Ecologically Modified Leather of Bacterial Origin.

The research presented here is an attempt to develop an innovative and environmentally friendly material based on bacterial nanocellulose (BNC), which will be able to replace both animal skins and synthetic polymer products. Bacterial nanocellulose becomes stiff and brittle when dried, so attempts have been made to plasticise this material so that BNC can be used in industry. The research presented here focuses on the ecological modification of bacterial nanocellulose with vegetable oils such as rapeseed oil, linseed oil, and grape seed oil. The effect of compatibilisers of a natural origin on the plasticisation process of BNC, such as chlorophyll, curcumin, and L-glutamine, was also evaluated. BNC samples were modified with rapeseed, linseed, and grapeseed oils, as well as mixtures of each of these oils with the previously mentioned additives. The modification was carried out by passing the oil, or oil mixture, through the BNC using vacuum filtration, where the BNC acted as a filter. The following tests were performed to determine the effect of the modification on the BNC: FTIR spectroscopic analysis, contact angle measurements, and static mechanical analysis. As a result of the modification, the BNC was plasticised. Rapeseed oil proved to be the best for this purpose, with the help of which a material with good strength and elasticity was obtained.

Potential of Bacterial Cellulose in Reconstructive Surgery of Body Integumentary System: Preliminary Studies in Animals.

The aim of the study is to present the preliminary results of the in vivo application of Komagataeibacter xylinum E25 bacterial cellulose (BC) as a replacement material for produced defects during operations. Three pigs (sus scrofa domestica) had the same defects in the ear cartilage (4 × 4 cm) and in the rectus abdominis muscle (6 × 10 cm) with BC membranes implanted into them. The time of observation of the condition of the animals was 3 months. Implantation sites did not show clinical signs of complications in the form of inflammation or necrosis. Histologically, a normal scar was produced as a result of the material healing into the host's body. In one case, no residual implant material was found at the site of implantation, and the remodeled scar confirmed healing. No systemic inflammatory reaction was observed in any of the animals. The host organism's reaction to the bacterial cellulose allows us to believe that it meets the expectations as a material that can be widely used in reconstructive surgery. Nevertheless, this requires further research on a larger group and also using other foreign bodies. The next step would be an experiment on a group consisting of people.

Highly Stretchable Bacterial Cellulose Produced by Komagataeibacter hansenii SI1.

A new strain of bacteria producing cellulose was isolated from Kombucha and identified as Komagataeibacter hansenii, named SI1. In static conditions, the strain synthesises bacterial nanocellulose with an improved ability to stretch. In this study, utilisation of various carbon and nitrogen sources and the impact of initial pH was assessed in terms of bacterial nanocellulose yield and properties. K. hansenii SI1 produces cellulose efficiently in glycerol medium at pH 5.0-6.0 with a yield of 3.20-3.60 g/L. Glucose medium led to the synthesis of membrane characterised by a strain of 77%, which is a higher value than in the case of another Komagataeibacter species. Supplementation of medium with vitamin C results in an enhanced porosity and improves the ability of bacterial nanocellulose to stretch (up to 123%). The properties of modified membranes were studied by scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction and mechanical tests. The results show that bacterial nanocellulose produced in SH medium and vitamin C-supplemented medium has unique properties (porosity, tensile strength and strain) without changing the chemical composition of cellulose. The method of production BNC with altered properties was the issue of Polish patent application no. P.431265.

Response surface methodology-based improvement of the yield and differentiation of properties of bacterial cellulose by metabolic enhancers.

This study aims to examine the effect of ethanol and lactic acid on the production of bacterial cellulose, and determine the optimal composition of a co-supplemented culture using response surface methodology. Both ethanol and lactic acid, when added separately or jointly, affected the yield and properties of the biomaterial. Optimization resulted in an increase of 470% in the yield, compared to the Schramm-Hestrin medium. Culture growth profiles, substrate consumption and by-products generation, were examined. The growth rate was increased for cultures supplemented with lactic acid and both lactic acid and ethanol, while the production of gluconic acid was diminished for all modified cultures. The properties of BNC, such as the structure, crystallinity, water holding capacity and tensile strength, were also determined. BNC produced in optimal conditions is more porous and characterized by wider fibers. Despite a decrease in crystallinity, by the addition of ethanol, lactic acid and both additives, the ratio of cellulose Iα was almost unchanged. The stress, strain, young modulus and toughness were improved 2.8-4.2 times, 1-1.9 times, 2.4-3.5 times and 2.5-6.8 times, respectively. The new approach to improving BNC yields and properties presented here could contribute to more economical production and wider application of this biopolymer.

Extracellular Nucleotides Affect the Proangiogenic Behavior of Fibroblasts, Keratinocytes, and Endothelial Cells.

Chronic wound healing is currently a severe problem due to its incidence and associated complications. Intensive research is underway on substances that retain their biological activity in the wound microenvironment and stimulate the formation of new blood vessels critical for tissue regeneration. This group includes synthetic compounds with proangiogenic activity. Previously, we identified phosphorothioate analogs of nucleoside 5'-O-monophosphates as multifunctional ligands of P2Y6 and P2Y14 receptors. The effects of a series of unmodified and phosphorothioate nucleotide analogs on the secretion of VEGF from keratinocytes and fibroblasts, as well as their influence on the viability and proliferation of keratinocytes, fibroblasts, and endothelial cells were analyzed. In addition, the expression profiles of genes encoding nucleotide receptors in tested cell models were also investigated. In this study, we defined thymidine 5'-O-monophosphorothioate (TMPS) as a positive regulator of angiogenesis. Preliminary analyses confirmed the proangiogenic potency of TMPS in vivo.

Towards control of cellulose biosynthesis by Komagataeibacter using systems-level and strain engineering strategies: current progress and perspectives.

The strains of the Komagataeibacter genus have been shown to be the most efficient bacterial nanocellulose producers. Although exploited for many decades, the studies of these species focused mainly on the optimisation of cellulose synthesis process through modification of culturing conditions in the industrially relevant settings. Molecular physiology of Komagataeibacter was poorly understood and only a few studies explored genetic engineering as a strategy for strain improvement. Only since recently the systemic information of the Komagataeibacter species has been accumulating in the form of omics datasets representing sequenced genomes, transcriptomes, proteomes and metabolomes. Genetic analyses of the mutants generated in the untargeted strain modification studies have drawn attention to other important proteins, beyond those of the core catalytic machinery of the cellulose synthase complex. Recently, modern molecular and synthetic biology tools have been developed which showed the potential for improving targeted strain engineering. Taking the advantage of the gathered knowledge should allow for better understanding of the genotype-phenotype relationship which is necessary for robust modelling of metabolism as well as selection and testing of new molecular engineering targets. In this review, we discuss the current progress in the area of Komagataeibacter systems biology and its impact on the research aimed at scaled-up cellulose synthesis as well as BNC functionalisation. Key points • The accumulated omics datasets advanced the systemic understanding of Komagataeibacter physiology at the molecular level. • Untargeted and targeted strain modification approaches have been applied to improve nanocellulose yield and properties. • The development of modern molecular and synthetic biology tools presents a potential for enhancing targeted strain engineering. • The accumulating omic information should improve modelling of Komagataeibacter's metabolism as well as selection and testing of new molecular engineering targets.

Evolved Fusarium oxysporum laccase expressed in Saccharomyces cerevisiae.

Fusarium oxysporum laccase was functionally expressed in Saccharomyces cerevisiae and engineered towards higher expression levels and higher reactivity towards 2,6-dimethoxyphenol, that could be used as a mediator for lignin modification. A combination of classical culture optimization and protein engineering led to around 30 times higher activity in the culture supernatant. The winner mutant exhibited three times lower Km, four times higher kcat and ten times higher catalytic efficiency than the parental enzyme. The strategy for laccase engineering was composed of a combination of random methods with a rational approach based on QM/MM MD studies of the enzyme complex with 2,6-dimethoxyphenol. Laccase mediator system with 2,6-dimethoxyphenol caused fulvic acids release from biosolubilized coal.

Effect of ethanol supplementation on the transcriptional landscape of bionanocellulose producer Komagataeibacter xylinus E25.

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.

Modification of bacterial nanocellulose properties through mutation of motility related genes in Komagataeibacter hansenii ATCC 53582.

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.

Structural changes of bacterial nanocellulose pellicles induced by genetic modification of Komagataeibacter hansenii ATCC 23769.

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.

Molecular aspects of bacterial nanocellulose biosynthesis.

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.

Comparative genomics of the Komagataeibacter strains-Efficient bionanocellulose producers.

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.

Stable composite of bacterial nanocellulose and perforated polypropylene mesh for biomedical applications.

The article presents the method of preparation of new, stable bacterial cellulose composites with perforated solid materials for biomedical applications, comprising reconstructive surgery of soft and hard tissues. The composites were obtained in specially designed bioreactors equipped with a set of perforated mesh stripes threaded vertically to the culture medium, ensuring perpendicular growth of bacterial nanocellulose synthesized by Komagataeibacter xylinus E25 in stationary culture. The developed biocomposites have been tested for stability and mechanical strength, as well as for their in vitro inflammatory responses shown as mast cell degranulation with N-acetyl-ß-d-hexosaminidase release and mast cell adhesion. The obtained results indicate that the composites components are well integrated after the process of cultivation and purification. Bacterial nanocellulose does not negatively influence mechanical properties of the polypropylene porous mesh, preserving its tensile strength, elasticity, and load. Moreover, application of bacterial cellulose makes the composites less immunogenic as compared to polypropylene itself. Therefore, the composites have the great potential of application in medicine, and depending on the applied porous material, might be used either in hernioplasty (if porous hernia mesh is used), cranioplasty (if perforated metal or polymeric cranial implant is applied), or as a protective barrier in any application that requires biocompatibility or antiadhesive properties improvement. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 978-987, 2019.

Scaffolds for Chondrogenic Cells Cultivation Prepared from Bacterial Cellulose with Relaxed Fibers Structure Induced Genetically.

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.

Improvement of efficiency of brown coal biosolubilization by novel recombinant Fusarium oxysporum laccase.

Clean coal technologies (e.g. coal biosolubilization) are of essential value, especially in Europe, where coal is the national wealth and other energy sources like crude oil are not available. Fusarium oxysporum LOCK 1134, the strain isolated from brown coal, efficiently biosolubilizes lignite. The obtained liquefied products contain 50% less sulfur and over 99% less mercury than the crude coal. Moreover, the liquefied coal can be modified further by laccase. In this study F. oxysporum laccase was expressed in Pichia pastoris for the first time and was assessed as an additional agent for coal degradation. The novel laccase contributes to humic and fulvic acids release from liquefied coal due to introduction of oxygen into coal structure. The effect is increased when a natural redox mediator, sinapic acid, is present in the reaction mixture-up to 30% and 80% respectively. Humic acids obtained by biological process are environmentally friendly fertilizers that may have stimulating effects on crop growth.

Diversity of laccase-coding genes in Fusarium oxysporum genomes.

Multiple studies confirm laccase role in fungal pathogenicity and lignocellulose degradation. In spite of broad genomic research, laccases from plant wilt pathogen Fusarium oxysporum are still not characterized. The study aimed to identify F. oxysporum genes that may encode laccases sensu stricto and to characterize the proteins in silico in order to facilitate further research on their impact on the mentioned processes. Twelve sequenced F. oxysporum genomes available on Broad Institute of Harvard and MIT (2015) website were analyzed and three genes that may encode laccases sensu stricto were found. Their amino acid sequences possess all features essential for their catalytic activity, moreover, the homology models proved the characteristic 3D laccase structures. The study shades light on F. oxysporum as a new source of multicopper oxidases, enzymes with possible high redox potential and broad perspective in biotechnological applications.

Complete genome sequence of Gluconacetobacter xylinus E25 strain--valuable and effective producer of bacterial nanocellulose.

This study reports the release of complete genome sequence of the producer of bacterial nanocellulose (BNC) - Gluconacetobacter xylinus E25, a vinegar-isolated strain. Preliminary sequence analysis revealed complexity of the genome structure and familiarized genetic basis of productive properties of E25 strain. The genome consists of one chromosome and five plasmids. Whole genome sequencing has opened up new perspectives for further bioinformatics and experimental studies allowing the elucidation of molecular mechanisms responsible for regulation of production of BNC - a valuable biomaterial.

Modified bacterial cellulose tubes for regeneration of damaged peripheral nerves.

INTRODUCTION: The subject of the experiment was bacterial nanocellulose, a natural polymer produced by bacteria - Gluconacetobacter xylinus. Following a specific modification process a cartilage-like material for restoration of damaged tissues may be produced. The obtained implants with excellent biocompatibility, mouldability, biophysical and chemical properties perfectly fit the needs of reconstructive surgery. The goal of the experiment was to develop and analyze cellulosic guidance channels in vivo for the reconstruction of damaged peripheral nerves. MATERIAL AND METHODS: The experiments were conducted on Wistar rats, femoral nerve. Cellulose was produced according to a self-patented method. In the experimental group tubulization was applied, whereas in the control traditional end-to-end connection was used. Observation time was 30, 60, 90, and 180 days. Results evaluation included histological analysis and postoperative observation of motor recovery. RESULTS: The overgrowth of connective tissue and disorganisation of neural structures was evident in 86.67% of control specimens, while for cellulosic group it was only 35% (p = 0.0022). Tubulization prevented the excessive proliferation of connective tissue and isolated from penetration with scar tissue. Autocannibalism, being probably an evidence of neurotrophic factors amassment, was observed in cellulosic group but not in the control one. Motor recovery did not differ significantly (p > 0.05). Biocompatibility of implants was affirmed by very small level of tissue response and susceptibility to vascularisation. CONCLUSIONS: Cellulosic neurotubes effectively prevent the formation of neuromas. They are of very good biocompatibility and allow the accumulation of neurotrophic factors inside, thus facilitating the process of nerve regeneration.

Crystal structures of the apo form of ß-fructofuranosidase from Bifidobacterium longum and its complex with fructose.

We solved the 1.8 Å crystal structure of ß-fructofuranosidase from Bifidobacterium longum KN29.1 - a unique enzyme that allows these probiotic bacteria to function in the human digestive system. The sequence of ß-fructofuranosidase classifies it as belonging to the glycoside hydrolase family 32 (GH32). GH32 enzymes show a wide range of substrate specificity and different functions in various organisms. All enzymes from this family share a similar fold, containing two domains: an N-terminal five-bladed ß-propeller and a C-terminal ß-sandwich module. The active site is located in the centre of the ß-propeller domain, in the bottom of a 'funnel'. The binding site, -1, responsible for tight fructose binding, is highly conserved among the GH32 enzymes. Bifidobacterium longum KN29.1 ß-fructofuranosidase has a 35-residue elongation of the N-terminus containing a five-turn α-helix, which distinguishes it from the other known members of the GH32 family. This new structural element could be one of the functional modifications of the enzyme that allows the bacteria to act in a human digestive system. We also solved the 1.8 Å crystal structure of the ß-fructofuranosidase complex with ß-D-fructose, a hydrolysis product obtained by soaking apo crystal in raffinose.