Philipp Laura-Alina, Kneuer Lukas, Mayer-Windhorst Carina, Jautelat Simon, Le Nhat Quang, Gescher Johannes
Institute of Technical Microbiology, Hamburg University of Technology, Hamburg, Germany.
Appl Environ Microbiol. 2025 Jun 18;91(6):e0068525. doi: 10.1128/aem.00685-25. Epub 2025 May 13.
Research in electro-microbiology provides unique opportunities to study and exploit microbial physiology. Several efforts have been made to transplant the extracellular electron transport chain from the native exoelectrogenic model organism into . However, systematic comparisons between donor and recipient strain configurations are largely missing. Hence, the proposed minimal protein set, consisting of the -type cytochromes cytoplasmic membrane protein A (CymA), small tetraheme cytochrome (STC), MtrA, and MtrC, as well as the -barrel protein MtrB, was heterologously expressed in in different expansion stages. These stages were compared to corresponding strains in terms of anthraquinone-2,6-disulfonate (AQDS) and ferric citrate reduction rates. This revealed that transplantation of heterologous extracellular electron transfer (EET) chains is associated with a tremendous decrease in electron transfer rates. As the acquired electron transfer rates were not competitive to , it was hypothesized that protein localization and maturation might be affected by heterologous expression. Hence, the type II secretion system from was also transplanted into an strain. The latter allowed the secretion of the terminal reductase MtrC onto the cell surface of for the first time. This was correlated with significantly increased but still insufficient extracellular electron transfer rates. Further experiments suggest that the correct folding of MtrB might be a further bottleneck.IMPORTANCEResearch on transplanting extracellular electron transfer (EET) chains into non-native exoelectrogens is vital for advancing bioenergy and bioremediation technologies. Enabling these organisms to transfer electrons to external surfaces like anodes can enhance microbial fuel cell efficiency and electricity generation from organic waste. This approach can broaden the range of substrates and products for biotechnological applications, offering innovative solutions for sustainable production. Our work shows that transplanting the EET chain of into is more complex than previously suggested. The heterologous expression of only -type cytochromes and the β-barrel protein MtrB is insufficient for competitive reduction rates. Predominantly, MtrC and MtrB require specific proteins for transport and folding, necessitating co-expression and maturation. We could identify the type II secretion system of as crucial for MtrC secretion in . Thereby, this work highlights the substrate specificity of bacterial type II secretion systems, suggesting methods to optimize protein production and secretion in bioelectrochemical applications.
电微生物学研究为研究和利用微生物生理学提供了独特的机会。人们已经做出了多项努力，将细胞外电子传递链从天然的产电模式生物移植到……中。然而，供体和受体菌株配置之间的系统比较在很大程度上缺失。因此，由 - 型细胞色素细胞质膜蛋白A（CymA）、小四血红素细胞色素（STC）、MtrA和MtrC以及 - 桶状蛋白MtrB组成的提议的最小蛋白集在不同的扩展阶段在……中进行了异源表达。根据蒽醌 - 2,6 - 二磺酸盐（AQDS）和柠檬酸铁还原率，将这些阶段与相应的……菌株进行了比较。这表明异源细胞外电子转移（EET）链的移植与电子转移速率的大幅下降有关。由于获得的电子转移速率与……不具竞争力，因此推测蛋白质定位和成熟可能受到异源表达的影响。因此，来自……的II型分泌系统也被移植到一个……菌株中。后者首次使末端还原酶MtrC分泌到……的细胞表面。这与显著增加但仍然不足的细胞外电子转移速率相关。进一步的实验表明MtrB的正确折叠可能是另一个瓶颈。
重要性
将细胞外电子转移（EET）链移植到非天然产电菌中的研究对于推进生物能源和生物修复技术至关重要。使这些生物体能够将电子转移到阳极等外表面可以提高微生物燃料电池的效率以及从有机废物中发电。这种方法可以拓宽生物技术应用的底物和产品范围，为可持续生产提供创新解决方案。我们的工作表明，将……的EET链移植到……中比之前认为的更为复杂。仅 - 型细胞色素和β - 桶状蛋白MtrB的异源表达不足以实现具有竞争力的还原速率。主要地，MtrC和MtrB需要特定的蛋白质进行运输和折叠，需要共表达和成熟。我们可以确定……的II型分泌系统对于……中MtrC的分泌至关重要。因此，这项工作突出了细菌II型分泌系统的底物特异性，提出了在生物电化学应用中优化蛋白质生产和分泌的方法。
