Abstract:

Electroactive microorganisms (EAMs) engage in bidirectional electron exchange with extracellular electron acceptors/donors through the extracellular electron transfer (EET) pathways, resulting in the generation or consumption of electric current. EAMs have been widely applied in many microbial electrochemical technologies, such as biogeochemical cycling of Earth elements, bioremediation of environmental pollutants, electricity production, biosensing, biomining, and microbial electrosynthesis of chemicals, rendering EAMs a focal point in the global pursuit of environmental conservation and low-carbon economy. However, there are still substantial limitations in the practical applications of EAMs. For example, microbial fuel cells encounter a capped upper limit in power density, and the CO₂ reduction rate in microbial electrosynthesis remains below the desired threshold for practical applications. Overcoming these challenges necessitates enhancement in the bidirectional EET rate of EAMs. Nonetheless, complex phenotypes such as elevated EET efficiency often correlate with the expression of multiple genes. To obtain high-performance EAM strains, a deep comprehension of the genotype-phenotype relationship in EAMs and more nuanced manipulation at the genomic level are imperative. This review provides a comprehensive summary of the latest advances in genome editing and transcriptional regulation in EAMs. The main focus is on the CRISPR (clustered regularly interspaced short palindromic repeat)-based biotechnologies developed in model EAMs, such as Shewanellaoneidensis and Geobactersulfurreducens, and a few other representative EAMs. The genome editing techniques to be discussed include (CRISPR-assisted) homologous recombination, CRISPR-associated transposase systems, and base editing. Similarly, transcriptional regulation tools involve CRISPR-based interference (CRISPRi) and activation (CRISPRa) systems. Strategies and advancements related to multiplexed editing and regulation are thoroughly summarized. Subsequently, the review delves into the applications of these technologies in both fundamental and applied scientific domains. On the fundamental science front, efforts are directed toward unveiling factors related to EET and uncovering hidden genotypes. In the realm of application, green electricity-producing microbial fuel cells, bioremediation of nuclear waste, heavy metals, and azo dyes are discussed. Finally, current challenges and future directions in the genetic engineering of EAMs are discussed.

Key words: electroactive microorganisms, gene editing, transcriptional regulation, CRISPR, extracellular electron transfer

摘要：

电活性微生物通过胞外电子传递通路与胞外电子受体/供体进行双向电子交换，产生或吞噬电流。电活性微生物已广泛应用于微生物电化学技术领域，涵盖了元素的生物地球化学循环、环境污染的生物处理与电能生产、生物传感、微生物冶金以及化学品的微生物电合成等多个领域，成为全球环境保护和低碳经济的研究热点。然而，这些微生物在实际应用中仍面临较大局限，如微生物燃料电池的输出功率密度存在一定的上限、微生物电合成技术中的CO₂还原速率尚未达到理想水平等。为了克服这些限制性因素，需要通过高效的基因编辑和转录调控策略来改变电活性微生物的遗传特性，提高其双向电子传递效率。本文首先总结了模式电活性微生物（希瓦氏菌和地杆菌）和其他代表性电活性微生物的基因编辑方法和利用CRISPR（clustered regularly interspaced short palindromic repeat）技术实现转录调控的策略。在基因编辑方面，涵盖了（CRISPR辅助的）同源重组、碱基编辑等方法；而在转录调控方面，包括了CRISPR介导的抑制和激活。此外，对于多基因编辑和调控的策略也进行了深入探讨。其次，综述了这些技术在环境、能源领域中的应用，包括微生物燃料电池、污染物生物处理和修复等。最后，讨论了目前电活性微生物工程改造所面临的挑战和未来的发展方向。

关键词: 电活性微生物, 基因编辑, 转录调控, CRISPR, 胞外电子传递