Twin-layer biosensor for real-time monitoring of alkane metabolism.

Intracellular metabolic sensors can be used for efficient screening and optimization of microbial cell factories. In particular, the sensors are useful in acquiring information about pathway dynamics and bottlenecks in a straightforward manner. Here, we developed a twin-layer biosensor that functions simultaneously at two levels: through transcription factor mediated sensing and enzyme-metabolite interaction, providing insights into the dynamics of alkane metabolism. In addition, the sensor can be used for monitoring either alkane degradation or biosynthesis, depending on the used cellular context. Alkanes are monitored using a fluorescent reporter green fluorescent protein placed under a native alkane-inducible promoter, whereas a bacterial luciferase producing bioluminescence signal enzymatically detects a specific metabolic intermediate in the alkane production/degradation pathway. First, we employed the sensor to investigate the native alkane degradation route in Acinetobacter baylyi ADP1. The highest fluorescence and luminescence signals were obtained for dodecane. Second, we constructed a non-native alkane synthesis pathway in A. baylyi ADP1, of which the functionality was confirmed with the sensor. The twin-layer approach provides convenient means to study and optimize the kinetics and performance of the heterologous pathway and will facilitate the development of an efficient cell factory.