the flux through the Leloir pathway, and through overexpression of PGM2 the galactose uptake rate could be increased by 70% compared to that of the reference strain. Based on our findings, we concluded that phosphoglucomutase plays a key role in controlling the flux through the Leloir pathway, probably due to increased conversion of glucose-l-phosphate to glucose-6-phosphate. This conclusion was supported by measurements of sugar phosphates, which showed that there were increased concentrations of glucose-6-phosphate, galactose-6-phosphate, and fructose-6-phosphate in the strain construct overexpressing PGM2.