Centre de Biologie Structurale (CBS), INSERM U1054, CNRS UMR5048, University of Montpellier, 29 Rue de Navacelles, Montpellier 34090, France.
ACS Synth Biol. 2021 Dec 17;10(12):3527-3536. doi: 10.1021/acssynbio.1c00456. Epub 2021 Dec 1.
Bacteria equipped with genetically encoded lactate biosensors are promising tools for biopharmaceutical production, diagnostics, and cellular therapies. However, many applications involve glucose-rich and anoxic environments, in which current whole-cell lactate biosensors show low performance. Here we engineer an optimized, synthetic lactate biosensor system by repurposing the natural LldPRD promoter regulated by the LldR transcriptional regulator. We removed glucose catabolite and anoxic repression by designing a hybrid promoter, containing LldR operators and tuned both regulator and reporter gene expressions to optimize biosensor signal-to-noise ratio. The resulting lactate biosensor, termed ALPaGA (A Lactate Promoter Operating in Glucose and Anoxia), can operate in glucose-rich, aerobic and anoxic conditions. We show that ALPaGA works reliably in the probiotic chassisNissle 1917 and can detect endogenous l-lactate produced by 3D tumor spheroids with an improved dynamic range. In the future, the ALPaGA system could be used to monitor bioproduction processes and improve the specificity of engineered bacterial cancer therapies by restricting their activity to the lactate-rich microenvironment of solid tumors.
配备基因编码乳酸生物传感器的细菌是生物制药生产、诊断和细胞治疗的有前途的工具。然而，许多应用涉及富含葡萄糖和缺氧的环境，当前的全细胞乳酸生物传感器在这些环境中的性能较低。在这里，我们通过重新利用受 LldR 转录调节剂调节的天然 LldPRD 启动子来设计优化的合成乳酸生物传感器系统。我们通过设计一个包含 LldR 操作子的混合启动子来消除葡萄糖分解代谢物和缺氧抑制，并且对调节剂和报告基因的表达进行了调整，以优化生物传感器的信噪比。由此产生的乳酸生物传感器，称为 ALPaGA（在葡萄糖和缺氧条件下工作的乳酸启动子），可以在富含葡萄糖、需氧和缺氧条件下工作。我们表明，ALPaGA 在益生菌底盘 Nissle 1917 中可靠地工作，并可以检测 3D 肿瘤球体产生的内源性 l-乳酸，具有改善的动态范围。将来，ALPaGA 系统可用于监测生物生产过程，并通过将其活性限制在实体瘤中富含乳酸的微环境中来提高工程细菌癌症疗法的特异性。
