Unusual 1-3 peptidoglycan cross-links in Acetobacteraceae are made by L,D-transpeptidases with a catalytic domain distantly related to YkuD domains.

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.

Investigating the Potential of Phenolic Compounds and Carbohydrates as Acceptor Substrates for Levansucrase-Catalyzed Transfructosylation Reaction.

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.

Novel Gluconobacter oxydans strains selected from Kombucha with potential postbiotic activity.

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.

Comparative studies on substrate specificity of succinic semialdehyde reductase from Gluconobacter oxydans and glyoxylate reductase from Acetobacter aceti.

Gluconobacter oxydans succinic semialdehyde reductase (GoxSSAR) and Acetobacter aceti glyoxylate reductase (AacGR) represent a novel class in the ß-hydroxyacid dehydrogenases superfamily. Kinetic analyses revealed GoxSSAR's activity with both glyoxylate and succinic semialdehyde, while AacGR is glyoxylate specific. GoxSSAR K167A lost activity with succinic semialdehyde but retained some with glyoxylate, whereas AacGR K175A lost activity. These findings elucidate differences between these homologous enzymes.

High-Yield Production of Dihydroxyacetone from Crude Glycerol in Fed-Batch Cultures of Gluconobacter oxydans.

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.

Screening of Gluconobacter oxydans in xylonic acid fermentation for tolerance of the inhibitors formed dilute acid pretreatment.

Pre-hydrolysate liquor, as an inevitable by-product, contains a large amount of xylose, and is therefore an inexpensive feedstock that can be upgraded to value-added chemical xylonic acid. However, inhibitors, simultaneously formed in lignocellulose pretreatment process, are regarded as the major obstacle for effectively bio-converting xylose in pre-hydrolysate into xylonic acid. In this study, Gluconobacter oxydans, with highly selective and efficient, was employed for xylonic acid production; the impacts of five typical toxic inhibitory compounds on xylonic acid productivity and the activity of the membrane-bound dehydrogenase were evaluated. The results revealed that the inhibitors showed different degrees of influence toward xylonic acid production, and the order of inhibitory effect for acidic inhibitors was formic acid > acetic acid > levulinic acid; the inhibitory effect of aldehyde inhibitors was furfural > 5-hydroxymethyl-furfural. This study provides an important basis of metabolic modification and detoxification process for enhancing inhibitor tolerance and xylonic acid productivity.

Kinetic modeling of xylonic acid production by Gluconobacter oxydans: effects of hydrodynamic conditions.

In this study, the synthesis of xylonic acid from xylose by Gluconobacter oxydans NL71 has been investigated. According to the relationship between oxygen transfer rate and oxygen uptake rate, three different kinetic models of product formation were established and the nonlinear fitting was carried out. The results showed that G. oxydans has critical dissolved oxygen under different strain concentrations, and the relationship between respiration intensity and dissolved oxygen conformed to the Monod equation [Formula: see text]. The maximum reaction rate per unit cell mass and the theoretical maximum specific productivity of G. oxydans obtained by the kinetic model are 0.042 mol/L/h and 6.97 g/gx/h, respectively. These results will assist in determining the best balance between the airflow rate and cell concentration in the reaction and improve the production efficiency of xylonic acid.

Microbial Interactions in a Vitamin C Industrial Fermentation System: Novel Insights and Perspectives.

In industrial production, the precursor of l-ascorbic acid (L-AA, also referred to as vitamin C), 2-keto-l-gulonic acid (2-KLG), is mainly produced using a classic two-step fermentation process performed by Gluconobacter oxydans, Bacillus megaterium, and Ketogulonicigenium vulgare. In the second step of the two-step fermentation process, the microbial consortium of K. vulgare and B. megaterium is used to achieve 2-KLG production. K. vulgare can transform l-sorbose to 2-KLG, but the yield of 2-KLG is much lower in the monoculture than in the coculture fermentation system. The relationship between the two strains is too diverse to analyze and has been a hot topic in the field of vitamin C fermentation. With the development of omics technology, the relationships between the two strains are well explained; nevertheless, the cell-cell communication is unclear. In this review, based on current omics results, the interactions between the two strains are summarized, and the potential cell-cell communications between the two strains are discussed, which will shed a light on the further understanding of synthetic consortia.

Overexpression of mGDH in Gluconobacter oxydans to improve D-xylonic acid production from corn stover hydrolysate.

BACKGROUND: D-Xylonic acid is a versatile platform chemical with broad potential applications as a water reducer and disperser for cement and as a precursor for 1,4-butanediol and 1,2,4-tributantriol. Microbial production of D-xylonic acid with bacteria such as Gluconobacter oxydans from inexpensive lignocellulosic feedstock is generally regarded as one of the most promising and cost-effective methods for industrial production. However, high substrate concentrations and hydrolysate inhibitors reduce xylonic acid productivity. RESULTS: The D-xylonic acid productivity of G. oxydans DSM2003 was improved by overexpressing the mGDH gene, which encodes membrane-bound glucose dehydrogenase. Using the mutated plasmids based on pBBR1MCS-5 in our previous work, the recombinant strain G. oxydans/pBBR-R3510-mGDH was obtained with a significant improvement in D-xylonic acid production and a strengthened tolerance to hydrolysate inhibitors. The fed-batch biotransformation of D-xylose by this recombinant strain reached a high titer (588.7 g/L), yield (99.4%), and volumetric productivity (8.66 g/L/h). Moreover, up to 246.4 g/L D-xylonic acid was produced directly from corn stover hydrolysate without detoxification at a yield of 98.9% and volumetric productivity of 11.2 g/L/h. In addition, G. oxydans/pBBR-R3510-mGDH exhibited a strong tolerance to typical inhibitors, i.e., formic acid, furfural, and 5-hydroxymethylfurfural. CONCLUSION: Through overexpressing mgdh in G. oxydans, we obtained the recombinant strain G. oxydans/pBBR-R3510-mGDH, and it was capable of efficiently producing xylonic acid from corn stover hydrolysate under high inhibitor concentrations. The high D-xylonic acid productivity of G. oxydans/pBBR-R3510-mGDH made it an attractive choice for biotechnological production.

Highly efficient fermentation of 5-keto-D-fructose with Gluconobacter oxydans at different scales.

BACKGROUND: The global market for sweeteners is increasing, and the food industry is constantly looking for new low-caloric sweeteners. The natural sweetener 5-keto-D-fructose is one such candidate. 5-Keto-D-fructose has a similar sweet taste quality as fructose. Developing a highly efficient 5-keto-D-fructose production process is key to being competitive with established sweeteners. Hence, the 5-keto-D-fructose production process was optimised regarding titre, yield, and productivity. RESULTS: For production of 5-keto-D-fructose with G. oxydans 621H ΔhsdR pBBR1-p264-fdhSCL-ST an extended-batch fermentation was conducted. During fructose feeding, a decreasing respiratory activity occurred, despite sufficient carbon supply. Oxygen and second substrate limitation could be excluded as reasons for the decreasing respiration. It was demonstrated that a short period of oxygen limitation has no significant influence on 5-keto-D-fructose production, showing the robustness of this process. Increasing the medium concentration increased initial biomass formation. Applying a fructose feeding solution with a concentration of approx. 1200 g/L, a titre of 545 g/L 5-keto-D-fructose was reached. The yield was with 0.98 g5-keto-d-fructose/gfructose close to the theoretical maximum. A 1200 g/L fructose solution has a viscosity of 450 mPa∙s at a temperature of 55 °C. Hence, the solution itself and the whole peripheral feeding system need to be heated, to apply such a highly concentrated feeding solution. Thermal treatment of highly concentrated fructose solutions led to the formation of 5-hydroxymethylfurfural, which inhibited the 5-keto-D-fructose production. Therefore, fructose solutions were only heated to about 100 °C for approx. 10 min. An alternative feeding strategy was investigated using solid fructose cubes, reaching the highest productivities above 10 g5-keto-d-fructose/L/h during feeding. Moreover, the scale-up of the 5-keto-D-fructose production to a 150 L pressurised fermenter was successfully demonstrated using liquid fructose solutions (745 g/L). CONCLUSION: We optimised the 5-keto-D-fructose production process and successfully increased titre, yield and productivity. By using solid fructose, we presented a second feeding strategy, which can be of great interest for further scale-up experiments. A first scale-up of this process was performed, showing the possibility for an industrial production of 5-keto-D-fructose.

A facile process for adipic acid production in high yield by oxidation of 1,6-hexanediol using the resting cells of Gluconobacter oxydans.

BACKGROUND: Adipic acid (AA) is one of the most important industrial chemicals used mainly for the production of Nylon 6,6 but also for making polyurethanes, plasticizers, and unsaturated polyester resins, and more recently as a component in the biodegradable polyester poly(butylene adipate terephthalate) (PBAT). The main route for AA production utilizes benzene as feedstock and generates copious amounts of the greenhouse gas NO2. Hence, alternative clean production routes for AA from renewable bio-based feedstock are drawing increasing attention. We have earlier reported the potential of Gluconobacter oxydans cells to oxidize 1,6-hexanediol, a potentially biobased diol to AA. RESULTS: The present report involves a study on the effect of different parameters on the microbial transformation of 1,6-hexanediol to adipic acid, and subsequently testing the process on a larger lab scale for achieving maximal conversion and yield. Comparison of three wild-type strains of G. oxydans DSM50049, DSM2003, and DSM2343 for the whole-cell biotransformation of 10 g/L 1,6-hexanediol to adipic acid in batch mode at pH 7 and 30 °C led to the selection of G. oxydans DSM50049, which showed 100% conversion of the substrate with over 99% yield of adipic acid in 30 h. An increase in the concentrations of the substrate decreased the degree of conversion, while the product up to 25 g/L in batch and 40 g/L in fed-batch showed no inhibition on the conversion. Moreover, controlling the pH of the reaction at 5-5.5 was required for the cascade oxidation reactions to work. Cell recycling for the biotransformation resulted in a significant decrease in activity during the third cycle. Meanwhile, the fed-batch mode of transformation by intermittent addition of 1,6-hexanediol (30 g in total) in 1 L scale resulted in complete conversion with over 99% yield of adipic acid (approximately 37 g/L). The product was recovered in a pure form using downstream steps without the use of any solvent. CONCLUSION: A facile, efficient microbial process for oxidation of 1,6-hexanediol to adipic acid, having potential for scale up was demonstrated. The entire process is performed in aqueous medium at ambient temperatures with minimal greenhouse gas emissions. The enzymes involved in catalyzing the oxidation steps are currently being identified.

A practical approach to producing isomaltomegalosaccharide using dextran dextrinase from Gluconobacter oxydans ATCC 11894.

Dextran dextrinase (DDase) catalyzes formation of the polysaccharide dextran from maltodextrin. During the synthesis of dextran, DDase also generates the beneficial material isomaltomegalosaccharide (IMS). The term megalosaccharide is used for a saccharide having DP = 10-100 or 10-200 (DP, degree of polymerization). IMS is a chimeric glucosaccharide comprising α-(1 → 6)- and α-(1 → 4)-linked portions at the nonreducing and reducing ends, respectively, in which the α-(1 → 4)-glucosyl portion originates from maltodextrin of the substrate. In this study, IMS was produced by a practical approach using extracellular DDase (DDext) or cell surface DDase (DDsur) of Gluconobacter oxydans ATCC 11894. DDsur was the original form, so we prepared DDext via secretion from intact cells by incubating with 0.5% G6/G7 (maltohexaose/maltoheptaose); this was followed by generation of IMS from various concentrations of G6/G7 substrate at different temperatures for 96 h. However, IMS synthesis by DDext was limited by insufficient formation of α-(1 → 6)-glucosidic linkages, suggesting that DDase also catalyzes elongation of α-(1 → 4)-glucosyl chain. For production of IMS using DDsur, intact cells bearing DDsur were directly incubated with 20% G6/G7 at 45 °C by optimizing conditions such as cell concentration and agitation efficiency, which resulted in generation of IMS (average DP = 14.7) with 61% α-(1 → 6)-glucosyl content in 51% yield. Increases in substrate concentration and agitation efficiency were found to decrease dextran formation and increase IMS production, which improved the reaction conditions for DDext. Under modified conditions (20% G6/G7, agitation speed of 100 rpm at 45 °C), DDext produced IMS (average DP = 14.5) with 65% α-(1 → 6)-glucosyl content in a good yield of 87%. KEY POINTS: • Beneficial IMS was produced using thermostabilized DDase. • Optimum conditions for reduced dextran formation were successfully determined. • A practical approach was established to provide IMS with a great yield of 87%.

Characterization of 3 phylogenetically distinct membrane-bound d-gluconate dehydrogenases of Gluconobacter spp. and their biotechnological application for efficient 2-keto-d-gluconate production.

We identified a novel flavoprotein-cytochrome c complex d-gluconate dehydrogenase (GADH) encoded by gndXYZ of Gluconobacter oxydans NBRC 3293, which is phylogenetically distinct from previously reported GADHs encoded by gndFGH and gndSLC of Gluconobacter spp. To analyze the biochemical properties of respective GADHs, Gluconobacter japonicus NBRC 3271 mutant strain lacking membranous d-gluconate dehydrogenase activity was constructed. All GADHs (GndFGH, GndSLC, and GndXYZ) were successfully overexpressed in the constructed strain. The optimal pH and KM value at that pH of GndFGH, GndSLC, and GndXYZ were 5, 6, and 4, and 8.82 ± 1.15, 22.9 ± 5.0, and 11.3 ± 1.5 m m, respectively. When the mutants overexpressing respective GADHs were cultured in d-glucose-containing medium, all of them produced 2-keto-d-gluconate, revealing that GndXYZ converts d-gluconate to 2-keto-d-gluconate as well as other GADHs. Among the recombinants, the gndXYZ-overexpressing strain accumulated the highest level of 2-keto-d-gluconate, suggesting its potential for 2-keto-d-gluconate production.

Periplasmic dehydroshikimate dehydratase combined with quinate oxidation in Gluconobacter oxydans for protocatechuate production.

Protocatechuate (3,4-dihydroxybenzoate) has antioxidant properties and is a raw material for the production of muconic acid, which is a key compound in the synthesis of polymers such as nylon and polyethylene terephthalate. Gluconobacter oxydans strain NBRC3244 has a periplasmic system for oxidation of quinate to produce 3-dehydroquinate. Previously, a periplasmic 3-dehydroshikimate production system was constructed by heterologously expressing Gluconacetobacter diazotrophicus dehydroquinate dehydratase in the periplasm of G. oxydans strain NBRC3244. 3-Dehydroshikimate is converted to protocatechuate by dehydration. In this study, we constructed a G. oxydans strain that expresses the Acinetobacter baylyi quiC gene, which encodes a dehydroshikimate dehydratase of which the subcellular localization is likely the periplasm. We attempted to produce protocatechuate by co-cultivation of two recombinant G. oxydans strains-one expressing the periplasmically targeted dehydroquinate dehydratase and the other expressing A. baylyi dehydroshikimate dehydratase. The co-cultivation system produced protocatechuate from quinate in a nearly quantitative manner.

Efficient aerobic fermentation of gluconic acid by high tension oxygen supply strategy with reusable Gluconobacter oxydans HG19 cells.

Gluconic acid is a widely used food and beverage additive, but its production suffers from low efficiency and high cost. In this study, a preferable gluconic acid biosynthesis method without repeated seed culture was proposed and developed using the superior performance of Gluconobacter oxydans. A high oxygen atmosphere satisfying the demand of bio-oxidation increased the productivity of gluconic acid up to ~ 32 g/L/h and the accumulation up to ~ 420 g/L within 24 h of fed-batch fermentation. However, the productivity remarkably decreased when the gluconic acid content was over 350 g/L. Therefore, a continuous fermentation was designed, which in combination with 5 runs of fed-batch fermentation resulted in the final production of 1700 g gluconic acid from 1750 g glucose within 60 h in a 3 L bioreactor. The results suggest that the validity of this model and can enable cost-competitive gluconic acid production in the industry.

The industrial versatility of Gluconobacter oxydans: current applications and future perspectives.

Gluconobacter oxydans is a well-known acetic acid bacterium that has long been applied in the biotechnological industry. Its extraordinary capacity to oxidize a variety of sugars, polyols, and alcohols into acids, aldehydes, and ketones is advantageous for the production of valuable compounds. Relevant G. oxydans industrial applications are in the manufacture of L-ascorbic acid (vitamin C), miglitol, gluconic acid and its derivatives, and dihydroxyacetone. Increasing efforts on improving these processes have been made in the last few years, especially by applying metabolic engineering. Thereby, a series of genes have been targeted to construct powerful recombinant strains to be used in optimized fermentation. Furthermore, low-cost feedstocks, mostly agro-industrial wastes or byproducts, have been investigated, to reduce processing costs and improve the sustainability of G. oxydans bioprocess. Nonetheless, further research is required mainly to make these raw materials feasible at the industrial scale. The current shortage of suitable genetic tools for metabolic engineering modifications in G. oxydans is another challenge to be overcome. This paper aims to give an overview of the most relevant industrial G. oxydans processes and the current strategies developed for their improvement.

The Auxiliary NADH Dehydrogenase Plays a Crucial Role in Redox Homeostasis of Nicotinamide Cofactors in the Absence of the Periplasmic Oxidation System in Gluconobacter oxydans NBRC3293.

Gluconobacter oxydans has the unique property of a glucose oxidation system in the periplasmic space, where glucose is oxidized incompletely to ketogluconic acids in a nicotinamide cofactor-independent manner. Elimination of the gdhM gene for membrane-bound glucose dehydrogenase, the first enzyme for the periplasmic glucose oxidation system, induces a metabolic change whereby glucose is oxidized in the cytoplasm to acetic acid. G. oxydans strain NBRC3293 possesses two molecular species of type II NADH dehydrogenase (NDH), the primary and auxiliary NDHs that oxidize NAD(P)H by reducing ubiquinone in the cell membrane. The substrate specificities of the two NDHs are different from each other: primary NDH (p-NDH) oxidizes NADH specifically but auxiliary NDH (a-NDH) oxidizes both NADH and NADPH. We constructed G. oxydans NBRC3293 derivatives defective in the ndhA gene for a-NDH, in the gdhM gene, and in both. Our ΔgdhM derivative yielded higher cell biomass on glucose, as reported previously, but grew at a lower rate than the wild-type strain. The ΔndhA derivative showed growth behavior on glucose similar to that of the wild type. The ΔgdhM ΔndhA double mutant showed greatly delayed growth on glucose, but its cell biomass was similar to that of the ΔgdhM strain. The double mutant accumulated intracellular levels of NAD(P)H and thus shifted the redox balance to reduction. Accumulated NAD(P)H levels might repress growth on glucose by limiting oxidative metabolisms in the cytoplasm. We suggest that a-NDH plays a crucial role in redox homeostasis of nicotinamide cofactors in the absence of the periplasmic oxidation system in G. oxydansIMPORTANCE Nicotinamide cofactors NAD+ and NADP+ mediate redox reactions in metabolism. Gluconobacter oxydans, a member of the acetic acid bacteria, oxidizes glucose incompletely in the periplasmic space-outside the cell. This incomplete oxidation of glucose is independent of nicotinamide cofactors. However, if the periplasmic oxidation of glucose is abolished, the cells oxidize glucose in the cytoplasm by reducing nicotinamide cofactors. Reduced forms of nicotinamide cofactors are reoxidized by NADH dehydrogenase (NDH) on the cell membrane. We found that two kinds of NDH in G. oxydans have different substrate specificities: the primary enzyme is NADH specific, and the auxiliary one oxidizes both NADH and NADPH. Inactivation of the latter enzyme in G. oxydans cells in which we had induced cytoplasmic glucose oxidation resulted in elevated intracellular levels of NAD(P)H, limiting cell growth on glucose. We suggest that the auxiliary enzyme is important if G. oxydans grows independently of the periplasmic oxidation system.

FNR-Type Regulator GoxR of the Obligatorily Aerobic Acetic Acid Bacterium Gluconobacter oxydans Affects Expression of Genes Involved in Respiration and Redox Metabolism.

Gene expression in the obligately aerobic acetic acid bacterium Gluconobacter oxydans responds to oxygen limitation, but the regulators involved are unknown. In this study, we analyzed a transcriptional regulator named GoxR (GOX0974), which is the only member of the fumarate-nitrate reduction regulator (FNR) family in this species. Evidence that GoxR contains an iron-sulfur cluster was obtained, suggesting that GoxR functions as an oxygen sensor similar to FNR. The direct target genes of GoxR were determined by combining several approaches, including a transcriptome comparison of a ΔgoxR mutant with the wild-type strain and detection of in vivo GoxR binding sites by chromatin affinity purification and sequencing (ChAP-Seq). Prominent targets were the cioAB genes encoding a cytochrome bd oxidase with low O2 affinity, which were repressed by GoxR, and the pnt operon, which was activated by GoxR. The pnt operon encodes a transhydrogenase (pntA1A2B), an NADH-dependent oxidoreductase (GOX0313), and another oxidoreductase (GOX0314). Evidence was obtained for GoxR being active despite a high dissolved oxygen concentration in the medium. We suggest a model in which the very high respiration rates of G. oxydans due to periplasmic oxidations cause an oxygen-limited cytoplasm and insufficient reoxidation of NAD(P)H in the respiratory chain, leading to inhibited cytoplasmic carbohydrate degradation. GoxR-triggered induction of the pnt operon enhances fast interconversion of NADPH and NADH by the transhydrogenase and NADH reoxidation by the GOX0313 oxidoreductase via reduction of acetaldehyde formed by pyruvate decarboxylase to ethanol. In fact, small amounts of ethanol were formed by G. oxydans under oxygen-restricted conditions in a GoxR-dependent manner.IMPORTANCEGluconobacter oxydans serves as a cell factory for oxidative biotransformations based on membrane-bound dehydrogenases and as a model organism for elucidating the metabolism of acetic acid bacteria. Surprisingly, to our knowledge none of the more than 100 transcriptional regulators encoded in the genome of G. oxydans has been studied experimentally until now. In this work, we analyzed the function of a regulator named GoxR, which belongs to the FNR family. Members of this family serve as oxygen sensors by means of an oxygen-sensitive [4Fe-4S] cluster and typically regulate genes important for growth under anoxic conditions by anaerobic respiration or fermentation. Because G. oxydans has an obligatory aerobic respiratory mode of energy metabolism, it was tempting to elucidate the target genes regulated by GoxR. Our results show that GoxR affects the expression of genes that support the interconversion of NADPH and NADH and the NADH reoxidation by reduction of acetaldehyde to ethanol.

Simultaneous transformation of five vectors in Gluconobacter oxydans.

Gluconobacter oxydans is an obligate Gram-negative bacterium that belongs to the family Acetobacteraceae. It is one of the most frequently used microorganisms in industrial biotechnology to produce chemicals related to incomplete oxidation. However, the fine-tuning of G. oxydans is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. Thus, a series of shuttle vectors for G. oxydans inspired by a series of wild-type plasmids in different G. oxydans strains were constructed. Fifteen shuttle vectors were employed to express mCherry in G. oxydans WSH-003 using the replication origin of these wild-type plasmids. Among them, the intensity of fluorescent proteins expressed by p15-K-mCherry was about 10 times that of fluorescent proteins expressed by p5-K-mCherry. Quantitative real-time polymerase chain reaction showed that the relative copy number of p15-K-mCherry reached 19 and had high stability. In contrast, some of the plasmids had a relative copy number of less than 10. The co-expression of multiple shuttle vectors revealed five shuttle vectors that could be transformed into G. oxydans WSH-003 and could express five different fluorescent proteins. The shuttle vectors will facilitate genetic operations for Gluconobacter strains to produce useful compounds more efficiently.

Bioconversion of recombinantly produced precursor peptide pqqA into pyrroloquinoline quinone (PQQ) using a cell-free in vitro system.

Pyrroloquinoline quinone (PQQ) has been recognized as the third class of redox cofactors in addition to the well-known nicotinamides (NAD(P)+) and flavins (FAD, FMN). It plays important physiological roles in various organisms and has strong antioxidant properties. The biosynthetic pathway of PQQ involves a gene cluster composed of 4-7 genes, named pqqA-G, among which pqqA is a key gene for PQQ synthesis, encoding the precursor peptide PqqA. To produce recombinant PqqA in E. coli, fusion tags were used to increase the stability and solubility of the peptide, as well simplify the scale-up of the fermentation process. In this paper, pqqA from Gluconobacter oxydans 621H was expressed in E. coli BL21 (DE3) as a fusion protein with SUMO and purified using a hexahistidine (His6) tag. The SUMO fusion protein and His6 tag were specifically recognized and cleaved by the SUMO specific ULP protease, and immobilized-metal affinity chromatography was used to obtain high-purity precursor peptide PqqA. Expression and purification of target proteins was confirmed by Tricine-SDS-PAGE. Finally, the synthesis of PQQ in a cell-free enzymatic reaction in vitro was confirmed by LC-MS.