Ruijter G J, Panneman H, Visser J
Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, The Netherlands.
Biochim Biophys Acta. 1997 Mar 15;1334(2-3):317-26. doi: 10.1016/s0304-4165(96)00110-9.
Phosphofructokinase and pyruvate kinase were overexpressed in the filamentous fungus Aspergillus niger. Moderate overexpression of these glycolytic enzymes in A. niger N400 (3-5-fold the wild-type level), either individually or simultaneously, did not increase citric acid production by the fungus significantly. Thus, phosphofructokinase and pyruvate kinase do not seem to contribute in a major way to flux control of the metabolism involved in the conversion of glucose to citric acid. Overexpression of phosphofructokinase and pyruvate kinase did not influence the activities of other enzymes in the pathway, nor did it change intermediary metabolite levels. However, in strains overexpressing phosphofructokinase, the level of fructose 2,6-bisphosphate, a positive allosteric effector of phosphofructokinase, was reduced almost 2-fold compared to the wild-type strain. Measurements with purified phosphofructokinase, using substrate, product and effector concentrations found intracellularly, showed that such a reduction in the fructose-2,6-bisphosphate level could decrease the specific activity of phosphofructokinase in the cell significantly. Thus, the fungus seems to adapt to overexpression of phosphofructokinase by decreasing the specific activity of the enzyme through a reduction in the level of fructose 2,6-bisphosphate.
磷酸果糖激酶和丙酮酸激酶在丝状真菌黑曲霉中过表达。在黑曲霉N400中适度过表达这些糖酵解酶（为野生型水平的3至5倍），无论是单独过表达还是同时过表达，均未显著增加该真菌的柠檬酸产量。因此，磷酸果糖激酶和丙酮酸激酶似乎对葡萄糖转化为柠檬酸所涉及的代谢通量控制没有主要贡献。磷酸果糖激酶和丙酮酸激酶的过表达既不影响该途径中其他酶的活性，也不改变中间代谢物水平。然而，在过表达磷酸果糖激酶的菌株中，磷酸果糖激酶的正别构效应剂果糖-2,6-二磷酸的水平与野生型菌株相比降低了近2倍。使用在细胞内发现的底物、产物和效应剂浓度对纯化的磷酸果糖激酶进行测量表明，果糖-2,6-二磷酸水平的这种降低可显著降低细胞中磷酸果糖激酶的比活性。因此，该真菌似乎通过降低果糖-2,6-二磷酸水平来降低该酶的比活性，从而适应磷酸果糖激酶的过表达。
