Gene expression patterns for metabolic pathway in pgi knockout Escherichia coli with and without phb genes based on RT-PCR.
Metabolic regulations were investigated from the viewpoint of gene expressions for Escherichia coli JM109 and pgi knockout E. coli with and without phb genes using RT-PCR. One of the main features of pgi knockout E. coli is the overproduction of NADPH produced in pentose phosphate (PP) pathway. NADPH overproduction in PP pathway in pgi mutant causes some reducing power imbalance that ultimately affects the cell growth. It was shown that this reducing power imbalance can be recovered to some extent by introducing NADPH absorbing pathway such as PHB synthetic pathway into pgi mutant E. coli. To get insight into the regulation mechanism of pgi mutant E. coli at the transcriptional level, 87 E. coli genes involved in central metabolic pathways and key regulatory mechanisms were investigated by semi-quantitative RT-PCR analysis. The analysis showed that pentose phosphate pathway genes and part of the glycolysis pathway genes were affected significantly by expression of phb genes in pgi mutant E. coli DF11/pAeKG1 as well as in pgi mutant E. coli DF11 as compared with those in E. coli JM109. In contrast, most of the TCA cycle genes except icdA were downregulated in both pgi mutants E. coli. The upregulation of icdA gene may be due to the positive regulation of fruR. Moreover, it was found that ack gene as well as aceA and aceB genes involved in the glyoxylate shunt were upregulated in pgi mutants while ppc gene was downregulated, indicating that pgi inactivation changes the anaplerotic pathway from ppc pathway to glyoxylate shunt. Enzyme activities of glk, zwf, tpiA, fbaA, ldhA, gltA, aceA, mdh and maeB were also measured and compared with the corresponding gene expressions. Most of them are well correlated except for aceA gene indicating that glyoxylate pathway is regulated on the protein level, not on the gene level.