Glycolysis has evolved to be a highly robust mechanism for maintaining the cellular metabolism of living organisms. However, relevant modifications of glycolytic activity are required to intentionally modulate cellular phenotypes. Here, we designed a platform that allows switching control of glycolysis in Escherichia coli in response to an environmental signal, in this case, temperature. This system functions by regulating the expression of gapA, which encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), one of the key glycolytic enzymes. Because a very low level of gapA expression is capable of maintaining cellular physiology, we also modified GAPDH through directed evolution to provide sensitive regulation of glycolytic activity. The switching control of glycolysis was successfully demonstrated by regulating the expression of engineered gapA through changes in temperature. This system offers potential control over the cell's central carbon-metabolism switch, providing the ability to perform reprogrammed tasks with desired timing depending on environmental signals.
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