Genomic characterisation of cellulose-producing Komagataeibacter strains KKR and KKO isolated from kombucha

Whole-genome-based comparative genomics of two efficient bacterial cellulose (BC)-producing strains, Komagataeibacter rhaeticus KKR and Komagataeibacter oboediens KKO, was performed to elucidate their genome architecture, functional subsystems, and polysaccharide biosynthesis potential. The genome of K. rhaeticus KKR was 3.78 Mbp in size with a GC content of 62.9%, whereas K. oboediens KKO possessed a slightly larger genome of 3.97 Mbp with a reduced GC content of 61.4%. Comparative genomic analysis with closely related reference strains revealed strong genomic conservation and clear species-level clustering. Orthologous Average Nucleotide Identity (OrthoANI) and in silico DNA-DNA hybridisation (dDDH) analyses supported their taxonomic affiliation, with OrthoANI values above 98% and dDDH values of 90.8% and 87.3% against their nearest phylogenetic neighbours. K. rhaeticus KKR and K. oboediens KKO possessed genes encoding key enzymes of the Embden-Meyerhof-Parnas (EMP), Pentose Phosphate (PP), and Entner-Doudoroff (ED) pathways, except for the phosphofructokinase gene, indicating the presence of central carbon metabolic routes that support bacterial cellulose biosynthesis and energy generation. Both strains harboured genes encoding enzymes involved in the assimilation of alternative carbon sources, including fructose, sucrose, mannitol and glycerol, indicating metabolic versatility. Functional analysis of genes involved in cellulose biosynthesis revealed 4 bcs operons in both genomes, including a fully conserved bcsI operon and other incomplete operons. According to comparative CAZyme analysis, the glycosyltransferase and glycoside hydrolase families linked to cellulose production and carbohydrate metabolism dominated the highly conserved carbohydrate-active enzyme repertoire in both strains. In addition to cellulose biosynthesis, both strains harboured a complete, syntenic acetan type I gene cluster. Notably, a levan biosynthesis locus (lsdA-lsdB) was also detected in K. oboediens KKO. Collectively, these results provide insights into the genetic features of kombucha-associated Komagataeibacter strains and emphasise their potential as biotechnologically relevant strains for the synthesis of industrial exopolysaccharides and sustainable bacterial cellulose.