Expanding the repertoire of counterselection markers for markerless gene deletion in the human gut bacterium Phocaeicola vulgatus.

OBJECTIVE: Phocaeicolavulgatus (formerly Bacteroides vulgatus) is a highly abundant and ubiquitous member of the human gut microbiota, associated with human health and disease, and therefore represents an important target for further investigations. In this study a novel gene deletion method was developed for P. vulgatus, expanding the tools available for genetic manipulation of members of the microbial order Bacteroidales. MATERIAL AND METHODS: The study used a combination of bioinformatics and growth experiments in interaction with molecular cloning to validate the applicability of SacB as a counterselection marker in P. vulgatus. RESULTS: In this study, the levansucrase gene sacB from Bacillussubtilis was verified as a functional counterselection marker for P. vulgatus, conferring a lethal sensitivity towards sucrose. Markerless gene deletion based on SacB was applied to delete a gene encoding a putative endofructosidase (BVU1663). The P. vulgatus Δbvu1663 deletion mutant displayed no biomass formation when grown on levan, inulin or their corresponding fructooligosaccharides. This system was also applied for the deletion of the two genes bvu0984 and bvu3649, which are involved in the pyrimidine metabolism. The resulting P. vulgatus Δ0984 Δ3649 deletion mutant no longer showed sensitivity for the toxic pyrimidine analogon 5-fluorouracil, allowing a counterselection with this compound in the double knockout strain. CONCLUSION: The genetic toolbox for P. vulgatus was expanded by a markerless gene deletion system based on SacB as an efficient counterselection marker. The system was employed to successfully delete three genes in P. vulgatus which all resulted in expected phenotypes as confirmed by subsequent growth experiments.

Genetic tools for the redirection of the central carbon flow towards the production of lactate in the human gut bacterium Phocaeicola (Bacteroides) vulgatus.

Species of the genera Bacteroides and Phocaeicola play an important role in the human colon. The organisms contribute to the degradation of complex heteropolysaccharides to small chain fatty acids, which are in part utilized by the human body. Furthermore, these organisms are involved in the synthesis of vitamins and other bioactive compounds. Of special interest is Phocaeicola vulgatus, originally classified as a Bacteroides species, due to its abundance in the human intestinal tract and its ability to degrade many plant-derived heteropolysaccharides. We analyzed different tools for the genetic modification of this microorganism, with respect to homologous gene expression of the ldh gene encoding a D-lactate dehydrogenase (LDH). Therefore, the ldh gene was cloned into the integration vector pMM656 and the shuttle vector pG106 for homologous gene expression in P. vulgatus. We determined the ldh copy number, transcript abundance, and the enzyme activity of the wild type and the mutants. The strain containing the shuttle vector showed an approx. 1500-fold increase in the ldh transcript concentration and an enhanced LDH activity that was about 200-fold higher compared to the parental strain. Overall, the proportion of lactate in the general catabolic carbon flow increased from 2.9% (wild type) to 28.5% in the LDH-overproducing mutant. This approach is a proof of concept, verifying the genetic accessibility of P. vulgatus and could form the basis for targeted genetic optimization. KEY POINTS: • A lactate dehydrogenase was overexpressed in Phocaeicola (Bacteroides) vulgatus. • The ldh transcript abundance and the LDH activity increased sharply in the mutant. • The proportion of lactate in the catabolic carbon flow increased to about 30%.

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.

Physiology and central carbon metabolism of the gut bacterium Prevotella copri.

The human gut microbiota is a crucial factor for the host's physiology with respect to health and disease. Metagenomic shotgun sequencing of microbial gut communities revealed that Prevotella copri is one of the most important players in the gastrointestinal tract of many individuals. Because of the importance of this bacterium we analyzed the growth behavior and the central metabolic pathways of P. copri. Bioinformatic data, transcriptome profiling and enzyme activity measurements indicated that the major pathways are based on glycolysis and succinate production from fumarate. In addition, pyruvate can be degraded to acetate and formate. Electron transport phosphorylation depends on fumarate respiration with NADH and reduced ferredoxin as electron donors. In contrast to Bacteroides vulgatus, P. copri showed a more pronounced dependency on the addition of CO2 or bicarbonate for biomass formation, which is a remarkable difference between P. copri and Bacteroides spp. with important implication in the context of gut microbial competition. The analysis of substrate consumption and product concentrations from many P. copri cultures with different optical densities allowed a prediction of the carbon and electron flow in the central metabolism and a detailed calculation of growth yields as well as carbon and redox balances.

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 200 mM 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%.

Growth Characteristics of Methanomassiliicoccus luminyensis and Expression of Methyltransferase Encoding Genes.

DNA sequence analysis of the human gut revealed the presence a seventh order of methanogens referred to as Methanomassiliicoccales. Methanomassiliicoccus luminyensis is the only member of this order that grows in pure culture. Here, we show that the organism has a doubling time of 1.8 d with methanol + H2 and a growth yield of 2.4 g dry weight/mol CH4. M. luminyensis also uses methylamines + H2 (monomethylamine, dimethylamine, and trimethylamine) with doubling times of 2.1-2.3 d. Similar cell yields were obtained with equimolar concentrations of methanol and methylamines with respect to their methyl group contents. The transcript levels of genes encoding proteins involved in substrate utilization indicated increased amounts of mRNA from the mtaBC2 gene cluster in methanol-grown cells. When methylamines were used as substrates, mRNA of the mtb/mtt operon and of the mtmBC1 cluster were found in high abundance. The transcript level of mtaC2 was almost identical in methanol- and methylamine-grown cells, indicating that genes for methanol utilization were constitutively expressed in high amounts. The same observation was made with resting cells where methanol always yielded the highest CH4 production rate independently from the growth substrate. Hence, M. luminyensis is adapted to habitats that provide methanol + H2 as substrates.

BEAP profiles as rapid test system for status analysis and early detection of process incidents in biogas plants.

A method was developed to quantify the performance of microorganisms involved in different digestion levels in biogas plants. The test system was based on the addition of butyrate (BCON), ethanol (ECON), acetate (ACON) or propionate (PCON) to biogas sludge samples and the subsequent analysis of CH4 formation in comparison to control samples. The combination of the four values was referred to as BEAP profile. Determination of BEAP profiles enabled rapid testing of a biogas plant's metabolic state within 24 h and an accurate mapping of all degradation levels in a lab-scale experimental setup. Furthermore, it was possible to distinguish between specific BEAP profiles for standard biogas plants and for biogas reactors with process incidents (beginning of NH4+-N inhibition, start of acidification, insufficient hydrolysis and potential mycotoxin effects). Finally, BEAP profiles also functioned as a warning system for the early prediction of critical NH4+-N concentrations leading to a drop of CH4 formation.

Methanosarcina flavescens sp. nov., a methanogenic archaeon isolated from a full-scale anaerobic digester.

A novel, strictly anaerobic, methanogenic archaeon, strain E03.2T, was isolated from a full-scale biogas plant in Germany. Cells were non-motile sarcina-like cocci, occurring in aggregates. Strain E03.2T grew autotrophically on H2 plus CO2, and additionally cells could utilize acetate, methanol, moni-, di- and trimethylamine as carbon and energy sources; however, growth or methanogenesis on formate was not observed. Yeast extract and vitamins stimulated growth but were not mandatory. The optimal growth temperature of strain E03.2T was approximately 45 °C; maximal growth rates were obtained at about pH 7.0 in the presence of approximately 6.8 mM NaCl. The DNA G+C content of strain E03.2T was 41.3 mol%. Phylogenetic analyses based on 16S rRNA gene and mcrA sequences placed strain E03.2T within the genus Methanosarcina. Based on 16S rRNA gene sequence similarity strain E03.2T was related to seven different species of the genus Methanosarcina, but most closely related to Methanosarcina thermophila TM-1T. Phenotypic, physiological and genomic characteristics indicated that strain E03.2T represents a novel species of the genus Methanosarcina, for which the name Methanosarcina flavescens sp. nov. is proposed. The type strain is E03.2T ( = DSM 100822T = JCM 30921T).

Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans.

Gluconobacter (G.) oxydans is able to incompletely oxidize various sugars and polyols for the production of biotechnologically important compound. Recently, we have shown that the organism produces and accumulates mannitol as compatible solute under osmotic stress conditions. The present study describes the role of two cytoplasmic mannitol dehydrogenases for osmotolerance of G. oxydans. It was shown that Gox1432 is a NADP+-dependent mannitol dehydrogenase (EC 1.1.1.138), while Gox0849 uses NAD+ as cofactor (EC 1.1.1.67). The corresponding genes were deleted and the mutants were analyzed for growth under osmotic stress and non-stress conditions. A severe growth defect was detected for Δgox1432 when grown in high osmotic media, while the deletion of gox0849 had no effect when cells were exposed to 450 mM sucrose in the medium. Furthermore, the intracellular mannitol content was reduced in the mutant lacking the NADP+-dependent enzyme Gox1432 in comparison to the parental strain and the Δgox0849 mutant under stress conditions. In addition, transcriptional analysis revealed that Gox1432 is more important for mannitol production in G. oxydans than Gox0849 as the transcript abundance of gene gox1432 was 30-fold higher than of gox0849. In accordance, the activity of the NADH-dependent enzyme Gox0849 in the cell cytoplasm was 10-fold lower in comparison to the NADPH-dependent mannitol dehydrogenase Gox1432. Overexpression of gox1432 in the corresponding deletion mutant restored growth of the cells under osmotic stress, further strengthening the importance of the NADP+-dependent mannitol dehydrogenase for osmotolerance in G. oxydans. These findings provide detailed insights into the molecular mechanism of mannitol-mediated osmoprotection in G. oxydans and are helpful engineering strains with improved osmotolerance for biotechnological applications.

Extracellular targeting of an active endoxylanase by a TolB negative mutant of Gluconobacter oxydans.

Gluconobacter (G.) oxydans strains have great industrial potential due to their ability to incompletely oxidize a wide range of carbohydrates. But there is one major limitation preventing their full production potential. Hydrolysis of polysaccharides is not possible because extracellular hydrolases are not encoded in the genome of Gluconobacter species. Therefore, as a first step for the generation of exoenzyme producing G. oxydans, a leaky outer membrane mutant was created by deleting the TolB encoding gene gox1687. As a second step the xynA gene encoding an endo-1,4-ß-xylanase from Bacillus subtilis was expressed in G. oxydans ΔtolB. More than 70 % of the total XynA activity (0.91 mmol h(-1) l culture(-1)) was detected in the culture supernatant of the TolB mutant and only 10 % of endoxylanase activity was observed in the supernatant of G. oxydans xynA. These results showed that a G. oxydans strain with an increased substrate spectrum that is able to use the renewable polysaccharide xylan as a substrate to produce the prebiotic compounds xylobiose and xylooligosaccharides was generated. This is the first report about the combination of the process of incomplete oxidation with the degradation of renewable organic materials from plants for the production of value-added products.

Bioenergetics and anaerobic respiratory chains of aceticlastic methanogens.

Methane-forming archaea are strictly anaerobic microbes and are essential for global carbon fluxes since they perform the terminal step in breakdown of organic matter in the absence of oxygen. Major part of methane produced in nature derives from the methyl group of acetate. Only members of the genera Methanosarcina and Methanosaeta are able to use this substrate for methane formation and growth. Since the free energy change coupled to methanogenesis from acetate is only -36kJ/mol CH4, aceticlastic methanogens developed efficient energy-conserving systems to handle this thermodynamic limitation. The membrane bound electron transport system of aceticlastic methanogens is a complex branched respiratory chain that can accept electrons from hydrogen, reduced coenzyme F420 or reduced ferredoxin. The terminal electron acceptor of this anaerobic respiration is a mixed disulfide composed of coenzyme M and coenzyme B. Reduced ferredoxin has an important function under aceticlastic growth conditions and novel and well-established membrane complexes oxidizing ferredoxin will be discussed in depth. Membrane bound electron transport is connected to energy conservation by proton or sodium ion translocating enzymes (F420H2 dehydrogenase, Rnf complex, Ech hydrogenase, methanophenazine-reducing hydrogenase and heterodisulfide reductase). The resulting electrochemical ion gradient constitutes the driving force for adenosine triphosphate synthesis. Methanogenesis, electron transport, and the structure of key enzymes are discussed in this review leading to a concept of how aceticlastic methanogens make a living. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Succinic semialdehyde reductase Gox1801 from Gluconobacter oxydans in comparison to other succinic semialdehyde-reducing enzymes.

Gluconobacter oxydans is an industrially important bacterium that possesses many uncharacterized oxidoreductases, which might be exploited for novel biotechnological applications. In this study, gene gox1801 was homologously overexpressed in G. oxydans and it was found that the relative expression of gox1801 was 13-fold higher than that in the control strain. Gox1801 was predicted to belong to the 3-hydroxyisobutyrate dehydrogenase-type proteins. The purified enzyme had a native molecular mass of 134 kDa and forms a homotetramer. Analysis of the enzymatic activity revealed that Gox1801 is a succinic semialdehyde reductase that used NADH and NADPH as electron donors. Lower activities were observed with glyoxal, methylglyoxal, and phenylglyoxal. The enzyme was compared to the succinic semialdehyde reductase GsSSAR from Geobacter sulfurreducens and the γ-hydroxybutyrate dehydrogenase YihU from Escherichia coli K-12. The comparison revealed that Gox1801 is the first enzyme from an aerobic bacterium reducing succinic semialdehyde with high catalytic efficiency. As a novel succinic semialdehyde reductase, Gox1801 has the potential to be used in the biotechnological production of γ-hydroxybutyrate.

Identification of mannitol as compatible solute in Gluconobacter oxydans.

Gluconobacter oxydans is an industrially important bacterium owing to its regio- and enantio-selective incomplete oxidation of various sugars, alcohols, and polyols. The complete genome sequence is available, but it is still unknown how the organism adapts to highly osmotic sugar-rich environments. Therefore, the mechanisms of osmoprotection in G. oxydans were investigated. The accumulation and transport of solutes are hallmarks of osmoadaptation. To identify potential osmoprotectants, G. oxydans was grown on a yeast glucose medium in the presence of 100 mM potassium phosphate (pH 7.0) along with various concentrations of sucrose (0-600 mM final concentration), which was not metabolized. Intracellular metabolites were analyzed by HPLC and (13)C NMR spectroscopy under stress conditions. Both of these analytical techniques highlighted the accumulation of mannitol as a potent osmoprotectant inside the stressed cells. This intracellular mannitol accumulation correlated with increased extracellular osmolarity of the medium. For further confirmation, the growth behavior of G. oxydans was analyzed in the presence of small amounts of mannitol (2.5-10 mM) and 300 mM sucrose. Growth under sucrose-induced osmotic stress conditions was almost identical to control growth when exogenous mannitol was added in low amounts. Thus, mannitol alleviates the osmotic stress of sucrose on cellular growth. Moreover, the positive effect of exogenous mannitol on the rate of glucose consumption and gluconate formation was also monitored. These results may be helpful to optimize the processes of industrial product formation in highly concentrated sugar solutions.

Acquisition of 1,000 eubacterial genes physiologically transformed a methanogen at the origin of Haloarchaea.

Archaebacterial halophiles (Haloarchaea) are oxygen-respiring heterotrophs that derive from methanogens--strictly anaerobic, hydrogen-dependent autotrophs. Haloarchaeal genomes are known to have acquired, via lateral gene transfer (LGT), several genes from eubacteria, but it is yet unknown how many genes the Haloarchaea acquired in total and, more importantly, whether independent haloarchaeal lineages acquired their genes in parallel, or as a single acquisition at the origin of the group. Here we have studied 10 haloarchaeal and 1,143 reference genomes and have identified 1,089 haloarchaeal gene families that were acquired by a methanogenic recipient from eubacteria. The data suggest that these genes were acquired in the haloarchaeal common ancestor, not in parallel in independent haloarchaeal lineages, nor in the common ancestor of haloarchaeans and methanosarcinales. The 1,089 acquisitions include genes for catabolic carbon metabolism, membrane transporters, menaquinone biosynthesis, and complexes I-IV of the eubacterial respiratory chain that functions in the haloarchaeal membrane consisting of diphytanyl isoprene ether lipids. LGT on a massive scale transformed a strictly anaerobic, chemolithoautotrophic methanogen into the heterotrophic, oxygen-respiring, and bacteriorhodopsin-photosynthetic haloarchaeal common ancestor.

Short-term effect of acetate and ethanol on methane formation in biogas sludge.

Biochemical processes in biogas plants are still not fully understood. Especially, the identification of possible bottlenecks in the complex fermentation processes during biogas production might provide potential to increase the performance of biogas plants. To shed light on the question which group of organism constitutes the limiting factor in the anaerobic breakdown of organic material, biogas sludge from different mesophilic biogas plants was examined under various conditions. Therefore, biogas sludge was incubated and analyzed in anaerobic serum flasks under an atmosphere of N2/CO2. The batch reactors mirrored the conditions and the performance of the full-scale biogas plants and were suitable test systems for a period of 24 h. Methane production rates were compared after supplementation with substrates for syntrophic bacteria, such as butyrate, propionate, or ethanol, as well as with acetate and H2+CO2 as substrates for methanogenic archaea. Methane formation rates increased significantly by 35 to 126 % when sludge from different biogas plants was supplemented with acetate or ethanol. The stability of important process parameters such as concentration of volatile fatty acids and pH indicate that ethanol and acetate increase biogas formation without affecting normally occurring fermentation processes. In contrast to ethanol or acetate, other fermentation products such as propionate, butyrate, or H2 did not result in increased methane formation rates. These results provide evidence that aceticlastic methanogenesis and ethanol-oxidizing syntrophic bacteria are not the limiting factor during biogas formation, respectively, and that biogas plant optimization is possible with special focus on methanogenesis from acetate.

Characterization of a periplasmic quinoprotein from Sphingomonas wittichii that functions as aldehyde dehydrogenase.

The α-proteobacterium Sphingomonas wittichii RW1 is known for its ability to degrade dioxins and related toxic substances. Bioinformatic analysis of the genome indicated that this organism may contain the largest number of pyrroloquinoline quinone-dependent dehydrogenases of any bacteria sequenced so far. Sequence analysis also showed that one of these genes (swit_4395) encodes an enzyme that belongs to the class of periplasmic glucose dehydrogenases. This gene was fused to a pelB signal sequence and a strep-tag coding region at the 5' and 3' ends, respectively. The fusion product was cloned into the broad-host range expression vector pBBR1p264-Streplong and the corresponding protein was heterologously produced in Escherichia coli, purified via Strep-Tactin affinity chromatography, and characterized. The protein Swit_4395 had a subunit mass of 39.3 kDa and formed active homooctamers and homododecamers. The enzyme showed the highest activities with short- and medium-chain aldehydes (chain length C1-C6) and ketoaldehydes, such as methylglyoxal and phenylglyoxal. Butyraldehyde was the best substrate, with V max and apparent K M values of 3,970 U/mg protein and 12.3 mM, respectively. Pyrroloquinoline quinone was detected using UV-Vis spectroscopy and was found to be a prosthetic group of the purified enzyme. Therefore, Swit_4395 was identified as a pyrroloquinoline quinone-dependent aldehyde dehydrogenase. The enzyme could be purified from the native host when the expression vector was introduced into S. wittichii RW1, indicating homologous protein production. Overproduction of Swit_4395 in S. wittichii RW1 dramatically increased the tolerance of the bacterium toward butyraldehyde and thus might contribute to the detoxification of toxic aldehydes.

Increase of methane formation by ethanol addition during continuous fermentation of biogas sludge.

Very recently, it was shown that the addition of acetate or ethanol led to enhanced biogas formation rates during an observation period of 24 h. To determine if increased methane production rates due to ethanol addition can be maintained over longer time periods, continuous reactors filled with biogas sludge were developed which were fed with the same substrates as the full-scale reactor from which the sludge was derived. These reactors are well reflected conditions of a full-scale biogas plant during a period of 14 days. When the fermenters were pulsed with 50-100 mM ethanol, biomethanation increased by 50-150 %, depending on the composition of the biogas sludge. It was also possible to increase methane formation significantly when 10-20 mM pure ethanol or ethanolic solutions (e.g. beer) were added daily. In summary, the experiments revealed that "normal" methane production continued to take place, but ethanol led to production of additional methane.

Effects of membrane-bound glucose dehydrogenase overproduction on the respiratory chain of Gluconobacter oxydans.

The acetic acid bacterium Gluconobacter oxydans incompletely oxidizes carbon sources as a natural part of its metabolism, and this feature has been exploited for many biotechnological applications. The most important enzymes used to harness the biocatalytic oxidative capacity of G. oxydans are the pyrroloquinoline quinone (PQQ)-dependent dehydrogenases. The membrane-bound PQQ-dependent glucose dehydrogenase (mGDH), encoded by gox0265, was used as model protein for homologous membrane protein production using the previously described Gluconobacter expression vector pBBR1p452. The mgdh gene had ninefold higher expression in the overproduction strain compared to the parental strain. Furthermore, membranes from the overexpression strain had a five- and threefold increase of mGDH activity and oxygen consumption rates, respectively. Oxygen consumption rate of the membrane fraction could not be increased by the addition of a substrate combination of glucose and ethanol in the overproduction strain, indicating that the terminal quinol oxidases of the respiratory chain were rate limiting. In contrast, addition of glucose and ethanol to membranes of the control strain increased oxygen consumption rates approaching the observed rates with G. oxydans overproducing mGDH. The higher glucose oxidation rates of the mGDH overproduction strain corresponded to a 70 % increase of the gluconate production rate compared to the control strain. The high rate of glucose oxidation may be useful in the industrial production of gluconates and ketogluconates, or as whole-cell biosensors. Furthermore, mGDH was purified to homogeneity by one-step strep-tactin affinity chromatography and characterized. To our knowledge, this is the first report of a membrane integral quinoprotein being purified by affinity chromatography and serves as a proof-of-principle for using G. oxydans as a host for membrane protein expression and purification.

Asymmetric reduction of diketones by two Gluconobacter oxydans oxidoreductases.

Two genes encoding recombinant cytosolic oxidoreductases from Gluconobacter oxydans, gox0313 and gox0646, were heterologously expressed in Escherichia coli and the resulting proteins were purified and characterized. GOX0313 was identified as a medium-chain alcohol dehydrogenase, whereas GOX0646 was classified as a ketocarbonyl reductase. GOX0313 had a broad substrate spectrum and oxidized various primary alcohols. However, GOX0313 had a preference for substrate reduction, reducing many aldehydes and α-diketones. In contrast, GOX0646 had a narrow substrate spectrum and reduced α-diketones, preferring short-chain ketocarbonyls. Both enzymes regio- and stereospecifically reduced α-diketones to the corresponding (S)-hydroxy ketone, as shown by NMR. These products are difficult to produce chemically, requiring complicated protecting group chemistry. Furthermore, hydroxy ketones find industrial application in the production of pheromones, fragrances, flavors, and pharmaceuticals. Hence, these enzymes are interesting biocatalysts for the production of enantiomerically pure building blocks that are difficult to prepare chemically.

Acetate activation in Methanosaeta thermophila: characterization of the key enzymes pyrophosphatase and acetyl-CoA synthetase.

The thermophilic methanogen Methanosaeta thermophila uses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction in Methanosarcina sp. is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction in Mt. thermophila seems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl-CoA, AMP, and pyrophosphate (PP(i)) and was only moderately inhibited by PP(i). The breakdown of PP(i) was performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part of PP(i) (K(M) = 0.27 ± 0.05 mM) that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides or PP(i). However, it cannot be excluded that other PP(i)-dependent enzymes take advantage of the remaining PP(i) and contribute to the energy balance of the cell.