Gluconobacter Strains Research Articles

Draft genome sequences of two Gluconobacter strains isolated from spoiled orange juice.

Here, we present the draft genome sequences of two Gluconobacter strains that were isolated from spoiled orange juice. Gluconobacter are members of the acetic acid bacteria and are known for their unique metabolism and use in industry. Understanding acetic acid bacteria diversity is essential for engineering further optimized industrial strains.

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.

Complete genome sequence of Gluconobacter frateurii ML.ISBL3, an endophytic strain isolated from aerial roots of Syngonium podophyllum.

The endophytic strain Gluconobacter frateurii ML.ISBL3 was isolated from aerial roots of Syngonium podophyllum in Hong Kong. Its complete genome, established through hybrid assembly, comprises a single chromosome of 3,309,710 bp (56.30% G+C).

Isolation and Identification of Acetobacter tropicalis From Selected Malaysian Local Fruits for Potential BC Production

Acetobacter spp. that are commonly found on fruits, can perform oxidation processes, resulting in acetic acid production in vinegar. Besides that, Acetobacter spp. able to produce bacterial cellulose (BC), which is an essential by-product. This present study was carried out to isolate Acetobacter spp. from selected local fruits. Species verification of the bacterial isolates was performed using molecular and bioinformatic approaches. A total of six local fruits (starfruit, jackfruit, watermelon, pineapple, honeydew & banana) were subjected to seven days of fermentation in a brown sugar solution. Acetobacter spp. were isolated from the fermented medium using bromocresol green ethanol agar as the selective medium. Thirteen bacterial isolates were obtained and subjected to molecular works, including DNA extraction and PCR amplification using universal primers, targeting the 16S rRNA genes. PCR-amplified products were selected for single-pass sequencing. BLASTn analysis of the sequencing results showed three isolates (23.1%) belonging to Acetobacter tropicalis and one isolate (7.7%) representing Gluconobacter oxydans might have potential in BC production. However, the remaining nine isolates (69.2%) hit the Lactobacillus genus. Morphological observation using FESEM showed that the BC produced by all the positive bacterial isolates is similar to dried nata de coco and BC produced by Acetobacter xylinum. In addition, four similar regions of -OH stretch (3400 - 3300 cm-1), -CH stretch (2970 to 2800 cm-1), -OH bending (1620 cm-1), and -COC stretch (1100 to 1073 cm-1) are identified in the BC samples. In the future, the isolated Acetobacter and Gluconobacter strains could be further utilized for large-scale BC production in a suitable fermentation medium.

Engineering Gluconobacter cerinus CGMCC 1.110 for direct 2-keto-L-gulonic acid production.

Gluconobacter is a potential strain for single-step production of 2-keto-L-gulonic acid (2-KLG), which is the direct precursor of vitamin C. Three dehydrogenases, namely, sorbitol dehydrogenase (SLDH), sorbose dehydrogenase (SDH), and sorbosone dehydrogenase (SNDH), are involved in the production of 2-KLG from D-sorbitol. In the present study, the potential SNDH/SDH gene cluster in the strain Gluconobacter cerinus CGMCC 1.110 was mined by genome analysis, and its function in transforming L-sorbose to 2-KLG was verified. Proteomic analysis showed that the expression level of SNDH/SDH had a great influence on the titer of 2-KLG, and fermentation results showed that SDH was the rate-limiting enzyme. A systematic metabolic engineering process, which was theoretically suitable for increasing the titer of many products involving membrane-bound dehydrogenase from Gluconobacter, was then performed to improve the 2-KLG titer in G. cerinus CGMCC 1.110 from undetectable to 51.9g/L in a 5-L bioreactor after fermentation optimization. The strategies used in this study may provide a reference for mining other potential applications of Gluconobacter. KEY POINTS: • The potential SNDH/SDH gene cluster in G. cerinus CGMCC 1.110 was mined. • A systematic engineering process was performed to improve the titer of 2-KLG. • The 2-KLG titer was successfully increased from undetectable to 51.9g/L.

Efficient 1,3-dihydroxyacetone biosynthesis in Gluconobacter oxydans using metabolic engineering and a fed-batch strategy

1,3-Dihydroxyacetone (DHA) is a commercially important chemical and widely used in cosmetics, pharmaceuticals, and food industries as it prevents excessive water evaporation, and provides anti-ultraviolet radiation protection and antioxidant activity. Currently, the industrial production of DHA is based on a biotechnological synthetic route using Gluconobacter oxydans. However, achieving higher production requires more improvements in the synthetic process. In this study, we compared DHA synthesis levels in five industrial wild-type Gluconobacter strains, after which the G. oxydans WSH-003 strain was selected. Then, 16 dehydrogenase genes, unrelated to DHA synthesis, were individually knocked out, with one strain significantly enhancing DHA production, reaching 89.49 g L−1 and 42.27% higher than the wild-type strain. By optimizing the culture media, including seed culture and fermentation media, DHA production was further enhanced. Finally, using an established fed-batch fermentation system, DHA production reached 198.81 g L−1 in a 5 L bioreactor, with a glycerol conversion rate of 82.84%.Graphical

Candidate Acetic Acid Bacteria Strains for Levan Production.

In this study, twelve strains of acetic acid bacteria (AAB) belonging to five different genera were tested for their ability to produce levan, at 70 and 250 g/L of sucrose concentration, respectively. The fructan produced by the bacterial strains was characterized as levan by NMR spectroscopy. Most of the strains produced levan, highlighting intra- and inter-species variability. High yield was observed for Neoasaia chiangmaiensis NBRC 101099 T, Kozakia baliensis DSM 14400 T and Gluconobacter cerinus DSM 9533 T at 70 g/L of sucrose. A 12-fold increase was observed for N. chiangmaiensis NBRC 101099 T at 250 g/L of sucrose concentration. Levan production was found to be affected by glucose accumulation and pH reduction, especially in Ko. baliensis DSM 14400 T. All the Gluconobacter strains showed a negative correlation with the increase in sucrose concentration. Among strains of Komagataeibacter genus, no clear effect of sucrose on levan yield was found. Results obtained in this study highlighted the differences in levan yield among AAB strains and showed interdependence between culture conditions, carbon source utilization, and time of incubation. On the contrary, the levan yield was not always related to the sucrose concentration.

The Novel Strain of Gluconobacter oxydans H32 Isolated from Kombucha as a Proposition of a Starter Culture for Sour Ale Craft Beer Production

Acetic acid bacteria (AAB) has found applications in food technology, including beverages and vinegar. Generally, AAB shows several beneficial properties and has technological usefulness. Properly selected and tested strains of this group of bacteria may constitute a new and interesting solution among starter cultures for functional food. Therefore, the study aimed to develop a sour beer technology, based on the novel strain Gluconobacter oxydans H32. The microbiological, physical-chemical (HPLC method), and sensory (QDP method) quality were determined during 6 months of storage of dark and light beer samples. The AAB count at the beginning of storage was approximately 8 log CFU mL−1, and 6 log CFU mL−1 after 6 months of storage. As a result of the metabolic activity, acetic acid, gluconic acid, and ascorbic acid were detected in the samples. The light beer had a significantly better sensory quality, especially sample BPGL with the addition of G. oxydans H32 starter culture. It was found that it is possible to develop a functional beer with the novel strain Gluconobacter oxydans H32. These Sour Ale craft beers were not only a good source of H32 strain but also its pro-health metabolites.

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.

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.

Gluconobacter aidae sp. nov., an acetic acid bacterium isolated from tropical fruits in Thailand.

Two bacterial strains, isolates AC10T and AC20, which were reported in a previous study on the diversity of acetic acid bacteria in Thailand, were subjected to a taxonomic study. The phylogenetic analysis based on the 16S rRNA gene sequences showed that the two isolates were located closely to the type strains of Gluconobacter oxydans and Gluconobacter roseus. However, the two isolates formed a separate cluster from the type strains of the two species. The genomic DNA of isolate AC10T was sequenced. The assembled genomes of the isolate were analysed for average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH). The results showed that the highest ANI and dDDH values between isolate AC10T and G. oxydans DSM 3503T were 91.15 and 68.2 %, which are lower than the suggested values for species delineation. The genome-based tree was reconstructed and the phylogenetic lineage based on genome sequences showed that the lineage of isolate AC10T was distinct from G. oxydans DSM 3503T and its related species. The two isolates were distinguished from G. oxydans and their relatives by their phenotypic characteristics and MALDI-TOF profiles. Therefore, the two isolates, AC10T (=BCC 15749T=TBRC 11329T=NBRC 103576T) and AC20 (=BCC 15759=TBRC 11330=NBRC 103579), can be assigned to an independent species within the genus Gluconobacter, and the name Gluconobacter aidae sp. nov. is proposed for the two isolates.

The membrane-bound sorbosone dehydrogenase of Gluconacetobacter liquefaciens is a pyrroloquinoline quinone-dependent enzyme

Membrane-bound sorbosone dehydrogenase (SNDH) of Gluconacetobacter liquefaciens oxidizes l-sorbosone to 2-keto-l-gulonic acid (2KGLA), a key intermediate in vitamin C production. We constructed recombinant Escherichia coli and Gluconobacter strains harboring plasmids carrying the sndh gene from Ga. liquefaciens strain RCTMR10 to identify the prosthetic group of SNDH. The membranes of the recombinant E. coli showed l-sorbosone oxidation activity, only after the holo-enzyme formation with pyrroloquinoline quinone (PQQ), indicating that SNDH is a PQQ-dependent enzyme. The sorbosone-oxidizing respiratory chain was thus heterologously reconstituted in the E. coli membranes. The membranes that contained SNDH showed the activity of sorbosone:ubiquinone analogue oxidoreductase. These results suggest that the natural electron acceptor for SNDH is membranous ubiquinone, and it functions as the primary dehydrogenase in the sorbosone oxidation respiratory chain in Ga. liquefaciens. A biotransformation experiment showed l-sorbosone oxidation to 2KGLA in a nearly quantitative manner. Phylogenetic analysis for prokaryotic SNDH homologues revealed that they are found only in the Proteobacteria phylum and those of the Acetobacteraceae family are clustered in a group where all members possess a transmembrane segment. A three-dimensional structure model of the SNDH constructed with an in silico fold recognition method was similar to the crystal structure of the PQQ-dependent pyranose dehydrogenase from Coprinopsis cinerea. The structural similarity suggests a reaction mechanism under which PQQ participates in l-sorbosone oxidation.

Synthesis of the alternative sweetener 5-ketofructose from sucrose by fructose dehydrogenase and invertase producing Gluconobacter strains

A promising alternative to high-calorie sugars and artificial sweeteners is the microbially produced fructose derivative 5-ketofructose (5-KF). The key enzyme for biotransformation, fructose dehydrogenase (Fdh), was overproduced in Gluconobacter (G.) oxydans and G. japonicus LMG 26773. Furthermore, the fdh genes were integrated into the chromosome of G. oxydans (G. oxydans Δmgdh::fdh). All mutants showed high fructose oxidation rates forming 5-KF. G. japonicus LMG 26773 fdh was selected for 5-KF production from the cost-efficient and renewable feedstock sucrose because the organism possessed both, a highly active Fdh and an enzyme able to cleave sucrose. However, 5-KF yield was low because the strain formed levan and consumed 5-KF in the second growth phase. Several Gluconobacter strains were screened for sucrose-hydrolyzing enzymes. One of these proteins (Inv1417) was characterized and it was found that the enzyme showed the highest specific activity compared to all mesophilic invertases described so far (Vmax = 2295 ± 243 U mg protein−1). The corresponding gene was expressed in G. oxydans Δmgdh::fdh. The results clearly indicated that both heterologously produced enzymes Fdh and Inv1417 were active in this single-strain system for 5-KF synthesis. Overall 84 ± 2% of the available fructose units of sucrose were converted to 5-KF.

Efficient glycerol transformation by resting Gluconobacter cells.

In the present work, glycerol biotransformation using Gluconobacter strains was studied with a process intensification perspective that facilitated the development of a cleaner and more efficient technology from those previously reported. Starting from the industrial by‐product, crude glycerol, resting cells of Gluconobacter frateurii and Gluconobacter oxydans were able to convert glycerol under batch reactor conditions in water with no other additive but for the substrate. The study of strains, biomass:solution ratio, pH, growth stage, and simplification of media composition in crude glycerol bioconversions facilitated productivities of glyceric acid of 0.03 g/L.h and 2.07 g/L.h (71.5 g/g % pure by NMR) of dihydroxyacetone (DHA). Productivities surmounted recent reported fermentative bioconversions of crude glycerol and were unprecedented for the use of cell suspended solely in water. This work proposes a novel approach that allows higher productivities, cleaner production, and reduction in water and energy consumption, and demonstrates the applicability of the proposed approach.

Integrative process for sugarcane bagasse biorefinery to co-produce xylooligosaccharides and gluconic acid

An integrated and green process for co-producing xylooligosaccharides (XOS) and gluconic acid (GA), was developed by utilizing sugarcane bagasse as starting material. In this study, the highest XOS yield of 39.1% obtained from the prehydrolysis was achieved with 10% acetic acid at 150 °C for 45 min. Subsequently, 88.6% conversion of cellulose was achieved in a fed-batch enzymatic hydrolysis using a solid loading of 15%. Results of glucose fermentation suggested that inherent regulatory system of strain Gluconobacter oxydans ATCC 621H boosted GA accumulation without the requirement of pH control, leading to a good 96.3% of GA yield. Great performance of this strain offer an economically feasible option for the large-scale sustainable GA production from biomass. Overall, approximately 105 g XOS and 340 g GA were co-produced from 1 kg of dried sugarcane bagasse as feedstock; this integrated process might be a cost-effective option for the comprehensive utilization of sugarcane bagasse.

Introducing a thermotolerant Gluconobacter japonicus strain, potentially useful for coenzyme Q10 production

In this report, Gluconobacter strains were screened for coenzyme Q10 (CoQ10) production. A thermotolerant strain, Gluconobacter japonicus FM10, was eventually employed for CoQ10 production optimization. To do so, a two-step optimization strategy was used. The first step focused on biomass increase and the second step focused on increase in CoQ10 production. Factors including temperature, pH, carbon, and nitrogen sources were optimized at the first step, and temperature, pH, and aeration were optimized at the second step. The batch culture fermentation was used with the optimized factors of the first phase (30°C, pH6.5, D-sorbitol, and yeast extract-peptone as the carbon and nitrogen sources). After 18h, the temperature, pH, and aeration were shifted to the optimized values of the second step (36°C, pH7, and no aeration). By this strategy, the dry cell mass (17.1g/L) and CoQ10 (23.2mg/L) were obtained after 20h, which the latter was 2.3 times higher than that of the first step of optimization. Among the conditions tested, carbon source was the most important factor on the cell growth at the first step while no aeration was the key factor for CoQ10 production in the second step of optimization.

Characterization of Bacterial Cellulose Produced using Media Containing Waste Apple Juice

The present study involves bacterial cellulose (BC) production using freshly prepared apple juice medium (AJM) and the bacterial strain Gluconobacter xylinum CCM 3611T. The AJM was modified with ammonium sulfate, dipotassium phosphate, sucrose, acetic acid, with and without ethanol. BC sheets (in the dry state) were analyzed on the basis of morphological, rheological and structural properties, thermal stability, water holding capacity (WHC) and water absorption capacity (WAC). Comparative X-ray diffractograms of BC using cobalt radiation is observed for the first time. The WAC analysis revealed that lyophilized BC samples had the higher WAC than the oven air-dried samples. There is an evidential structural difference observed in BC prepared from different AJM. Moreover, the AJM modified with only ethanol, exhibited quite a significant yield of BC. BC produced from the medium without ethanol had the highest thermal stability, viscoelasticity, and WHC.

Improved heterologous expression of the membrane-bound quinoprotein quinate dehydrogenase from Gluconobacter oxydans

Gluconobacter oxydans produces 3-dehydroquinate by oxidation of quinate through a reaction catalyzed by the quinate dehydrogenase (QDH), membrane-bound, pyrroloquinoline quinone (PQQ)-dependent dehydrogenase. We previously reported the nucleotide and deduced amino acid sequence of QDH and constructed a heterologous expression system of QDH in Pseudomonas sp. (A.S. Vangnai, W. Promden, W. De-Eknamkul, K. Matsushita, H. Toyama, Biochemistry (Moscow) 75:452–459, 2010). Through this study, we aim to update the sequences of QDH and improve the heterologous expression of QDH in Gluconobacter strains using a broad-host-range plasmid. Expression of QDH using a plasmid containing a long 5′-UTR was higher than that using a plasmid with a short 5′-UTR. In addition, the usage of the putative promoter region of the membrane-bound, alcohol dehydrogenase (ADH) of Gluconobacter resulted in higher expression levels compared to the usage of the lacZ promoter. Base substitution experiments allowed to identify the correct TTG initiation codon between two possibilities, and the result of these experiments were consistent with the N-terminal amino acid sequence of the expressed QDH. However, change of the TTG codon to ATG did not increase QDH expression. Therefore, the optimal plasmid for QDH expression included the structural gene with a long 5′-UTR and the ADH promoter. Cell membrane of the recombinant Gluconobacter strain presented approximately 10-times higher specific QDH activity than that observed in the wild-type strain.

Production of 5-ketofructose from fructose or sucrose using genetically modified Gluconobacter oxydans strains.

The growing consumer demand for low-calorie, sugar-free foodstuff motivated us to search for alternative non-nutritive sweeteners. A promising sweet-tasting compound is 5-keto-D-fructose (5-KF), which is formed by membrane-bound fructose dehydrogenases (Fdh) in some Gluconobacter strains. The plasmid-based expression of the fdh genes in Gluconobacter (G.) oxydans resulted in a much higher Fdh activity in comparison to the native host G. japonicus. Growth experiments with G. oxydans fdh in fructose-containing media indicated that 5-KF was rapidly formed with a conversion efficiency of 90%. 5-KF production from fructose was also observed using resting cells with a yield of about 100%. In addition, a new approach was tested for the production of the sweetener 5-KF by using sucrose as a substrate. To this end, a two-strain system composed of the fdh-expressing strain and a G. oxydans strain that produced the sucrose hydrolyzing SacC was developed. The strains were co-cultured in sucrose medium and converted 92.5% of the available fructose units into 5-KF. The glucose moiety of sucrose was converted to 2-ketogluconate and acetate. With regard to the development of a sustainable and resource-saving process for the production of 5-KF, sugar beet extract was used as substrate for the two-strain system. Fructose as product from sucrose cleavage was mainly oxidized to 5-KF which was detected in a concentration of over 200mM at the end of the fermentation process. In summary, the two-strain system was able to convert fructose units of sugar beet extract to 5-KF with an efficiency of 82 ± 5%.

Expression of membrane-bound dehydrogenases from a mother of vinegar metagenome in Gluconobacter oxydans.

Acetic acid bacteria are well-known for their membrane-bound dehydrogenases rapidly oxidizing a variety of substrates in the periplasm. Since many acetic acid bacteria have not been successfully cultured in the laboratory yet, studying membrane-bound dehydrogenases directly from a metagenome of vinegar microbiota seems to be a promising way to identify novel variants of these enzymes. To this end, DNA from a mother of vinegar was isolated, sequenced, and screened for membrane-bound dehydrogenases using an in silico approach. Six metagenomic dehydrogenases were successfully expressed using an expression vector with native promoters in the acetic acid bacterium strain Gluconobacter oxydans BP.9, which is devoid of its major native membrane-bound dehydrogenases. Determining the substrates converted by these enzymes, using a whole-cell DCPIP assay, revealed one glucose dehydrogenase with an enlarged substrate spectrum additionally oxidizing aldoheptoses, D-ribose and aldotetroses, one polyol dehydrogenase with an extreme diminished spectrum but distinguishing cis and trans-1,2-cyclohexandiol and a completely new secondary alcohol dehydrogenase, which oxidizes secondary alcohols with a hydroxyl group at position 2, as long as no primary hydroxyl group is present. Three further dehydrogenases were found with substrate spectra similar to known dehydrogenases of G. oxydans 621H.