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Since the first six of those genes catalyze reversible reactions, their upregulation enhances the flux in the glycolytic direction, which is much greater than the gluconeogenic flux during growth in glucose. The down regulation of pykF in both E. Indeed, pykF, but not pykA, has been regarded to be negatively regulated by fruR , a global regulatory gene that was found to be upregulated in both E. Interestingly, however, ppsA coding for phosphoenol pyruvate synthase was upregulated Symbols for genes inside the boxes follow the E.
Symbols for genes inside the boxes follow E. Red represents upregulation, blue represents downregulation, and black represents no change 32 K. Red represents upregulation, blue represents downregulation, and black represents no change Metabolic Flux Analysis Based on 13C-Labeling Experiments 33 The gene glk was upregulated by 1. The decreases in the ptsG and ptsH transcript levels were consistent with the reduced specific glucose consumption rates.
Recent Progress of Biochemical and Biomedical Engineering in Japan II
The increase in glk transcript level corroborates a previous report which shows that glucokinase takes over the major role from the glucose-PTS to supply G6P under increased protein synthesis conditions , although the mechanism for this induction is still unknown. Examination of the operational catabolic pathways and their flux ratios using [UC6] glucose-labeling experiments and metabolic flux ratio analysis provide evidence for the PP pathway as the primary route of glucose catabolism in pgi mutant E.
Among the PP pathway genes, most of the gene transcripts in both E. The expression of gnd, which is regulated by the growth rate  clearly decreased in pgi mutants as the growth rate reduced as compared with that of E. By contrast, talA was upregulated in pgi mutant E. In addition, edd was upregulated in both pgi mutants, although eda was downregulated significantly as was shown in Figs.
Significant involvement of the malic enzyme in NADPH reoxidation can be excluded, since virtually no pyruvate originates from malate , and this result was consistent with our measurement of no malic enzyme activity. However, expression of maeB was moderately downregulated in both pgi mutants E.
Similarly, pntAB is unlikely to catalyze sufficient reoxidation of NADPH since pgi pntAB double mutant grows as slowly as the pgi mutant , which means that additional knockout of pntAB does not further reduce the rate of glucose catabolism. Expression level of pntA and pntB were upregulated in both pgi mutants compared to those in control strain see Figs. Overexpression of soluble transhydrogenase udhA in pgi mutant E. The down regulation of 34 K.
It was observed from our experimental results that the expression of phb genes in pgi knockout E.
Since glucose is metabolized exclusively via PP pathway in pgi mutant, the important glycolysis metabolite GAP is supplied from PP pathway. GAP is one of the important precursor metabolites for the synthesis of isopentenyl diphosphate IPP that acts as a carrier in the synthesis of a number of cell envelopes and extracellular polymers e. As a result, large fraction of GAP is being consumed to maintain the glycolytic flux for biomass production and to provide the precursor metabolites for PHB synthesis. Therefore, the expression of the gene gapA The possible reason of ppsA upregulation in E.
Another gene pckA also upregulated in E. It may be noted that ppsA upregulation might also be due to the effect of global regulatory gene, fruR since ppsA was found to be positively regulated by fruR . The ppc gene was downregulated in both E. The regulation of these genes has not been fully elucidated, although it has been shown that ppc overexpression affects acetate production . Thus, the downregulation of ppc expression that occurs during PHB synthesis may result in an increase in the acetate production flux.
Recent Progress of Biochemical and Biomedical Engineering in Japan II | SpringerLink
Furthermore, during growth in glucose, ppc is the major route to the aspartate family of amino acids. Therefore, downregulation of ppc has to be compensated by another anaplerotic pathway. The pyruvate dehydrogenase complex is composed of multiple copies of three separate enzymes: pyruvate dehydrogenase encoded by aceE , dihydrolipoate transacetylase encoded by aceF and lipoamide dehydrogenase encoded by lpdA. The reactions of the PDH complex serve to interconnect the metabolic pathways of glycolysis and fatty acid synthesis to the TCA cycle as well as to the PHB synthetic route.
Although the activity of the PDH complex is highly regulated by a variety of allosteric effectors and by covalent modification, it can be expected that a reduced level of expression of these genes, i. This downregulation was expected since most of the TCA cycle genes such as gltA , sucA , sdhC , fumA , and mdh  are known to be regulated by several regulators such as fnr and arcA that were found to be upregulated in both pgi strains. The upregulation of icdA may be due to the positive regulation of fruR . In addition, the genes involved in the glyoxylate shunt aceA and aceB were upregulated in both pgi mutants E.
Among the fermentative genes, fdhF, coding for formyl dehydrogenase, increased in E. The upregulation of ackA in both pgi mutants is of interest. Pta and ackA genes were thought to be involved in both acetate synthesis and consumption, and constitutively expressed in the cell . The upregulation of ackA in pgi mutants indicates that although both genes are present in the same operon, they are regulated differentially through different promoters. Therefore, the transcript levels of many heat shock proteins such as dnaK, dnaJ, groS, grpE, htpG, ibpA, and ibpB were all upregulated significantly in recombinant pgi mutant E.
Moreover, although those heat-shock genes except ibpA and ibpB were also found to be upregulated in E. Furthermore, the transcript level of groL and lon were slightly upregulated in both pgi mutants E. The expression level of the genes for the protein secretion and disulfide bond formation, secA, lepB, and dsbA, were increased significantly in E.
This result implies that PHB accumulation in pgi mutant E. The expression ratio of the translation elongation factor gene tufA was significantly higher in E. Expression of uspA coding for universal stress protein was upregulated in both pgi mutants 36 K.
Shimizu E. The significant upregulation of the gene fkpA in E. Furthermore, the takeover of the cytosolic space by PHB granules may disturb the normal intracellular architecture such as chromosomal attachment and may consequently result in the heat shock response. Certainly, this will become a major stress on the cells for several possible reasons including protein denaturation on the surface of PHB granules. This unfavorable condition generated by PHB accumulation might have resulted in the highest expression of rpsA coding 30S ribosomal protein S1 in E. Moreover, protein S1 has been reported to be necessary in some cases for translation initiation  and for translation elongation .
However, expression of the gene rpsA could be the effect of acetate accumulation as was observed by Arnold et al. In our study, highest expression of the gene rpsA in E. Therefore, the highest amount of soluble protein synthesis in pgi mutant having phb genes was due to the effect of PHB accumulation that ultimately resulted in the highest expression of the gene rpsA.
This phenomenon implies that protein synthesis capacity in E. The semiquantitative RT-PCR results reflect the relative abundance of the transcript, which is influenced by both transcriptional activity and the mRNA stability.
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In contrast, the activity of enzymes can be regulated by reversible binding of effectors, covalent modification and alteration of enzyme concentration. However, to a large extent, the mRNA expression level reflects the enzyme activity . We compared the enzyme activities with gene expressions for three strains.
Figure 9 shows how those are correlated except for aceA. Those results show that gene expression data obtained by semi-quantitative RT-PCR were reasonably consistent with the data obtained from enzyme activities except for the gene aceA indicating a different level of regulation for that gene after transcription initiation. This phenomenon indicated that aceA was expressed in gene level but glyoxylate pathway enzyme was not active for E. The growth rate of mutant was a little lower as compared with that of the wild type E. During exponential growth, acetic acid was 38 K.
Shimizu also produced but its formation rate was significantly lower as compared with that of the wild type E.
It was found that the CO2 evolution rate was lower for the pykF— mutant as compared with that of the wild type. These results are consistent with other researches made by Ponce et al. Several enzyme activities for glycolytic enzymes Hxk, Pgi, Pfk, Tpi, and Pyk were measured for both the wild type and pykF— mutant of E. The enzyme activity of phosphoglucose transferase system was detected only in the presence of glucose, confirming its efficient induction by glucose.
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