As the highest-demand vitamin, the development of a one-step vitamin C synthesis process has been slow for a long time. In previous research, a Gluconobacter oxydans strain (GKLG9) was constructed that can directly synthesize 2-keto-L-gulonic acid (2-KLG) from glucose, but carbon source utilization remained low. Therefore, this study first identified the gene 4kas (4-keto-D-arabate synthase) to reduce the loss of extracellular carbon and inhibit the browning of fermentation broth. Then, promoter engineering was conducted to enhance the intracellular glucose transport pathway and concentrate intracellular glucose metabolism on the pentose phosphate pathway to provide more reducing power. Finally, by introducing the D-sorbitol pathway, the titer of 2-KLG was increased to 38.6 g/L within 60 h in a 5-L bioreactor, with a glucose-to-2-KLG conversion rate of about 46 %. This study is an important step in the development of single-bacterial one-step fermentation to produce 2-KLG.

The strain Gluconobacter oxydans LMG 1385 was used for the bioconversion of crude glycerol to dihydroxyacetone. The suitability of fed-batch cultures for the production of dihydroxyacetone was determined, and the influence of the pH of the culture medium and the initial concentration of glycerol on maximizing the concentration of dihydroxyacetone and on the yield and speed of obtaining dihydroxyacetone by bioconversion was examined. The feeding strategy of the substrate (crude glycerol) during the process was based on measuring the dissolved oxygen tension of the culture medium. The highest concentration of dihydroxyacetone PK = 175.8 g·L-1 and the highest yield YP/Sw = 94.3% were obtained when the initial concentration of crude glycerol was S0 = 70.0 g·L-1 and the pH of the substrate was maintained during the process at level 5.0.

Acetic acid bacteria are used in many industrial processes such as the production of vinegar, vitamin C, the antidiabetic drug miglitol, and various artificial flavorings. These industrially important reactions are primarily carried out by an arsenal of periplasmic-facing membrane-bound dehydrogenases that incompletely oxidize their substrates and shuttle electrons directly into the respiratory chain. Among these dehydrogenases, GOX1969 in Gluconobacter oxydans was predicted to be a pyrroloquinoline quinone-dependent dehydrogenase of unknown function. However, after multiple analysis by a number of labs, no dehydrogenase activity has been detected. Reanalysis of GOX1969 sequence and structure reveals similarities to Escherichia coli BamB, which functions as a subunit of the β-barrel assembly machinery complex that is responsible for the assembly of β-barrel outer membrane proteins in Gram-negative bacteria. To test if the physiological function of GOX1969 is similar to BamB in E. coli, we introduced the gox1969 gene into an E. coli ∆bamB mutant. Growth deficiencies in the ∆bamB mutant were restored when gox1969 was expressed on the plasmid pGox1969. Furthermore, increased membrane permeability conferred by bamB deletion was restored upon gox1969 expression, which suggests a direct link between GOX1969 and a role in maintaining outer membrane stability. Together, this evidence strongly suggests that GOX1969 is functionally acting as a BamB in G. oxydans. As such, functional information on uncharacterized genes will provide new insights that will allow for more accurate modeling of acetic acid bacterial metabolism and further efforts to design rational strains for industrial use.IMPORTANCEGluconobacter oxydans is an industrially important member of the acetic acid bacteria. Experimental characterization of putative genes is necessary to identify targets for further engineering of rational acetic acid bacteria strains that can be used in the production of vitamin C, antidiabetic compounds, artificial flavorings, or novel compounds. In this study, we have identified an undefined dehydrogenase GOX1969 with no known substrate and defined structural similarities to outer membrane biogenesis protein BamB in E. coli K12. Furthermore, we demonstrate that GOX1969 is capable of complementing bamB knockout phenotypes in E. coli K12. Taken together, these findings enhance our understanding of G. oxydans physiology and expand the list of potential targets for future industrial strain design.

Chemical production wastewater contains large amounts of organic solvents (OSs), which pose a significant threat to the environment. In this study, a 10 g·L-1 styrene oxide tolerant strain with broad-spectrum OSs tolerance was obtained via adaptive laboratory evolution. The mechanisms underlying the high OS tolerance of tolerant strain were investigated by integrating physiological, multi-omics, and genetic engineering analyses. Physiological changes are one of the main factors responsible for the high OS tolerance in mutant strains. Moreover, the P-type ATPase GOX_RS04415 and the LysR family transcriptional regulator GOX_RS04700 were also verified as critical genes for styrene oxide tolerance. The tolerance mechanisms of OSs can be used in biocatalytic chassis cell factories to synthesize compounds and degrade environmental pollutants. This study provides new insights into the mechanisms underlying the toxicological response to OS stress and offers potential targets for enhancing the solvent tolerance of G. oxydans.

Pichia yeasts are capable of forming biofilms during vinegar production and causing spoilage in various beverages. In addition, there exists a significant likelihood of encountering yeast contamination which can prevent vinegar production. The present study investigates the detection and characterization of the Pichia manshurica (P. manshurica) biofilm on traditionally produced homemade apple vinegar. The unique characteristics of vinegar were analyzed with a focus on the constituent, known as the \"mother of vinegar\", whose composition is comprised of cellulosic biofilm and acetic acid bacteria, including Gluconobacter oxydans (G. oxydans) Briefly, P. manshurica was isolated from apple vinegar and characterized in terms of the effect of biofilm formation on the surface of the cellulosic film on vinegar production. Microbial identification of vinegar with/without contamination by P. manshurica was analyzed through MALDI-TOF mass spectrometry (MS), and biofilm was characterized by Fourier transform infrared spectroscopy (FT-IR), Scanning electron microscopy (SEM), and crystal violet staining. Accordingly, MS spectrum of isolates was identified as G. oxydans and P. manshurica with a ratio of 2.01 and 1.94, respectively. The FTIR analysis indicated that the peaks within the range of 1150-900 cm-1 revealed a high content of polysaccharide in P. manchuria-contaminated biofilm, which is attributed to the stretching vibration of C-C and C-O bonds. The spectral region from 2921.51 to 2853.71 cm-1 exhibited the characteristic of lipids in bacterial cell walls and membranes. SEM images of bacterial biofilms revealed a three-dimensional network composed of ultrafine fibers with a ribbon-like shape; however, the condensed reticulated structure was observed in contaminated biofilms. The presence of two microbial populations was detected regarding the morphological analysis. Crystal violet staining of contaminated-cellulosic biofilms visualized bacterial and yeast colonization. Concisely, this study emphasizes that the proliferation of Pichia during apple fermentation has the potential to adversely affect the quality of the homemade vinegar, due to its distinct biofilm characteristics.

This study characterizes the acceptor specificity of levansucrases (LSs) from Gluconobacter oxydans (LS1), Vibrio natriegens (LS2), Novosphingobium aromaticivorans (LS3), and Paraburkholderia graminis (LS4) using sucrose as fructosyl donor and selected phenolic compounds and carbohydrates as acceptors. Overall, V. natriegens LS2 proved to be the best biocatalyst for the transfructosylation of phenolic compounds. More than one fructosyl unit could be attached to fructosylated phenolic compounds. The transfructosylation of epicatechin by P. graminis LS4 resulted in the most diversified products, with up to five fructosyl units transferred. In addition to the LS source, the acceptor specificity of LS towards phenolic compounds and their transfructosylation products were found to greatly depend on their chemical structure: the number of phenolic rings, the reactivity of hydroxyl groups and the presence of aliphatic chains or methoxy groups. Similarly, for carbohydrates, the transfructosylation yield was dependent on both the LS source and the acceptor type. The highest yield of fructosylated-trisaccharides was Erlose from the transfructosylation of maltose catalyzed by LS2, with production reaching 200 g/L. LS2 was more selective towards the transfructosylation of phenolic compounds and carbohydrates, while reactions catalyzed by LS1, LS3 and LS4 also produced fructooligosaccharides. This study shows the high potential for the application of LSs in the glycosylation of phenolic compounds and carbohydrates.

A novel conductive composite based on PEDOT:PSS, BSA, and Nafion for effective immobilization of acetic acid bacteria on graphite electrodes as part of biosensors and microbial fuel cells has been proposed. It is shown that individual components in the composite do not have a significant negative effect on the catalytic activity of microorganisms during prolonged contact. The values of heterogeneous electron transport constants in the presence of two types of water-soluble mediators were calculated. The use of the composite as part of a microbial biosensor resulted in an electrode operating for more than 140 days. Additional modification of carbon electrodes with nanomaterial allowed to increase the sensitivity to glucose from 1.48 to 2.81 μA × mM-1 × cm-2 without affecting the affinity of bacterial enzyme complexes to the substrate. Cells in the presented composite, as part of a microbial fuel cell based on electrodes from thermally expanded graphite, retained the ability to generate electricity for more than 120 days using glucose solution as well as vegetable extract solutions as carbon sources. The obtained data expand the understanding of the composition of possible matrices for the immobilization of Gluconobacter bacteria and may be useful in the development of biosensors and biofuel cells.

Gastric and colorectal cancer are among the most frequently diagnosed malignancies of the gastrointestinal tract. Searching for methods of therapy that complements treatment or has a preventive effect is desirable. Bacterial metabolites safe for human health, which have postbiotic effect, are of interest recently. The study aimed to preliminary assessment of the safety, antimicrobial, and anti-cancer activity of cell-free metabolites of Gluconobacter oxydans strains isolated from Kombucha beverages as an example of the potential postbiotic activity of acetic acid bacteria (AAB). The study material consisted of five AAB strains of Kombucha origin and three human cell lines (gastric adenoma-AGS, colorectal adenoma-HT-29, and healthy cells derived from the endothelium of the human umbilical vein-HUVEC). Results of the study confirms the health safety and functional properties of selected AAB strains, including their potential postbiotic properties. The best potential anticancer activity of the AAB cell-free supernatants was demonstrated against AGS gastric adenoma cells. The conducted research proves the postbiotic potential of selected acetic acid bacteria, especially the KNS30 strain. KEY POINTS: •The beneficial and application properties of acetic acid bacteria are poorly studied. •Gluconobacter oxydans from Kombucha show a postbiotic activity. •The best anticancer activity of the G. oxydans showed against gastric adenoma.

Biosensors based on an oxygen electrode, a mediator electrode, and a mediator microbial biofuel cell (MFC) using the bacteria Gluconobacter oxydans B-1280 were formed and tested to determine the integral toxicity. G. oxydans bacteria exhibited high sensitivity to the toxic effects of phenol, 2,4-dinitrophenol, salicylic and trichloroacetic acid, and a number of heavy metal ions. The system \"G. oxydans bacteria-ferrocene-graphite-paste electrode\" was superior in sensitivity to biosensors formed using an oxygen electrode and MFC, in particular regarding heavy metal ions (EC50 of Cr3+, Mn2+, and Cd2+ was 0.8 mg/dm3, 0.3 mg/dm3 and 1.6 mg/dm3, respectively). It was determined that the period of stable functioning of electrochemical systems during measurements was reduced by half (from 30 to 15 days) due to changes in the enzyme system of microbial cells when exposed to toxicants. Samples of the products made from polymeric materials were analyzed using developed biosensor systems and standard biotesting methods based on inhibiting the growth of duckweed Lemna minor, reducing the motility of bull sperm, and quenching the luminescence of the commercial test system \"Ecolum\". The developed bioelectrocatalytic systems were comparable in sensitivity to commercial biosensors, which made it possible to correlate the results and identify, by all methods, a highly toxic sample containing diphenylmethane-4,4\'-diisocyanate according to GC-MS data.

Peptidoglycan is an essential component of the bacterial cell envelope that contains glycan chains substituted by short peptide stems. Peptide stems are polymerized by D,D-transpeptidases, which make bonds between the amino acid in position four of a donor stem and the third residue of an acceptor stem (4-3 cross-links). Some bacterial peptidoglycans also contain 3-3 cross-links that are formed by another class of enzymes called L,D-transpeptidases which contain a YkuD catalytic domain. In this work, we investigate the formation of unusual bacterial 1-3 peptidoglycan cross-links. We describe a version of the PGFinder software that can identify 1-3 cross-links and report the high-resolution peptidoglycan structure of Gluconobacter oxydans (a model organism within the Acetobacteraceae family). We reveal that G. oxydans peptidoglycan contains peptide stems made of a single alanine as well as several dipeptide stems with unusual amino acids at their C-terminus. Using a bioinformatics approach, we identified a G. oxydans mutant from a transposon library with a drastic reduction in 1-3 cross-links. Through complementation experiments in G. oxydans and recombinant protein production in a heterologous host, we identify an L,D-transpeptidase enzyme with a domain distantly related to the YkuD domain responsible for these non-canonical reactions. This work revisits the enzymatic capabilities of L,D-transpeptidases, a versatile family of enzymes that play a key role in bacterial peptidoglycan remodelling.