Summary: Bacterial nanocellulose ( BNC ) produced by aerobic bacteria is a biopolymer with sophisticated technical properties. Although the potential for economically relevant applications is huge, the cost of BNC still limits its application to a few biomedical devices and the edible product Nata de Coco, made available by traditional fermentation methods in Asian countries. Thus, a wider economic relevance of BNC is still dependent on breakthrough developments on the production technology. On the other hand, the development of modified strains able to overproduce BNC with new properties – e.g. porosity, density of fibres crosslinking, mechanical properties, etc. – will certainly allow to overcome investment and cost production issues and enlarge the scope of BNC applications. This review discusses current knowledge about the molecular basis of BNC biosynthesis, its regulations and, finally, presents a perspective on the genetic modification of BNC producers made possible by the new tools available for genetic engineering.

Komagataeibacter species are well‐recognized bionanocellulose ( BNC ) producers. This bacterial genus, formerly assigned to Gluconacetobacter , is known for its phenotypic diversity manifested by strain‐dependent carbon source preference, BNC production rate, pellicle structure, and strain stability. Here, we performed a comparative study of nineteen Komagataeibacter genomes, three of which were newly contributed in this work. We defined the core genome of the genus, clarified phylogenetic relationships among strains, and provided genetic evidence for the distinction between the two major clades, the K . xylinus and the K. hansenii . We found genomic traits, which likely contribute to the phenotypic diversity between the Komagataeibacter strains. These features include genome flexibility, carbohydrate uptake and regulation of its metabolism, exopolysaccharides synthesis, and the c‐di‐ GMP signaling network. In addition, this work provides a comprehensive functional annotation of carbohydrate metabolism pathways, such as those related to glucose, glycerol, acetan, levan, and cellulose. Findings of this multi‐genomic study expand understanding of the genetic variation within the Komagataeibacter genus and facilitate exploiting of its full potential for bionanocellulose production at the industrial scale.

Ethanol exerts a strong positive effect on the cellulose yields from the widely exploited microbial producers of the Komagataeibacter genus. Ethanol is postulated to provide an alternative energy source, enabling effective use of glucose for cellulose biosynthesis rather than for energy acquisition. In this paper, we investigate the effect of ethanol supplementation on the global gene expression profile of Komagataeibacter xylinus E25 using RNA sequencing technology (RNA-seq). We demonstrate that when ethanol is present in the culture medium, glucose metabolism is directed towards cellulose production due to the induction of genes related to UDP-glucose formation and the repression of genes involved in glycolysis and acetan biosynthesis. Transcriptional changes in the pathways of cellulose biosynthesis and c-di-GMP metabolism are also described. The transcript level profiles suggest that Schramm-Hestrin medium supplemented with ethanol promotes bacterial growth by inducing protein biosynthesis and iron uptake. We observed downregulation of genes encoding transposases of the IS110 family which may provide one line of evidence explaining the positive effect of ethanol supplementation on the genotypic stability of K. xylinus E25. The results of this study increase knowledge and understanding of the regulatory effects imposed by ethanol on cellulose biosynthesis, providing new opportunities for directed strain improvement, scaled-up bionanocellulose production, and wider industrial exploitation of the Komagataeibacter species as bacterial cellulose producers.

Bacterial nanocellulose (BNC) produced by Komagataeibacter hansenii has received significant attention due to its unique supernetwork structure and properties. It is nevertheless necessary to modify bacterial nanocellulose to achieve materials with desired properties and thus with broader areas of application. The aim here was to influence the 3D structure of BNC by genetic modification of the cellulose producing K. hansenii strain ATCC 53582. Two genes encoding proteins with homology to the MotA and MotB proteins, which participate in motility and energy transfer, were selected for our studies. A disruption mutant of one or both genes and their respective complementation mutants were created. The phenotype analysis of the disruption mutants showed a reduction in motility, which resulted in higher compaction of nanocellulose fibers and improvement in their mechanical properties. The data strongly suggest that these genes play an important role in the formation of BNC membrane by Komagataeibacter species.

Bacterial nanocellulose (BNC) synthesized by Komagataeibacter hansenii is a polymer that recently gained an attention of tissue engineers, since its features make it a suitable material for scaffolds production. Nevertheless, it is still necessary to modify BNC to improve its properties in order to make it more suitable for biomedical use. One approach to address this issue is to genetically engineer K. hansenii cells towards synthesis of BNC with modified features. One of possible ways to achieve that is to influence the bacterial movement or cell morphology. In this paper, we described for the first time, K. hansenii ATCC 23769 motA+ and motB+ overexpression mutants, which displayed elongated cell phenotype, increased motility, and productivity. Moreover, the mutant cells produced thicker ribbons of cellulose arranged in looser network when compared to the wild-type strain. In this paper, we present a novel development in obtaining BNC membranes with improved properties using genetic engineering tools.

Development of three-dimensional scaffolds mimicking in vivo cells’ environment is an ongoing challenge for tissue engineering. Bacterial nano-cellulose (BNC) is a well-known biocompatible material with enormous water-holding capacity. However, a tight spatial organization of cellulose fibers limits cell ingrowth and restricts practical use of BNC-based scaffolds. The aim of this study was to address this issue avoiding any chemical treatment of natural nanomaterial. Genetic modifications of Komagataeibacter hansenii ATCC 23769 strain along with structural and mechanical properties characterization of obtained BNC membranes were conducted. Furthermore, the membranes were evaluated as scaffolds in in vitro assays to verify cells viability and glycosaminoglycan synthesis by chondrogenic ATDC5 cells line as well as RBL-2H3 mast cells degranulation. K. hansenii mutants with increased cell lengths and motility were shown to produce BNC membranes with increased pore sizes. Novel, BNC membranes with relaxed fiber structure revealed superior properties as scaffolds when compared to membranes produced by a wild-type strain. Obtained results confirm that a genetic modification of productive bacterial strain is a plausible way of adjustment of bacterial cellulose properties for tissue engineering applications without the employment of any chemical modifications.