Climate change arising from increasingly excessive consumption of fossil fuels has necessitated a rethinking of transportation infrastructure (Keasling et al., 2021; Pfleger and Takors, 2023). Inclusive focus was given to greener transport fuel to cut down the net carbon emissions. Biofuels produced by microorganisms promise to replace carbon-polluting fuels (Ashokkumar et al., 2022; Lin and Lu, 2021). Compared to the well-established biofuel ethanol, advanced biofuels like butanol, isobutanol, fatty acid derivatives and isoprenoid-derivatives are more suitable alternatives to replace fossil fuels due to similar energy density, transportation, and storage characteristics (Das et al., 2020; Liu et al., 2022a; Rana et al., 2022). Thus, the biosynthesis of advanced biofuels is of great benefit in tackling the economic and environmental threats caused by reliance on fossil fuels.

The feasibility of microbial-assisted bioconversion relies on the inherent metabolic facet of the host strain. An ideal microorganism is supposed to have high productivity, high yield, and high titer. However, some advanced fuels are naturally produced in low concentration or low efficiency by the exogenous pathway (Ruffing et al., 2022). These shortcomings can be tackled by tailoring or redesigning metabolic pathways in the host strains (Madhavan et al., 2023; Zhang et al., 2021a). The synthetic biology “design-build-test-learn” principle enables rapid design and construction of vast microbial cell libraries, while the test phase remains a bottleneck for lacking effective high-throughput approaches to associate genotype with phenotype (Patwari et al., 2023). Genetically encoded biosensors based on transcription factors (TFs) stand out for realizing cheaper and faster in-situ high-throughput screening of desired properties by transforming the intracellular concentration of a target metabolite to a detectable signal.

The components of a TF-based biosensor encompass a TF that senses environment stimuli (end product, precursor, or other metabolites), a TF binding promoter which is regulated by TF-inducer complex, and a reporter gene under the control of cognate promoter (Yu et al., 2023). Upon the existence of biofuels or their precursors, TFs undergo conformation change and bind to the transcription factor binding site (TFBS) of the cognate promoter, controlling the expression level of downstream reporter genes (Liu et al., 2022b). With the development of synthetic biology, plenty of TFs have been discovered and developed as biosensors for the biosynthesis of biofuels (Morgan et al., 2016). However, wild-type biosensors rarely meet the practical requirements because of the low sensitivity, specificity, and dynamic range. These properties can be enhanced by modifying the TF and promoter of biosensors (Gao et al., 2022; Gong et al., 2022). Coupling with different output reporters, the optimized biosensor satisfies various applications, including precise product detection and dynamic metabolic flow regulation, thereby further increasing the production of advanced biofuels and other chemicals (Ding et al., 2021).

Herein, we summarized TF-based biosensors that detect advanced biofuels or their intermediate metabolites biosynthesis. The principles of construction and validation of the genetic circuits were highlighted, especially the selection and optimization of genetic parts. We also reviewed the applications of biosensors in enhancing the production of advanced biofuels by mining potential engineering targets, high-throughput screening evolved enzymes and hyperproducers, confirming functional proteins and fine-tuning metabolic flux. Lastly, this review discusses the current limitation of exploring and optimizing genetic encoded biosensor and outlines the potential solution in the future.