The use of cell factories for the production of bulk and value-added compounds is nowadays an advantageous alternative to the traditional petrochemical methods. Nevertheless, the efficiency and productivity of several of these processes can improve with the implementation of micro-oxic or anoxic conditions. In the industrial setting, laccases are appealing catalysts that can oxidize a wide range of substrates and reduce O₂ to H₂O. In this work, several laccase-based devices were designed and constructed to modulate the intracellular oxygen concentration in bacterial chassis. These oxygen consuming devices (OCDs) included Escherichia coli’s native laccase (CueO) and three variants of this protein obtained by directed evolution. The OCDs were initially characterized in vitro using E. coli DH5α protein extracts and subsequently using extracts obtained from other E. coli strains and in vivo. Upon induction of the OCDs, no major effect on growth was observed in four of the strains tested, and analysis of the cell extract protein profiles revealed increased levels of laccase. Moreover, oxygen consumption associated with the OCDs occurred under all of the conditions tested, but the performance of the devices was shown to be strain-dependent, highlighting the importance of the genetic background even in closely related strains. One of the laccase variants showed 13- and 5-fold increases in oxidase activity and O₂ consumption rate, respectively. Furthermore, it was also possible to demonstrate O₂ consumption in vivo using l-DOPA as the substrate, which represents a proof of concept that these OCDs generate an intracellular oxygen sink, thereby manipulating the redox status of the cells. In addition, the modularity and orthogonality principles used for the development of these devices allow easy reassembly and fine-tuning, foreseeing their introduction into other chassis/systems.

中文翻译：

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使用耗氧设备调节大肠杆菌中细胞内O _2的浓度

如今，使用电池工厂生产散装和高附加值化合物是传统石化方法的一种有利替代方案。然而，随着微氧或缺氧条件的实施，其中一些工艺的效率和生产率可以提高。在工业设置中，漆酶是吸引人的是可以氧化的宽范围的底物，并减少O催化剂₂至H ₂ O.在这项工作中，多个基于漆酶装置被设计和构造成调制细菌底盘细胞内的氧浓度。这些耗氧设备（OCD）包括大肠杆菌的天然漆酶（CueO）和通过定向进化获得的该蛋白的三个变体。最初使用大肠杆菌DH5α蛋白提取物在体外对OCD进行表征，然后使用从其他大肠杆菌菌株中获得的提取物进行体内表征。诱导OCD后，在所测试的四个菌株中未观察到对生长的主要影响，并且对细胞提取物蛋白质谱的分析显示漆酶水平升高。此外，与强迫症相关的耗氧量在所有测试条件下均发生，但该装置的性能显示出依赖于菌株，从而突出显示了遗传背景的重要性，即使在密切相关的菌株中也是如此。漆酶变体之一显示氧化酶活性和O ₂消耗速率分别增加13倍和5倍。此外，还可能通过使用L来证明体内O _2的消耗。-DOPA作为底物，代表了概念证明，这些OCD产生细胞内的氧沉，从而操纵细胞的氧化还原状态。另外，用于开发这些设备的模块化和正交性原理允许轻松地重新组装和微调，并预见将它们引入其他机箱/系统中。