Distinct Physiological Roles of Two Membrane-Bound Dehydrogenases Responsible for D-Sorbitol Oxidation in Gluconobacter frateurii

Two different membrane-bound enzymes oxidizing D-sorbitol are found in Gluconobacter frateurii THD32: pyroloquinoline quinone-dependent glycerol dehydrogenase (PQQ-GLDH) and FAD-dependent D-sorbitol dehydrogenase (FAD-SLDH). In this study, FAD-SLDH appeared to be induced by L-sorbose. A mutant defective in both enzymes grew as well as the wild-type strain did, indicating that both enzymes are dispensable for growth on D-sorbitol. The strain defective in PQQ-GLDH exhibited delayed L-sorbose production, and lower accumulation of it, corresponding to decreased oxidase activity for D-sorbitol in spite of high D-sorbitol dehydrogenase activity, was observed. In the mutant strain defective in PQQ-GLDH, oxidase activity with D-sorbitol was much more resistant to cyanide, and the H⁺/O ratio was lower than in either the wild-type strain or the mutant strain defective in FAD-SLDH. These results suggest that PQQ-GLDH connects efficiently to cytochrome bo ₃ terminal oxidase and that it plays a major role in L-sorbose production. On the other hand, FAD-SLDH linked preferably to the cyanide-insensitive terminal oxidase, CIO.     

Molecular properties of membrane-bound FAD-containing D-sorbitol dehydrogenase from thermotolerant Gluconobacter frateurii isolated from Thailand

There are two types of membrane-bound D-sorbitol dehydrogenase (SLDH) reported: PQQ-SLDH, having pyrroloquinoline quinone (PQQ), and FAD-SLDH, containing FAD and heme c as the prosthetic groups. FAD-SLDH was purified and characterized from the PQQ-SLDH mutant strain of a thermotolerant Gluconobacter frateurii, having molecular mass of 61.5 kDa, 52 kDa, and 22 kDa. The enzyme properties were quite similar to those of the enzyme from mesophilic G. oxydans IFO 3254. This enzyme was shown to be inducible by D-sorbitol, but not PQQ-SLDH. The oxidation product of FAD-SLDH from D-sorbitol was identified as L-sorbose. The cloned gene of FAD-SLDH had three open reading frames (sldSLC) corresponding to the small, the large, and cytochrome c subunits of FAD-SLDH respectively. The deduced amino acid sequences showed high identity to those from G. oxydans IFO 3254: SldL showed to other FAD-enzymes, and SldC having three heme c binding motives to cytochrome c subunits of other membrane-bound dehydrogenases.     

Optimized synthesis of L-sorbose by C(5)-dehydrogenation of D-sorbitol with Gluconobacter oxydans

The optimization of L-sorbose synthesis by regiospecific dehydrogenation of D-sorbitol using Gluconobacter oxydans is reported. The current L-sorbose production processes that are based on G. oxydans and other bacterial strains are suboptimal as to yield and rate of L-sorbose synthesis. One reason for these problems is the toxicity that is induced by the substrate D-sorbitol when used in concentrations of >10% (w/v). This phenomenon significantly limits the potentials of L-sorbose production from an industrial point of view. The goal of this study was to develop a fast production process that yields L-sorbose in stoichiometric amounts starting from D-sorbitol concentrations that exceed 10% (w/v). A gradual improvement of the inoculum build-up procedure, culture medium composition, and process parameters ultimately led to a theoretically maximal L-sorbose productivity (200 g L(-1) of L-sorbose from 200 g L(-1) of D-sorbitol in 28 h of fermentation) using a Gluconobacter oxydans mutant strain that was selected under conditions of substrate inhibition. Because the D-sorbitol/L‐sorbose bioconversion is used to mass-produce vitamin C, the procedure reported here will contribute to a more efficient and more economic synthesis of vitamin C.     

Isolation and characterization of thermotolerant Gluconobacter strains catalyzing oxidative fermentation at higher temperatures

Thermotolerant acetic acid bacteria belonging to the genus Gluconobacter were isolated from various kinds of fruits and flowers from Thailand and Japan. The screening strategy was built up to exclude Acetobacter strains by adding gluconic acid to a culture medium in the presence of 1% D-sorbitol or 1% D-mannitol. Eight strains of thermotolerant Gluconobacter were isolated and screened for D-fructose and L-sorbose production. They grew at wide range of temperatures from 10 degrees C to 37 degrees C and had average optimum growth temperature between 30-33 degrees C. All strains were able to produce L-sorbose and D-fructose at higher temperatures such as 37 degrees C. The 16S rRNA sequences analysis showed that the isolated strains were almost identical to G. frateurii with scores of 99.36-99.79%. Among these eight strains, especially strains CHM16 and CHM54 had high oxidase activity for D-mannitol and D-sorbitol, converting it to D-fructose and L-sorbose at 37 degrees C, respectively. Sugar alcohols oxidation proceeded without a lag time, but Gluconobacter frateurii IFO 3264T was unable to do such fermentation at 37 degrees C. Fermentation efficiency and fermentation rate of the strains CHM16 and CHM54 were quite high and they rapidly oxidized D-mannitol and D-sorbitol to D-fructose and L-sorbose at almost 100% within 24 h at 30 degrees C. Even oxidative fermentation of D-fructose done at 37 degrees C, the strain CHM16 still accumulated D-fructose at 80% within 24 h. The efficiency of L-sorbose fermentation by the strain CHM54 at 37 degrees C was superior to that observed at 30 degrees C. Thus, the eight strains were finally classified as thermotolerant members of G. frateurii.     

酮古龙酸菌山梨醇脱氢酶的原核表达及活性检测

目的：克隆酮古龙酸菌Y25的山梨醇脱氢酶基因sldh,在大肠杆菌中进行表达并检测表达产物的活性。方法：以酮古龙酸菌Y25基因组DNA为模板,PCR扩增sldh基因,连接到表达载体pTIG,转入大肠杆菌BL21（DE3）,IPTG诱导表达;取表达菌体、菌体裂解上清和沉淀进行SDS-PAGE分析;以山梨醇为底物,通过活性电泳、体外转化及休止细胞转化进行sldh基因表达产物的活性检测。结果：扩增得到1740 bp的山梨醇脱氢酶基因,构建了表达质粒pTIG-sldh并在大肠杆菌中获得表达,SDS-PAGE结果显示表达产物为可溶性形式,相对分子质量约58×10^3;活性电泳结果说明表达产物在以山梨醇为底物时表现出脱氢酶活性,而经体外转化和休止细胞转化后薄层层析检测出转化产物山梨糖的存在。结论：在大肠杆菌中实现了酮古龙酸菌山梨醇脱氢酶的可溶性表达,且表达的重组脱氢酶能将山梨醇脱氢生成山梨糖。     

Cloning and nucleotide sequencing of the membrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO 12258 and its expression in Gluconobacter oxydans.

Cloning and expression of the gene encoding Acetobacter liquefaciens IFO 12258 membrane-bound L-sorbosone dehydrogenase (SNDH) were studied. A genomic library of A. liquefaciens IFO 12258 was constructed with the mobilizable cosmid vector pVK102 (mob+) in Escherichia coli S17-1 (Tra+). The library was transferred by conjugal mating into Gluconobacter oxydans OX4, a mutant of G. oxydans IFO 3293 that accumulates L-sorbosone in the presence of L-sorbose. The transconjugants were screened for SNDH activity by performing a direct expression assay. One clone harboring plasmid p7A6 converted L-sorbosone to 2-keto-L-gulonic acid (2KGA) more rapidly than its host did and also converted L-sorbose to 2KGA with no accumulation of L-sorbosone. The insert (25 kb) of p7A6 was shortened to a 3.1-kb fragment, in which one open reading frame (1,347 bp) was found and was shown to encode a polypeptide with a molecular weight of 48,222. The SNDH gene was introduced into the 2KGA-producing strain G. oxydans IFO 3293 and its derivatives, which contained membrane-bound L-sorbose dehydrogenase. The cloned SNDH was correctly located in the membrane of the host. The membrane fraction of the clone exhibited almost stoichiometric formation of 2KGA from L-sorbosone and L-sorbose. Resting cells of the clones produced 2KGA very efficiently from L-sorbosone and L-sorbose, but not from D-sorbitol; the conversion yield from L-sorbosone was improved from approximately 25 to 83%, whereas the yield from L-sorbose was increased from 68 to 81%. Under fermentation conditions, cloning did not obviously improve the yield of 2KGA from L-sorbose.     

Main polyol dehydrogenase of Gluconobacter suboxydans IFO 3255, membrane-bound D-sorbitol dehydrogenase,that needs product of upstream gene,sldB, for activity

The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions. The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.0, indicating that the dehydrogenase was the main D-sorbitol-oxidizing enzyme in this bacterium. The cells did not produce D-fructose from D-mannitol or dihydroxyacetone from glycerol. The disruption of the sldB gene resulted in undetectable oxidation of D-sorbitol, D-mannitol, or glycerol, although the cells produced the dehydrogenase. The cells with the sldB gene disrupted produced more of what might be signal-unprocessed SldA than the wild-type cells did. SldB may be a chaperone-like component that assists signal processing and folding of the SldA polypeptide to form active D-sorbitol dehydrogenase.     

Membrane-bound D-sorbitol dehydrogenase of Gluconobacter suboxydans IFO 3255--enzymatic and genetic characterization

Gluconobacter strains effectively produce L-sorbose from D-sorbitol because of strong activity of the D-sorbitol dehydrogenase (SLDH). L-sorbose is one of the important intermediates in the industrial vitamin C production process. Two kinds of membrane-bound SLDHs, which consist of three subunits, were reportedly found in Gluconobacter strains [Agric. Biol. Chem. 46 (1982) 135,FEMS Microbiol. Lett. 125 (1995) 45]. We purified a one-subunit-type SLDH (80 kDa) from the membrane fraction of Gluconobacter suboxydans IFO 3255 solubilized with Triton X-100 in the presence of D-sorbitol, but the cofactor could not be identified from the purified enzyme. The SLDH was active on mannitol, glycerol and other sugar alcohols as well as on D-sorbitol to produce respective keto-aldoses. Then, the SLDH gene (sldA) was cloned and sequenced. It encodes the polypeptide of 740 residues, which contains a signal sequence of 24 residues. SLDH had 35-37% identity to those of membrane-bound quinoprotein glucose dehydrogenases (GDHs) from Escherichia coli, Gluconobacter oxydans and Acinetobacter calcoaceticus except the N-terminal hydrophobic region of GDH. Additionally, the sldB gene located just upstream of sldA was found to encode the polypeptide consisting of 126 very hydrophobic residues that is similar to the one-sixth N-terminal region of the GDH. Development of the SLDH activity in E. coli required co-expression of the sldA and sldB genes and the presence of PQQ. The sldA gene disruptant showed undetectable oxidation activities on D-sorbitol in growing culture, and resting-cell reaction (pH 4.5 and 7); in addition, they showed undetectable activities on D-mannitol and glycerol. The disruption of the sldB gene by a gene cassette with a downward promoter to express the sldA gene resulted in formation of a larger size of the SLDH protein and in undetectable oxidation of the polyols. In conclusion, the SLDH of the strain 3255 functions as the main polyol dehydrogenase in vivo. The sldB polypeptide possibly has a chaperone-like function to process the SLDH polypeptide into a mature and active form.     

Effect of toluene-permeabilization on oxidation of D-sorbitol to L-sorbose by Gluconobacter suboxydans cells immobilized in calcium alginate

Summary The effect of permeabilization of G. suboxydans cells with toluene on the oxidation of D-sorbitol to L-sorbose was investigated. Treatment of the cells with 10% toluene resulted in a three fold increase in the specific sorbitol dehydrogenase activity and a two fold increase in the efficiency of D-sorbitol conversion to L-sorbose of the free cell suspension. When the permeabilized cells were immobilized in calcium alginate, the operational stability during air-lift reactor operation was also found to increase with up to three times longer half-life(44 days) of catalytic activity compared with immobilized intact cells.     

Control of meta-cleavage degradation of 4-hydroxyphenylacetate in Pseudomonas putida.

Synthesis of enzymes of the 4-hydroxyphenylacetate meta-cleavage pathway was studied in Pseudomonas putida wild-type strain P23X1 (NCIB 9865) and mutant strains which had either structural or regulatory gene mutations. Induction studies with mutant strains each defective in an enzyme of the pathway showed that 4-hydroxyphenylacetate induced the hydroxylase and that 3,4-dihydroxyphenylacetate induced the 2,3-oxygenase, aldehyde dehydrogenase, isomerase, decarboxylase, and hydratase. This showed that the hydroxylase structural gene does not exist in an operon that contains any other structural gene of this meta pathway. Studies of mutant strains that synthesized constitutively the 2,3-oxygenase and subsequent enzymes suggested that the regulation of synthesis of these enzymes was coincident, and, in such strains, the hydroxylase was inducible only. Observations made with a putative polarity mutant that lacked 2,3-oxygenase activity suggested that the structural genes encoding this enzyme and subsequent enzymes of the pathway exist in the same operon. Studies of a regulatory mutant strain that was defective in the induction of the 2,3-oxygenase and subsequent enzymes suggest that the 2,3-oxygenase operon is under positive control.     

Constitutive biosynthesis and localization of alcohol oxidase in the ethanol-insensitive catabolite repression mutant ecr1 of the yeast Pichia methanolica

The activity and localization of alcohol oxidase (EC 1.1.3.13) have been studied in the Pichia methanolica mutant ecr1 defective in ethanol-induced catabolite repression of enzymes of methanol utilization. Ultrasctuctural, immunocytochemical, and biochemical analyses revealed the presence of peroxisomes containing active alcohol oxidase in the mutant grown in media with methanol, ethanol, and a mixture of both substrates. No alcohol oxidase was detected in the wild-type cells (ECR1) grown on ethanol-containing media. Mutant ecr1 growing in medium containing a mixture of different alcohols and the wild-type strain growing on methanol demonstrated similar buoyant density of peroxisomes (1.24-1.27 g/cm3)during isopicnic centrifugation of the organelles in sucrose density gradients. The integrated genetic, immunocytochemical, and biochemical data are in agreement with the model that synthesis, translocation into peroxisomes, and assembly of alcohol oxidase in P. methanolica may not require any regulatory signals induced by methanol.     

Direct fermentation of 2-keto-L-gulonic acid in recombinant Gluconobacter oxydans

We isolated Gluconobacter oxydans T-100 that had an activity to produce 2-KLGA from D-sorbitol; however, the yield of 2-KLGA was quite insufficient. Therefore, enzymes involved in the biosynthesis of L-sorbosone and 2-KLGA, L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH), respectively, were purified from G. oxydans T-100. A genomic library of G. oxydans T-100 was screened to clone both genes for SDH and SNDH based on their amino acid sequences. SNDH and SDH were encoded in sequential open reading frames with 1497 and 1596 nucleotides, respectively, which were verified by the expression in Escherichia coli. The amino acid sequence of SDH and SNDH showed close similarity with E. coli choline dehydrogenase (CDH) and betaine-aldehyde dehydrogenase (BADH), respectively, which cooperatively play a key role for conferring osmotic tolerance. Because the yield of 2-KLGA by G. oxydans introduced with the genes for SDH and SNDH were insufficient, replacement of the promoter with that of Escherichia coli tufB1 in combination with chemical mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine resulted in improvement of the production level.     

Application of random mutagenesis to enhance the production of polyhydroxyalkanoates by Cupriavidus necator H16 on waste frying oil

Using random chemical mutagenesis we obtained the mutant of Cupriavidus necator H16 which was capable of improved (about 35 %) production of poly(3-hydroxybuytrate) (PHB) compared to the wild-type strain. The mutant exhibited significantly enhanced specific activities of enzymes involved in oxidative stress response such as malic enzyme, NADP-dependent isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase. Probably, due to the activation of these enzymes, we also observed an increase of NADPH/NADP⁺ ratio. It is likely that as a side effect of the increase of NADPH/NADP⁺ ratio the activity of PHB biosynthetic pathway was enhanced, which supported the accumulation of PHB. Furthermore, the mutant was also able to incorporate propionate into copolymer poly(3-hydroxybuytyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] more efficiently than the wild-type strain (Y3HV/prec = 0.17 and 0.29 for the wild-type strain and the mutant, respectively)). We assume that it may be caused by lower availability of oxaloacetate for the utilization of propionyl-CoA in 2-methylcitrate cycle due to increased action of malic enzyme. Therefore, propionyl-CoA was incorporated into copolymer rather than transformed to pyruvate via 2-methylcitrate cycle. Thus, the mutant was capable of the utilization of waste frying oils and the production of P(3HB-co-3HV) with better yields and improved content of 3HV resulting in better mechanical properties of copolymer than the wild-type strain. The results of this work may be used for the development of innovative fermentation strategies for the production of PHA and also it might help to define novel targets for the genetic manipulations of PHA producing bacteria.     

生黑醋酸杆菌在高浓度山梨醇下的半连续发酵特性

目的：调节生黑醋酸杆菌生物和代谢特性,以提高发酵效率。方法：通过改变种液特性,采用半连续培养的方式,对生黑醋酸杆菌在高醇浓度下的生长特性进行了研究。结果：通过优化可以提高VC一步发酵底物山梨醇浓度达38%,32h左右发酵率达95%,山梨糖产量达360mg/ml,半连续培养连续5批之间产糖稳定,没有明显差别。结论：通过优化,有效地提高了山梨糖的产率。     

Multienzyme complexes of fatty acid oxidation from Escherichia coli K12 and from a mutant with a defective L-3-hydroxyacyl coenzyme A dehydrogenase

An Escherichia coli mutant (fadB64), with a defective L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) which is unable to grow on long-chain fatty acids as the sole carbon source, was shown to possess a fatty acid oxidation complex that contains five beta-oxidation enzymes, including L-3-hydroxyacyl-CoA dehydrogenase. A comparative study of the complexes from the mutant, from its parental strain and from wild-type E. coli B demonstrated the immunological and gross structural identity of all three fatty acid oxidation complexes. A kinetic evaluation of the complexes led to the suggestion that the mutation may have affected the active site of L-3-hydroxyacyl-CoA dehydrogenase so that it is inactive with acetoacetyl-CoA as a substrate, but exhibits an increasing percentage of the parental dehydrogenase activity with increasing chain length of the substrate.     

Xanthine dehydrogenase from Drosophila melanogaster: purification and properties of the wild-type enzyme and of a variant lacking iron-sulfur centers.

Xanthine dehydrogenase has been purified to homogeneity by conventional procedures from the wild-type strain of the fruit fly Drosophila melanogaster, as well as from a rosy mutant strain (E89----K, ry5231) known to carry a point mutation in the iron-sulfur domain of the enzyme. The wild-type enzyme had all the specific properties that are peculiar to the molybdenum-containing hydroxylases. It had normal contents of molybdenum, the pterin molybdenum cofactor, FAD, and iron-sulfur centers. EPR studies showed its molybdenum center to be quite indistinguishable from that of milk xanthine oxidase. As isolated, only about 10% of the enzyme was present in the functional form, with most or all of the remainder as the inactive desulfo form. It is suggested that this may be present in vivo. Extensive proteolysis accompanied by the development of oxidase activity took place during isolation, but dehydrogenase activity was retained. EPR properties of the reduced iron-sulfur centers, Fe-SI and Fe-SII, in the enzyme are very similar to those of the corresponding centers in milk xanthine oxidase. The E89----K mutant enzyme variant was in all respects closely similar to the wild-type enzyme, with the exception that it lacked both of the iron-sulfur centers. This was established both by its having the absorption spectrum of a simple flavoprotein and by the complete absence of EPR signals characteristic of iron-sulfur centers in the reduced enzyme. Despite the lack of iron-sulfur centers, the mutant enzyme had xanthine:NAD+ oxidoreductase activity indistinguishable from that of the wild-type enzyme. Stopped-flow measurements indicated that, as for the wild-type enzyme, reduction of the mutant enzyme was rate-limiting in turnover. Thus, the iron-sulfur centers appear irrelevant to the normal turnover of the wild-type enzyme with these substrates. However, activity to certain oxidizing substrates, particularly phenazine methosulfate, is abolished in the mutant enzyme variant. This is one of the first examples of deletion by genetic means of iron-sulfur centers from an iron-sulfur protein. The relevance of our findings both to the roles of iron-sulfur centers in other systems and to the nature of the oxidizing substrate for the Drosophila enzyme in vivo are briefly discussed.     

Microbial production of L-ascorbic acid from D-sorbitol, L-sorbose, L-gulose, and L-sorbosone by Ketogulonicigenium vulgare DSM 4025

Ketogulonicigenium vulgare DSM 4025, known as a 2-keto-L-gulonic acid producing strain from L-sorbose via L-sorbosone, surprisingly produced L-ascorbic acid from D-sorbitol, L-sorbose, L-gulose, and L-sorbosone as the substrate under a growing or resting condition. As the best result, K. vulgare DSM 4025 produced 1.37 g per liter of L-AA from 5.00 g per liter of L-sorbosone during 4 h incubation time at 30 degrees C under the resting cell condition having 5.70 g per liter of wet cells. The precursor of L-AA formation from D-sorbitol and L-sorbose, except for L-gulose, was thought to be the putative furanose form of L-sorbosone. This is the first time it is reported that bacteria can produce vitamin C via L-sorbosone.     

New quinoproteins in oxidative fermentation

Several quinoproteins have been newly indicated in acetic acid bacteria, all of which can be applied to fermentative or enzymatic production of useful materials by means of oxidative fermentation. (1) D-Arabitol dehydrogenase from Gluconobacter suboxydans IFO 3257 was purified from the bacterial membrane and found to be a versatile enzyme for oxidation of various substrates to the corresponding oxidation products. It is worthy of notice that the enzyme catalyzes D-gluconate oxidation to 5-keto-D-gluconate, whereas 2-keto-D-gluconate is produced by a flavoprotein D-gluconate dehydrogenase. (2) Membrane-bound cyclic alcohol dehydrogenase was solubilized and purified for the first time from Gluconobacter frateurii CHM 9. When compared with the cytosolic NAD-dependent cyclic alcohol dehydrogenase crystallized from the same strain, the reaction rate in cyclic alcohol oxidation by the membrane enzyme was 100 times stronger than the cytosolic NAD-dependent enzyme. The NAD-dependent enzyme makes no contribution to cyclic alcohol oxidation but contributes to the reduction of cyclic ketones to cyclic alcohols. (3) Meso-erythritol dehydrogenase has been purified from the membrane fraction of G. frateurii CHM 43. The typical properties of quinoproteins were indicated in many respects with the enzyme. It was found that the enzyme, growing cells and also the resting cells of the organism are very effective in producing L-erythrulose. Dihydroxyacetone can be replaced by L-erythrulose for cosmetics for those who are sensitive to dihydroxyacetone. (4) Two different membrane-bound D-sorbitol dehydrogenases were indicated in acetic acid bacteria. One enzyme contributing to L-sorbose production has been identified to be a quinoprotein, while another FAD-containing D-sorbitol dehydrogenase catalyzes D-sorbitol oxidation to D-fructose. D-Fructose production by the oxidative fermentation would be possible by the latter enzyme and it is superior to the well-established D-glucose isomerase, because the oxidative fermentation catalyzes irreversible one-way oxidation of D-sorbitol to D-fructose without any reaction equilibrium, unlike D-glucose isomerase. (5) Quinate dehydrogenase was found in several Gluconobacter strains and other aerobic bacteria like Pseudomonas and Acinetobacter strains. It has become possible to produce dehydroquinate, dehydroshikimate, and shikimate by oxidative fermentation. Quinate dehydrogenase was readily solubilized from the membrane fraction by alkylglucoside in the presence of 0.1 M KCl. A simple purification by hydrophobic chromatography gave a highly purified quinate dehydrogenase that was monodispersed and showed sufficient purity. When quinate dehydrogenase purification was done with Acinetobacter calcoaceticus AC3, which is unable to synthesize PQQ, purified inactive apo-quinate dehydrogenase appeared to be a dimer and it was converted to the monomeric active holo-quinate dehydrogenase by the addition of PQQ.