Bioluminescent monitoring of recombinant lactic acid bacteria and their products.
Bioluminescent monitoring of recombinant lactic acid bacteria and their products.
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促进健康的生物治疗策略，包括应用工程微生物来提供治疗分子，具有很强的前景。然而，如果没有精密的工具来检测治疗微生物及其产物，我们的监测和微调治疗递送的能力就会受到阻碍。在这里，我们采用了一种用于乳酸菌的生物发光肽标记系统，乳酸菌是一组生物，其成员通常被用作治疗药物和疫苗的递送载体。为了验证这一概念，我们开发了多种罗伊氏乳酸杆菌菌株，每种菌株都能产生具有11个氨基酸标签的重组治疗蛋白，这是产生发光信号所必需的。重组蛋白荧光定量比市售免疫分析法更敏感。此外，我们证明了生物发光肽标记系统允许在连续培养平行生物反应器系统中原位检测重组蛋白。这提供了一个令人兴奋的机会，以确定重组蛋白生产动态响应不同的刺激。最后，在口服重组微生物后，肠道和粪便样品中的发光使得快速检测微生物具有与传统平板计数相同的灵敏度。由于我们在包括9个属的12个物种中证明了这种生物发光肽标记系统的功能，我们的方法将在乳酸菌研究中创造以前未开发的机会。乳酸菌是一种遗传多样性的微生物，在食品工业、生物技术、农业和医药领域发挥着重要作用。对不同环境下细菌生理的核心理解对于选择和开发用于工业和医疗应用的细菌至关重要。然而，缺乏通用的工具来跟踪（重组）乳酸菌的蛋白质生产。在这项研究中，我们采用了一种基于多肽的生物发光标记系统，该系统在多个属和物种中都具有功能。该系统可以在体外和原位跟踪标记的蛋白质，同时它也可以用于枚举来自小鼠胃肠道的重组细菌，其准确性与传统的平板计数相当。我们的工作扩展了乳酸菌遗传工具箱，将为工业界和学术界的研究人员提供在不同生理相关条件下监测微生物和蛋白质的机会。
Biotherapeutic strategies to promote health, including the application of engineered microbes to deliver therapeutic molecules, hold strong promise. However, without precision tools to detect therapeutic microbes and their products, we are hampered in our ability to monitor and fine-tune therapeutic delivery. Here, we adapted a bioluminescent peptide tagging system for use in lactic acid bacteria, a group of organisms whose members are commonly exploited as delivery vehicles of therapeutics and vaccines. As a proof of concept, we developed various Limosilactobacillus reuteri strains that each produced a recombinant therapeutic protein with an 11 amino acid tag, which is essential to yield a luminescent signal. Luminescent-based quantification of recombinant protein was more sensitive than commercially available immunoassays. In addition, we demonstrated that the bioluminescent peptide tagging system allows in situ recombinant protein detection in a continuous-culture parallel bioreactor system. This presents an exciting opportunity to determine recombinant protein production dynamics in response to different stimuli. Finally, following oral administration of recombinant microbes, luminescence in intestinal and fecal samples allowed for rapid detection of microbes with equal sensitivity to conventional plate count. Because we demonstrated the functionality of this bioluminescent peptide tagging system in 12 species encompassing nine genera, our approach will create previously unexplored opportunities in lactic acid bacteria research. Lactic acid bacteria constitute a genetically diverse group of microorganisms with significant roles in the food industry, biotechnology, agriculture, and medicine. A core understanding of bacterial physiology in diverse environments is crucial to select and develop bacteria for industrial and medical applications. However, there is a lack of versatile tools to track (recombinant) protein production in lactic acid bacteria. In this study, we adapted a peptide-based bioluminescent tagging system that is functional across multiple genera and species. This system enables tracking of tagged proteins both in vitro and in situ, while it also can be used to enumerate recombinant bacteria from the mouse gastrointestinal tract with accuracy comparable to that of conventional plate counts. Our work expands the lactic acid bacteria genetic toolbox and will facilitate researchers in industry and academia with opportunities to monitor microbes and proteins under different physiologically relevant conditions.
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