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To date, many metabolic engineering tools and strategies have been developed, including tools for cofactor engineering, which is a common strategy for bioproduct synthesis. Cofactor engineering is used for the regulation of pyridine nucleotides, including NADH/NAD +  and NADPH/NADP + , and adenosine triphosphate/adenosine diphosphate (ATP/ADP), which is crucial for maintaining redox and energy balance. However, the intracellular levels of NADH/NAD + , NADPH/NADP + , and ATP/ADP cannot be monitored in real time using traditional methods. Recently, many biosensors for detecting, monitoring, and regulating the intracellular levels of NADH/NAD + , NADPH/NADP + , and ATP/ADP have been developed. Although cofactor biosensors have been mainly developed for use in mammalian cells, the potential application of cofactor biosensors in metabolic engineering in bacterial and yeast cells has received recent attention. Coupling cofactor biosensors with genetic circuits is a promising strategy in metabolic engineering for optimizing the production of biochemicals. In this review, we focus on the development of biosensors for NADH/NAD + , NADPH/NADP + , and ATP/ADP and the potential application of these biosensors in metabolic engineering. We also provide critical perspectives, identify current research challenges, and provide guidance for future research in this promising field.

Genetically encoded ATP and NAD(P)H biosensors: potential tools in metabolic engineering