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

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