Abstract
Bacterial cellulose (BC) is an extracellular polysaccharide produced by bacteria that has wide applications in the food industry, tissue engineering, and battery manufacturing. Genome editing of BC-producing Komagataeibacter species is expected to optimize BC production and its properties. However, the available technology can target only one gene at a time and requires foreign DNA templates, which may present a regulatory hurdle for genetically modified organisms. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-guided base editing method for Komagataeibacter species using Cas9 nickase and cytidine deaminase. Without foreign DNA templates, C-to-T conversions were performed within an 8 bp editing window with 90% efficiency. Double- and triple-gene editing was achieved with 80%-90% efficiency. Fusing uracil-DNA glycosylase with the base editor enabled C-to-G editing. The base editor worked efficiently with various Komagataeibacter species. Finally, mannitol metabolic genes were investigated using base-editing-mediated gene inactivation. This study provides a powerful tool for multiplex genome editing of Komagataeibacter species. IMPORTANCE: Komagataeibacter, a bacterial genus belonging to the family Acetobacteraceae, has important applications in food and material biosynthesis. However, the genome editing of Komagataeibacter relies on traditional homologous recombination methods. Therefore, only one gene can be manipulated in each round using foreign DNA templates, which may present a regulatory hurdle for genetically modified organisms when microorganisms are used in the food industry. In this study, a powerful base editing technology was developed for Komagataeibacter species. C-to-T and C-to-G base conversions were efficiently implemented at up to three loci in the Komagataeibacter genome. This base editing system is expected to accelerate basic and applied research on Komagataeibacter species.