[P001] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P01-055] Shifting the detection range of cell biosensors toward high concentrations using ligand-related exporters for applications

○Jiawei Li¹ (1. Shanghai Jiao Tong University (China))

Keywords:

high-concentration detection,exporter,quorum sensing,nisin A,two-component system

[Purpose] Cell biosensors are widely used for chemical detection, strain screening, and in situ dynamic regulation, but their performance is often limited by saturation and toxicity at high ligand concentrations. This work aims to systematically shift biosensor detection ranges toward higher ligand concentrations by regulating intracellular ligand levels via ligand-related exporters, enabling robust function in high-concentration industrial applications. [Method] We engineered exporter-assisted biosensors using two complementary strategies. When a dedicated exporter exists, we integrated a specific exporter into a two-component system (TCS) biosensor, exemplified by incorporating the nisin A exporter NisFEG into a Lactococcus lactis nisin A TCS biosensor (NisK/NisR with a Pnis reporter). When no known specific exporter is available, we screened nonspecific exporters and implemented regulatory control to tune efflux, exemplified by identifying AcrAB-TolC as an exporter for the QS ligand OHC14 and activating it through MarA overexpression. Exporter expression levels were tuned to balance sensitivity, range shifting, and host fitness. [Results] For the nisin A biosensor, NisFEG shifted the detection range upward and mitigated toxicity, expanding the upper detection limit from 5 to 500 ng/mL, enabling accurate measurement at high concentrations. The optimized biosensor supported high-throughput screening of a library of >5,000 nisin-producer mutants, yielding stable high-yield strains (1.5–2-fold increases). A top mutant achieved 400,000 ng/mL nisin A in fed-batch fermentation. For QS biosensors, MarA-activated AcrAB-TolC reduced intracellular OHC14 and shifted the operational range from 0.1–5 μM to 0.5–10 μM, enabling delayed quorum activation. This range shifting improved control of the QS biosensor-based autolysis system, supporting high-density autolysis and high-level protein production (RFP up to 3.7 g/L). The system further enabled efficient cell-free bioconversion from lignin-derived substrates (e.g., producing 4-vinylphenol at high titer from raw p-CA). [Consideration] Exporter-mediated tuning is modular and generalizable but requires careful calibration to avoid over-efflux that suppresses biosensor response or causes metabolic burden. Nonspecific exporters may introduce off-target transport effects; promoter/TF tuning and exporter engineering can mitigate interference. Environmental conditions can modulate regulatory efficacy, suggesting the need for application-specific optimization and robustness testing for scale-up. [Conclusion] Integrating ligand-related exporters provides a practical and extensible strategy to shift cell biosensor detection ranges toward high ligand concentrations while reducing toxicity. By combining specific exporters (e.g., NisFEG) and regulated nonspecific exporters (e.g., AcrAB-TolC), this approach expands biosensor utility for high-yield strain screening, programmable autolysis, and industrial bioconversion.