Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany.
Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.
Microb Biotechnol. 2024 Jul;17(7):e14527. doi: 10.1111/1751-7915.14527.
Formaldehyde is a key metabolite in natural and synthetic one-carbon metabolism. To facilitate the engineering of formaldehyde-producing enzymes, the development of sensitive, user-friendly, and cost-effective detection methods is required. In this study, we engineered Escherichia coli to serve as a cellular biosensor capable of detecting a broad range of formaldehyde concentrations. Using both natural and promiscuous formaldehyde assimilation enzymes, we designed three distinct E. coli growth biosensor strains that depend on formaldehyde for cell growth. These strains were engineered to be auxotrophic for one or several essential metabolites that could be produced through formaldehyde assimilation. The respective assimilating enzyme was expressed from the genome to compensate the auxotrophy in the presence of formaldehyde. We first predicted the formaldehyde dependency of the biosensors by flux balance analysis and then analysed it experimentally. Subsequent to strain engineering, we enhanced the formaldehyde sensitivity of two biosensors either through adaptive laboratory evolution or modifications at metabolic branch points. The final set of biosensors demonstrated the ability to detect formaldehyde concentrations ranging approximately from 30 μM to 13 mM. We demonstrated the application of the biosensors by assaying the in vivo activity of different methanol dehydrogenases in the most sensitive strain. The fully genomic nature of the biosensors allows them to be deployed as "plug-and-play" devices for high-throughput screenings of extensive enzyme libraries. The formaldehyde growth biosensors developed in this study hold significant promise for advancing the field of enzyme engineering, thereby supporting the establishment of a sustainable one-carbon bioeconomy.
甲醛是天然和合成一碳代谢中的关键代谢物。为了促进甲醛产生酶的工程改造，需要开发灵敏、易用且具有成本效益的检测方法。在这项研究中，我们对大肠杆菌进行了工程改造，使其成为一种能够检测广泛甲醛浓度的细胞生物传感器。我们利用天然和混杂的甲醛同化酶，设计了三种不同的大肠杆菌生长生物传感器菌株，这些菌株依赖甲醛进行细胞生长。这些菌株被设计为对一种或几种必需代谢物呈营养缺陷型，这些代谢物可以通过甲醛同化产生。相应的同化酶从基因组中表达，以在存在甲醛的情况下补偿营养缺陷型。我们首先通过通量平衡分析预测了生物传感器对甲醛的依赖性，然后进行了实验分析。在菌株工程改造之后，我们通过适应性实验室进化或在代谢分支点进行修改，增强了两个生物传感器的甲醛敏感性。最终的生物传感器集能够检测约 30μM 至 13mM 的甲醛浓度。我们通过在最敏感的菌株中测定不同甲醇脱氢酶的体内活性来演示了生物传感器的应用。生物传感器的全基因组性质允许它们作为“即插即用”设备，用于广泛酶库的高通量筛选。本研究中开发的甲醛生长生物传感器在推进酶工程领域具有重要意义，从而支持建立可持续的一碳生物经济。
