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CRISPR-based base editor (BE) offer diverse editing options for genetic engineering of microorganisms, but its application is limited by protospacer adjacent motif (PAM) sequences, context preference, editing window, and off-target effects. Here, a series of iteratively improved cytosine base editors (CBEs) are constructed using the FrCas9 nickase (FrCas9n) with the unique PAM palindromic structure (NNTA) to alleviate these challenges. The deaminase domain-inlaid FrCas9n exhibits an editing range covering 38 nucleotides upstream and downstream of the palindromic PAM, without context preference, which is 6.3 times larger than that of traditional CBEs. Additionally, lower off-target editing is achieved when incorporating high-fidelity mutations at R61A and Q964A in FrCas9n, while maintaining high editing efficiency. The final CBE, HF-ID824-evoCDA-FrCas9n demonstrates broad applicability across different microbes such as Escherichia coli MG1655, Shewanella oneidensis MR-1, and Pseudomonas aeruginosa PAO1. Collectively, this tool offers robust gene editing for facilitating mechanistic studies, functional exploration, and protein evolution in microbes. Cytosine base editors still face several limitations in practical microbial genome engineering. Here, the authors develop a high-efficiency, unbiased, wide-window, and low off-target cytosine base editor based on FrCas9 protein, demonstrating its effective application in various microorganisms. [ABSTRACT FROM AUTHOR]

Bacteria capable of direct ammonia oxidation (Dirammox) play important roles in global nitrogen cycling and nutrient removal from wastewater. Dirammox process, NH3 → NH2OH → N2, first defined in Alcaligenes ammonioxydans HO‐1 and encoded by dnf gene cluster, has been found to widely exist in aquatic environments. However, because of multidrug resistance in Alcaligenes species, the key genes involved in the Dirammox pathway and the interaction between Dirammox process and the physiological state of Alcaligenes species remain unclear. In this work, ammonia removal via the redistribution of nitrogen between Dirammox and microbial growth in A. ammonioxydans HO‐1, a model organism of Alcaligenes species, was investigated. The dnfA, dnfB, dnfC, and dnfR genes were found to play important roles in the Dirammox process in A. ammonioxydans HO‐1, while dnfH, dnfG, and dnfD were not essential genes. Furthermore, an unexpected redistribution phenomenon for nitrogen between Dirammox and cell growth for ammonia removal in HO‐1 was revealed. After the disruption of the Dirammox in HO‐1, more consumed NH4+ was recovered as biomass‐N via rapid metabolic response and upregulated expression of genes associated with ammonia transport and assimilation, tricarboxylic acid cycle, sulfur metabolism, ribosome synthesis, and other molecular functions. These findings deepen our understanding of the molecular mechanisms for Dirammox process in the genus Alcaligenes and provide useful information about the application of Alcaligenes species for ammonia‐rich wastewater treatment. [ABSTRACT FROM AUTHOR]