Hauf J, Zimmermann FK, Müller S
Institut für Mikrobiologie und Genetik, Technische Universität Darmstadt, Schnittspahnstrasse 10, D-64287, Darmstadt, Germany
Enzyme Microb Technol. 2000 Jun 1;26(9-10):688-698. doi: 10.1016/s0141-0229(00)00160-5.
Fusions of the glycolytic genes TPI1, PGK1, ENO1, PYK1, PDC1, and ADH1 with the lacZ reporter gene of Escherichia coli and a lacZ fusion construct of a 390-bp fragment from the promoter of the HXT7 gene were assayed for beta-galactosidase activity. The glycolytic promoters were induced after addition of glucose to ethanol-grown cells, whereas the HXT7 promoter fragment showed a constitutive beta-galactosidase expression on both carbon sources. The genes coding for the seven enzymes of lower glycolysis Tdh, Pgk, Gpm, Eno, Pyk, Pdc, and Adh were simultaneously put under the control of the same strong promoter, a truncated HXT7 promoter that is constitutively active on ethanol as well as on glucose medium. Genomic expression of the glycolytic genes under the control of this promoter, resulted in an at least 2-fold overexpression. The gene MSG5 was isolated, coding for a protein phosphatase normally involved in cell cycle regulation, as a factor that possibly influences the expression of the HXT7 gene. However, overexpression of MSG5 had no effect on the expression of the HXT7/lacZ fusion, whereas a deletion of this gene resulted in a decreased expression of beta-galactosidase.
将糖酵解基因TPI1、PGK1、ENO1、PYK1、PDC1和ADH1与大肠杆菌的lacZ报告基因进行融合，并对来自HXT7基因启动子的390bp片段的lacZ融合构建体进行β-半乳糖苷酶活性检测。在向乙醇培养的细胞中添加葡萄糖后，糖酵解启动子被诱导，而HXT7启动子片段在两种碳源上均表现出组成型β-半乳糖苷酶表达。编码低水平糖酵解的七种酶Tdh、Pgk、Gpm、Eno、Pyk、Pdc和Adh的基因同时置于相同强启动子的控制下，该启动子是截短的HXT7启动子，在乙醇和葡萄糖培养基上均组成型活跃。在该启动子控制下糖酵解基因的基因组表达导致至少2倍的过表达。分离出编码通常参与细胞周期调控的蛋白磷酸酶的基因MSG5，作为可能影响HXT7基因表达的一个因子。然而，MSG5的过表达对HXT7/lacZ融合体的表达没有影响，而该基因的缺失导致β-半乳糖苷酶表达降低。
