Gluconobacter Oxydans Research Articles

Relocation of dehydroquinate dehydratase to the periplasmic space improves dehydroshikimate production with Gluconobacter oxydans strain NBRC3244.

3-Dehydroshikimate (3-DHS) is a key intermediate for the synthesis of various compounds, including the antiviral drug oseltamivir. The Gluconobacter oxydans strain NBRC3244 intrinsically oxidizes quinate to produce 3-dehydroquinate (3-DHQ) in the periplasmic space. Even though a considerable activity is detected in the recombinant G. oxydans homologously overexpressing type II dehydroquinate dehydratase (DHQase) encoded in the aroQ gene at a pH where it grows, an alkaline shift of the culture medium is required for 3-DHS production in the middle of cultivation. Here, we attempted to adopt type I DHQase encoded in the aroD gene of Gluconacetobacter diazotrophicus strain PAL5 because the type I DHQase works optimally at weak acid, which is preferable for growth conditions of G. oxydans. In addition, we anticipated that subcellular localization of DHQase is the cytoplasm, and therefore, transports of 3-DHQ and 3-DHS across the cytoplasmic membrane are rate-limiting steps in the biotransformation. The Sec- and TAT-dependent signal sequences for secretion were attached to the N terminus of AroD to change the subcellular localization. G. oxydans that expresses the TAT-AroD derivative achieved 3-DHS production at a tenfold higher rate than the reference strain that expresses wild-type AroD even devoid of alkaline shift. Enzyme activity with the intact cell suspension and signal sequence cleavage supported the relocation of AroD to the periplasmic space. The present study suggests that the relocation of DHQase improves 3-DHS production in G. oxydans and represents a proof of concept for the potential of enzyme relocation in metabolic engineering. KEY POINTS: • Type-I dehydroquinate dehydratase (DHQase) was expressed in Gluconobacter oxydans. • Cytoplasmic DHQase was relocated to the periplasmic space in G. oxydans. • Relocation of DHQase in G. oxydans improved 3-dehydroshikimate production.

Coculture of Gluconobacter oxydans and Escherichia coli for 3,4-Dihydroxybutyric Acid Production from Xylose

Coculture of <i>Gluconobacter oxydans</i> and <i>Escherichia coli</i> for 3,4-Dihydroxybutyric Acid Production from Xylose

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.

A SacB-based system for diverse and multiple genome editing in Gluconobacter oxydans

Gluconobacter oxydans is an important industrial bacterial strain widely used to produce a lot of useful products. However, very few gene editing tools are available for G. oxydans. This study aimed to develop an efficient genome editing method for G. oxydans using SacB as a counter-selectable marker. A plasmid that could express the kanamycin resistance gene in both E. coli and G. oxydans was constructed using the screened shuttle promoter P116. After optimizing the genome editing conditions, the derivative plasmids could be effectively utilized for diverse genome editing, such as gene deletion, insertion, replacement, and in situ modification in G. oxydans WSH-003. In addition, the SacB-based system also achieved multiple gene editing in G. oxydans. Moreover, the genome of the industrial strain G. oxydans WSH-003 was modified and the growth rate and substrate conversion rate of the strain successfully increased using this system. The system could also have potential to be applied in different G. oxydans strains. The process established in this study also provides a reference for constructing genetic tools for many other genetically recalcitrant bacteria.

Towards Bioleaching of a Vanadium Containing Magnetite for Metal Recovery.

Vanadium – a transition metal – is found in the ferrous-ferric mineral, magnetite. Vanadium has many industrial applications, such as in the production of high-strength low-alloy steels, and its increasing global industrial consumption requires new primary sources. Bioleaching is a biotechnological process for microbially catalyzed dissolution of minerals and wastes for metal recovery such as biogenic organic acid dissolution of bauxite residues. In this study, 16S rRNA gene amplicon sequencing was used to identify microorganisms in Nordic mining environments influenced by vanadium containing sources. These data identified gene sequences that aligned to the Gluconobacter genus that produce gluconic acid. Several strategies for magnetite dissolution were tested including oxidative and reductive bioleaching by acidophilic microbes along with dissimilatory reduction by Shewanella spp. that did not yield significant metal release. In addition, abiotic dissolution of the magnetite was tested with gluconic and oxalic acids, and yielded 3.99 and 81.31% iron release as a proxy for vanadium release, respectively. As a proof of principle, leaching via gluconic acid production by Gluconobacter oxydans resulted in a maximum yield of 9.8% of the available iron and 3.3% of the vanadium. Addition of an increased concentration of glucose as electron donor for gluconic acid production alone, or in combination with calcium carbonate to buffer the pH, increased the rate of iron dissolution and final vanadium recoveries. These data suggest a strategy of biogenic organic acid mediated vanadium recovery from magnetite and point the way to testing additional microbial species to optimize the recovery.

Gluconobacter oxydans의 당 전이효소 활성을 이용한 다당류의 제조 및 배양액의 품질 특성

Dextran, a homologous polysaccharide with the main chain of glucose units composed by an α-1,6 glycosidic bond, is synthesized from dextrin by dextran-dextrinase (DDase), a transglucosidase, derived from acetic acid bacteria Gluconobacter oxydans and lactic acid bacteria (LAB). The effective culture conditions were examined for producing dextran via bio-conversion with acetic acid bacteria (AAB) demonstrating DDase activity in various medium compositions during 0 to 7 days with or without glycerol addition (2%, v/v) and different degrees of dextrin polymerization (D.E.) based on the addition level (1,5%, w/v). On day 7, the G. oxydans growth was almost tripled in presence of glycerol as observed via a cell growth curve (OD). After culturing for 7 days, the pH decreased from 6 to 3.1-3.5, and the acidity increased from 0.12% to 0.4-0.62%, depending on the dextrin D.E. and the addition level. The reducing sugar decreased continuously. The medium containing 5% dextrin showed shear-thinning characteristics. The apparent viscosity of the 5% dextrin DE4-7 culture solution was 5.6 mPa·s, which was similar to that of the 20% dextran aqueous solution. The analysis of constituent saccharides contained in the culture medium (HPAEC-PAD) showed a substance with a high degree of polymerization. 1H-NMR analysis showed that α-1,6 glycosidic bond existed as the intermolecular bond of this substance. Therefore, efficient production of dextran was possible by culturing in a medium containing 5% dextrin and glycerol during culture of AAB.

The first in-depth exploration of the genome of the engineered bacterium, Gluconobacter thailandicus.

Glycerol is an abundant byproduct of biodiesel production that has significant industrial value and can be converted into dihydroxyacetone (DHA). DHA is widely used for the production of various chemicals, pharmaceuticals, and food additives. Gluconobacter can convert glycerol to DHA through two different pathways, including membrane-bound dehydrogenases with pyrroloquinoline quinone (PQQ) and NAD(P)+ -dependent enzymes. Previous work has indicated that membrane-bound dehydrogenases are present in Gluconobacter oxydans and Gluconobacter frateurii, but the metabolic mechanism of Gluconobacter thailandicus's glycerol conversion is still not clear. Through in-depth analysis of the G. thailandicus genome and annotation of its metabolic pathways, we revealed the existence of both PQQ and NAD(P)+ -dependent enzymes in G. thailandicus. In addition, this study provides important information related to the tricarboxylic acid cycle, glycerol dehydrogenase level, and phylogenetic relationships of this important species.

Application of immersed silicone rubber membrane module for biocatalytic production of 2-phenylethanol and phenylacetic acid

Natural phenylacetic acid can be produced by the biotransformation of 2-phenylethanol by bacterial strains, such as Gluconobacter oxydans, with incomplete oxidative metabolism. 2-Phenylethanol can be produced by the biotransformation from L-phenylalanine using ordinary yeasts such as Saccharomyces cerevisiae. Both compounds are valuable components in flavor industry, where the demand for substances of natural origin has been increasing in recent years and can be satisfied by microbial production. In this work, we proposed two membrane hybrid systems for extractive bioproduction of 2-phenylethanol consisting of a mechanically stirred tank bioreactor, immersed silicone rubber membrane module for product removal, and equipment for the regeneration of water extractant from the membrane module applying two approaches: 1. extract was led to a fixed bed adsorption column for 2-phenylethanol accumulation; 2. extract was led to an airlift bioreactor with the bacterial strain Gluconobacter oxydans, where 2-phenylethanol was oxidized to phenylacetic acid. Biotransformation experiments performed in the proposed hybrid systems proved its functionality and capability to overcome product inhibition and to provide the desired product in high purity. In second approach, phenylacetic acid, as a product of higher value, was produced directly from L-phenylalanine using two strains of microorganisms in a unique double bioreactor membrane hybrid system.

Direct Bioelectrocatalytic Oxidation of Glucose by Gluconobacter oxydans Membrane Fractions in PEDOT:PSS/TEG-Modified Biosensors.

Recent years have witnessed an ever-increasing interest in developing electrochemical biosensors based on direct electron transfer-type bioelectrocatalysis. This work investigates the bioelectrocatalytic oxidation of glucose by membrane fractions of Gluconobacter oxydans cells on screen-printed electrodes modified with thermally expanded graphite and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS). Electrooxidation of glucose was shown to occur without the presence of electron transport mediators. Chronoamperometric and cyclic voltametric characteristics showed an increase of anodic currents at electrode potentials of 0–500 mV relative to the reference electrode (Ag/AgCl). The direct electron transfer effect was observed for non-modified PEDOT:PSS as well as for PEDOT:PSS linked with crosslinkers and conductive fillers such as polyethylene glycol diglycidyl or dimethyl sulfoxide. Bioelectrodes with this composite can be successfully used in fast reagent-free glucose biosensors.

Distribution of scandium in red mud and extraction using Gluconobacter oxydans

The aim was to study bioleaching of rare earth elements from red mud by a chemoorganotrophic microorganism, and to investigate the microbe-mineral interactions. Red mud is a promising resource for scandium, whose concentration was 92 and 54 mg/kg in two samples from Germany and India, respectively; however, both showed appreciably high concentration of zirconium. A gluconic acid-producing bacterium, Gluconobacter oxydans (DSMZ 46616), was used in the bioleaching experiments under parametric variation of inoculum concentration, pulp density, and culture adaptation. At a solid load of 10% (w/v) red mud with 10% (v/v) bacterial inoculum at 37 °C, a maximum solubilisation of 83% and 94% Sc was observed after 18–20 d of incubation for Indian and German red mud, respectively. The total amount of gluconic acid excreted by the bacteria increased with an increase in pulp density up to 10% (w/v). After bioleaching, SEM-EDAX analysis of the solid residue depicted enrichment of zirconium associated with the silicate matrix, and the bacterial cells were adhered to the major mineral matrix.

A cost-practical cell-recycling process for xylonic acid bioproduction from acidic lignocellulosic hydrolysate with whole-cell catalysis of Gluconobacter oxydans

Xylonic acid (XA), as a bio-based platform chemical, is of considerable interest for xylose bioconversion. The whole-cell catalysis of Gluconobacter oxydans presents a promising application potential, while the hard works of cell culture still severely hinder XA business from the crude toxics-containing lignocellulosic hydrolysate. Hence, the bacterial cells should be recycled to reduce commercial production cost. The implementation of diatomite detoxification not only absorbs most of the degraded inhibitors in hydrolysate, but also confines the sugar contents loss with 10% and allows the bacterial cells to maintain 90% bioconversion performance during cell-recycling operation. Additionally, a scale-up of XA bioproduction was achieved in a sealed oxygen supply fermenter. Finally, 210 g XA was produced from 1000 g corncob originated hydrolysate within 24 h of whole-cell catalysis. Diatomite treatment provides an efficient and cost-practical approach for the commercial bioproduction of biochemicals like XA from lignocellulosic biomass.

Selective Production of Xylooligosaccharides by Xylan Hydrolysis Using a Novel Recyclable and Separable Furoic Acid.

Xylooligosaccharides (XOS) have gained considerable attention worldwide as prebiotics due to their immune-strengthening activity and beneficial gut bacteria development and can be produced from xylan-rich resources by acid hydrolysis. The present study proved the organic acid hydrolysis to be beneficial for XOS yield. In this study, a recyclable and separable organic acid, i.e., furoic acid, was used for hydrolyzing xylan to produce XOS, and the response surface methodology design was applied to maximize the XOS yield; the results indicated that the quadratic model terms of the interaction between reaction temperature and hydrolysis time showed the most significant impact on XOS yields (P < 0.05). The predicted maximum yield of XOS was 49.0% with 1.2% furoic acid at 167°C for 33 min, being close to the experimental value (49.2%), indicating that the fitted models were in good agreement with the experimental results. Meanwhile, the primary byproducts, including xylose and furfural, were concurrently bio-oxidized into xylonic acid and furoic acid by Gluconobacter oxydans and separated by electrodialysis. Subsequently, the furoic acid with low solubility (<3.7%, 25°C) was recovered by natural crystallization. The above results indicate that the use of multi-steps contributes to sustainable XOS production by furoic acid.

Identification of Gradient Promoters of Gluconobacter oxydans and Their Applications in the Biosynthesis of 2-Keto-L-Gulonic Acid

The acetic acid bacterium Gluconobacter oxydans is known for its unique incomplete oxidation and therefore widely applied in the industrial production of many compounds, e.g., 2-keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C. However, few molecular tools are available for metabolically engineering G. oxydans, which greatly limit the strain development. Promoters are one of vital components to control and regulate gene expression at the transcriptional level for boosting production. In this study, the low activity of SDH was found to hamper the high yield of 2-KLG, and enhancing the expression of SDH was achieved by screening the suitable promoters based on RNA sequencing data. We obtained 97 promoters from G. oxydans’s genome, including two strong shuttle promoters and six strongest promoters. Among these promoters, P3022 and P0943 revealed strong activities in both Escherichia coli and G. oxydans, and the activity of the strongest promoter (P2703) was about threefold that of the other reported strong promoters of G. oxydans. These promoters were used to overexpress SDH in G. oxydans WSH-003. The titer of 2-KLG reached 3.7 g/L when SDH was under the control of strong promoters P2057 and P2703. This study obtained a series of gradient promoters, including two strong shuttle promoters, and expanded the toolbox of available promoters for the application in metabolic engineering of G. oxydans for high-value products.

Cultivable microbial ecology and aromatic profile of “mothers” for Vino cotto wine production

The aim of the present study was to assess the cultivable microbiota of “mothers” of Vino cotto collected from production of different years 1890, 1895, 1920, 1975, 2008. A total of 73 yeasts and 81 bacteria were isolated. Starmerella lactis-condensi, Starmerella bacillaris, Hanseniaspora uvarum, Saccharomyces cerevisiae, Hanseniaspora guillermondi and Metschnikowia pulcherrima were identified. Bacteria isolates belonged to lactic acid bacteria (Lactiplantibacillus plantarum and Pediococcus pentosaceus) and acetic acid bacteria (Gluconobacter oxydans). Remarkable biodiversity was observed for Starm. bacillaris, as well as L. plantarum and G. oxydans. Organic acids and volatile compounds were also determined. Malic and succinic acids were the main ones with values ranging from 8.49 g/L to 11.76 g/L and from 4.15 g/L to 7.73 g/L respectively, while citric acid was present at low concentrations (<0.2 g/L) in all samples. Esters and higher alcohols were the main volatile compounds detected followed by alkanes. This study permits to better understand the microbial communities associated to this product and could be considered a starting point for the definition of tailored starter cultures to improve the quality of Vino cotto preserving its typical traits.

Elemental composition, heat capacity from 2 to 300 K and derived thermodynamic functions of 5 microorganism species

Detailed elemental analysis and low-temperature calorimetric measurement results are reported for the first time for Gram-positive bacteria, Gram-negative bacteria and mold fungi. Microorganism unit carbon formulas (empirical formulas) were calculated. Standard molar heat capacity and entropy were found to be C⁰p,m = 38.200 J/C-mol K and S⁰m = 31.234 J/C-mol K for Escherichia coli, C⁰p,m = 54.188 J/C-mol K and S⁰m = 47.141 J/C-mol K for Gluconobacter oxydans, C⁰p,m = 31.475 J/C-mol K and S⁰m = 33.222 J/C-mol K for Pseudomonas fluorescens, C⁰p,m = 38.118 J/C-mol K and S⁰m = 37.042 J/C-mol K for Streptococcus thermophilus, and C⁰p,m = 35.470 J/C-mol K and S⁰m = 34.393 J/C-mol K for Penicillium chrysogenum. Microorganism heat capacities below 10 K were best described by an expanded Debye-T³ law. Based on the collected data, empirical formulas and entropies per C-mole of the analyzed organisms were determined. The measured heat capacities were compared to predictions of Kopp’s rule and Hurst-Harrison equation, both of which were found to be able to give reasonably accurate predictions. The determined entropies were compared to predictions of Battley and Roels models. The Battley model was found to be more accurate. The measured microorganism entropies lay between the values of their principal macromolecular constituents: DNA, and globular and fibrillar proteins. This indicates that self-assembly of the macromolecular components into cellular structures does not lead to decrease in thermal entropy.

Crystal structure of the 6-phosphogluconate dehydrogenase from Gluconobacter oxydans reveals tetrameric 6PGDHs as the crucial intermediate in the evolution of structure and cofactor preference in the 6PGDH family

Background: The enzyme 6-phosphogluconate dehydrogenase (6PGDH) is the central enzyme of the oxidative pentose phosphate pathway. Members of the 6PGDH family belong to different classes: either homodimeric enzymes assembled from long-chain subunits or homotetrameric ones assembled from short-chain subunits. Dimeric 6PGDHs bear an internal duplication absent in tetrameric 6PGDHs and distant homologues of the β-hydroxyacid dehydrogenase (βHADH) superfamily. Methods: We use X-ray crystallography to determine the structure of the apo form of the 6PGDH from Gluconobacter oxydans (Go6PGDH). We carried out a structural and phylogenetic analysis of short and long-chain 6PGDHs. We put forward an evolutionary hypothesis explaining the differences seen in oligomeric state vs. dinucleotide preference of the 6PGDH family. We determined the cofactor preference of Go6PGDH at different 6-phosphogluconate concentrations, characterizing the wild-type enzyme and three-point mutants of residues in the cofactor binding site of Go6PGDH. Results: The structural comparison suggests that the 6PG binding site initially evolved by exchanging C-terminal α-helices between subunits. An internal duplication event changed the quaternary structure of the enzyme from a tetrameric to a dimeric arrangement. The phylogenetic analysis suggests that 6PGDHs have spread from Bacteria to Archaea and Eukarya on multiple occasions by lateral gene transfer. Sequence motifs consistent with NAD+- and NADP+-specificity are found in the β2-α2 loop of dimeric and tetrameric 6PGDHs. Site-directed mutagenesis of Go6PGDH inspired by this analysis fully reverses dinucleotide preference. One of the mutants we engineered has the highest efficiency and specificity for NAD+ so far described for a 6PGDH. Conclusions: The family 6PGDH comprises dimeric and tetrameric members whose active sites are conformed by a C-terminal α-helix contributed from adjacent subunits. Dimeric 6PGDHs have evolved from the duplication-fusion of the tetrameric C-terminal domain before independent transitions of cofactor specificity. Changes in the conserved β2-α2 loop are crucial to modulate the cofactor specificity in Go6PGDH.

Leaching of rare earth elements and base metals from spent NiMH batteries using gluconate and its potential bio-oxidation products

Gluconate is known to mediate metal leaching. However, during bioleaching by e.g., Gluconobacter oxydans, gluconate can be oxidized to 2-ketogluconate and 5-ketogluconate. The impact of bio-oxidation of gluconate on metal leaching has not been investigated. Therefore, the aim of this study was to investigate leaching of rare earth elements (REEs) and base metals from spent nickel-metal-hydride (NiMH) batteries using gluconate, 2-ketogluconate and 5-ketogluconate. Batch leaching assays were conducted under controlled and uncontrolled pH conditions for 14 days using 60 mM of either the individual leaching agents or their various combinations. At target pH of 6.0 ± 0.1 and 9.0 ± 0.1 and without pH control, complexolysis was the dominating leaching mechanism and higher REE leaching efficiency was obtained with gluconate, while 5-ketogluconate enabled more efficient base metal leaching. At target pH of 3.0 ± 0.1, acidolysis dominated, and the base metal and REE leaching yields with all the tested leaching agents were higher than under the other studied pH conditions. The highest base metal and REE leaching yields (%) were obtained using gluconate at target pH of 3.0 ± 0.1 being 100.0 Mn, 90.3 Fe, 89.5 Co, 58.5 Ni, 24.0 Cu, 29.3 Zn and 56.1 total REEs. The obtained results are useful in optimization of heterotrophic bioleaching.

Impacts of Phosphorous Source on Organic Acid Production and Heterotrophic Bioleaching of Rare Earth Elements and Base Metals from Spent Nickel-Metal-Hydride Batteries

This study investigated heterotrophic bioleaching of rare earth elements (REEs) and base metals from spent nickel-metal-hydride (NiMH) batteries. Furthermore, the impacts of phosphorous source [Ca3(PO4)2, KH2PO4 and K2HPO4] and its concentration on organic acid production by Gluconobacter oxydans and Streptomyces pilosus were evaluated. Phosphorous source affected microbial acid production and metal leaching. Among the studied phosphorous sources, use of K2HPO4 resulted in highest organic acid production by both bacteria. Increasing K2HPO4 concentration from 2.7 to 27 mM enhanced pyruvic acid production by S. pilosus from 2.2 to 10.7 mM. However, no metal was leached from the spent NiMH batteries with S. pilosus using either one-step, two-step or spent-medium bioleaching. With G. oxydans, highest gluconic acid concentration of 45.0 mM was produced at the lowest K2HPO4 concentration of 2.7 mM. When using two-step bioleaching with G. oxydans, higher leaching efficiencies were obtained for base metals (88.0% vs. 68.0% Fe, 41.5% vs. 35.5% Co, 18.5% vs 16.5% Ni), while more REEs were leached using spent-medium bioleaching (9.0% vs. 6.0% total REEs). With both bioleaching methods, base metals leaching was faster than that of REEs. Surplus of phosphorous should be avoided in bioleaching cultures as precipitation especially with REEs is possible.Graphic

Perspective of Using Gluconacetobacter sucrofermentas VKPM B-11267 in Biofuel Cells

The bioelectrochemical and spectral properties of immobilized Gluconacetobacter sucrofermentas VKPM B-11267 bacteria were studied in the presence and absence of multiwalled carbon nanotubes (MWCNTs). The obtained characteristics were compared with the characteristics of Gluconobacter oxydans, which are phylogenetically close to them and are widely used in bioelectrochemistry. It was shown that modification of the bioelectrode with carbon nanotubes leads to a significant increase in the current level (by 2.5–3 times), as well as to a decrease in the total resistance both in the absence of substrates and in their presence. The potential use of immobilized G. sucrofermentas cells as part of a microbial fuel cell (MFC) was considered. The specific electrical power of an MFC based on immobilized G. sucrofermentas cells was lower than that of an MFC based on G. oxydans cells. Nevertheless, the results obtained indicate that G. sucrofermentas VKPM B-11267 cells can serve as a biocatalyst in MFCs.

Degradation of the low-calorie sugar substitute 5-ketofructose by different bacteria

There is an increasing public awareness about the danger of dietary sugars with respect to their caloric contribution to the diet and the rise of overweight throughout the world. Therefore, low-calorie sugar substitutes are of high interest to replace sugar in foods and beverages. A promising alternative to natural sugars and artificial sweeteners is the fructose derivative 5-keto-D-fructose (5-KF), which is produced by several Gluconobacter species. A prerequisite before 5-KF can be used as a sweetener is to test whether the compound is degradable by microorganisms and whether it is metabolized by the human microbiota. We identified different environmental bacteria (Tatumella morbirosei, Gluconobacter japonicus LMG 26773, Gluconobacter japonicus LMG 1281, and Clostridium pasteurianum) that were able to grow with 5-KF as a substrate. Furthermore, Gluconobacter oxydans 621H could use 5-KF as a carbon and energy source in the stationary growth phase. The enzymes involved in the utilization of 5-KF were heterologously overproduced in Escherichia coli, purified and characterized. The enzymes were referred to as 5-KF reductases and belong to three unrelated enzymatic classes with highly different amino acid sequences, activities, and structural properties. Furthermore, we could show that 15 members of the most common and abundant intestinal bacteria cannot degrade 5-KF, indicating that this sugar derivative is not a suitable growth substrate for prokaryotes in the human intestine.Key points• Some environmental bacteria are able to use 5-KF as an energy and carbon source.• Four 5-KF reductases were identified, belonging to three different protein families.• Many gut bacteria cannot degrade 5-KF.