Pyrroloquinoline quinone-dependent dehydrogenases from Ketogulonicigenium vulgare catalyze the direct conversion of L-sorbosone to L-ascorbic acid

A novel enzyme, L-sorbosone dehydrogenase 1 (SNDH1), which directly converts L-sorbosone to L-ascorbic acid (L-AA), was isolated from Ketogulonicigenium vulgare DSM 4025 and characterized. This enzyme was a homooligomer of 75-kDa subunits containing pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups. Two isozymes of SNDH, SNDH2 consisting of 75-kDa and 55-kDa subunits and SNDH3 consisting of 55-kDa subunits, were also purified from the bacterium. All of the SNDHs produced L-AA, as well as 2-keto-L-gulonic acid (2KGA), from L-sorbosone, suggesting that tautomerization of L-sorbosone causes the dual conversion by SNDHs. The sndH gene coding for SNDH1 was isolated and analyzed. The N-terminal four-fifths of the SNDH amino acid sequence exhibited 40% identity to the sequence of a soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. The C-terminal one-fifth of the sequence exhibited similarity to a c-type cytochrome with a heme-binding motif. A lysate of Escherichia coli cells expressing sndH exhibited SNDH activity in the presence of PQQ and CaCl2. Gene disruption analysis of K. vulgare indicated that all of the SNDH proteins are encoded by the sndH gene. The 55-kDa subunit was derived from the 75-kDa subunit, as indicated by cleavage of the C-terminal domain in the bacterial cells.     

醇醛脱氢酶的分离纯化及其基因文库的构建和筛选

在维生素C的发酵生产过程中,普通生酮基古龙酸菌S2(Ketogulonigenium vulgare)能产生醇醛脱氢酶,将L-山梨糖转化为VC的前体2-酮基-L-古龙酸(2-KLG)。通过超声波破碎菌体、硫酸铵分级沉淀、DEAE Sepharose Fast Flow阴离子交换层析,QSepharose High Performance柱层析等过程,从普通生酮基古龙酸菌S2发酵液中分离纯化了醇醛脱氢酶,并用该纯化酶免疫新西兰兔制备出了合格抗血清。同时,普通生酮基古龙酸菌S2基因组DNA经Sau3AⅠ部分酶切后,与黏粒载体pKC505连接,用包装蛋白进行包装,转染大肠杆菌DH5浕,构建了基因组文库。最后应用免疫酶斑点技术(Dot-ELISA)从12000个克隆子中筛选得到一个阳性克隆K719#。通过检测该基因工程菌的活性,表明K719#具有使L-山梨糖转化为2-KLG的功能,从而使醇醛脱氢酶在大肠杆菌中获得了高效表达,这为简化VC的生产工艺奠定了基础。     

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.     

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.     

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。     

Continuous 2-Keto-l-gulonic acid fermentation by mixed culture of Ketogulonicigenium vulgare DSM 4025 and Bacillus megaterium or Xanthomonas maltophilia

The fermentation process of 2-keto-L-gulonic acid (2KGA) from L-sorbose was developed using a two-stage continuous fermentation system. The mixed culture of Ketogulonicigenium vulgare DSM 4025 and Bacillus megaterium DSM 4026 produced 90 g/L of 2KGA from 120 g/L of L-sorbose at the dilution rate of 0.01 h⁻¹ in a single-stage continuous fermentation process. But after the production period was beyond 150 h, the significant decrease of 2KGA productivity was observed. When the non-spore forming bacteria Xanthomonas maltophilia IFO 12692 was used instead of B. megaterium DSM 4026 as a partner strain for K. vulgare DSM 4025, the 2KGA productivity was significantly improved in a two-stage continuous culture mode, in which two fermentors of the same size and volume were connected in series. In this mode, with two sets of 3-L jar fermentors, the steady state could be continued to over 1,331.5 h at least, when the dilution rates were 0.0382 h⁻¹ and 0.0380 hour⁻¹, respectively, for the first and second fermentors. The overall productivity was calculated to be 2.15 g/L/h at 113.1 g/L and a molar conversion yield of 90.1%. In the up-scaling fermentation to 30-L jar fermentors, 118.5 g/L of 2KGA was produced when dilution rates in both stages were 0.0430 hour⁻¹, and the overall productivity was calculated to be 2.55 g/L/h.     

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.     

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.     

New mechanisms for the biosynthesis and metabolism of 2-keto-L-gulonic acid in bacteria.

L-Sorbose is oxidized to 2-keto-L-gulonic acid (KGA) via the following sequence of reactions which we call the "sorbosone pathway": L-sorbose in equilibrium L-sorbosone leads to KGA. The first step is reversible and is mediated by enzymes found in a soluble fraction obtained from Pseudomonas putida ATCC 21812. Although no cofactor requirements were found for the forward reaction, the reverse reaction clearly required NADH. Enzymes for this NADH-dependent synthesis of L-sorbose could be differentiated on the basis of molecular weights. The second step in the sorbosone pathway is catalyzed by a particulate enzyme found in extracts from P. putida and Gluconobacter melanogenus IFO 3293. The rate limiting reaction in the sorbosone pathway is the synthesis of L-sorbosone. In addition to P. putida, Klebsiella pneumoniae (ATCC 27858) and Serratia marcescens (ATCC 27857) also contain the enzymes which catalyze the reactions of the sorbosone pathway. Two of the bacteria studied, P. putida and G. melanogenus, also contain an enzyme involved in the further metabolism of KGA to L-idonic acid. This enzyme, referred to as KGA-reductase, is found in the soluble fraction of cell-free extracts and is dependent on NADH or NADPH.     

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.     

New developments in oxidative fermentation

Oxidative fermentations have been well established for a long time, especially in vinegar and in L-sorbose production. Recently, information on the enzyme systems involved in these oxidative fermentations has accumulated and new developments are possible based on these findings. We have recently isolated several thermotolerant acetic acid bacteria, which also seem to be useful for new developments in oxidative fermentation. Two different types of membrane-bound enzymes, quinoproteins and flavoproteins, are involved in oxidative fermentation, and sometimes work with the same substrate but produce different oxidation products. Recently, there have been new developments in two different oxidative fermentations, D-gluconate and D-sorbitol oxidations. Flavoproteins, D-gluconate dehydrogenase, and D-sorbitol dehydrogenase were isolated almost 2 decades ago, while the enzyme involved in the same oxidation reaction for D-gluconate and D-sorbitol has been recently isolated and shown to be a quinoprotein. Thus, these flavoproteins and a quinoprotein have been re-assessed for the oxidation reaction. Flavoprotein D-gluconate dehydrogenase and D-sorbitol dehydrogenase were shown to produce 2-keto- D-gluconate and D-fructose, respectively, whereas the quinoprotein was shown to produce 5-keto- D-gluconate and L-sorbose from D-gluconate and D-sorbitol, respectively. In addition to the quinoproteins described above, a new quinoprotein for quinate oxidation has been recently isolated from Gluconobacter strains. The quinate dehydrogenase is also a membrane-bound quinoprotein that produces 3-dehydroquinate. This enzyme can be useful for the production of shikimate, which is a convenient salvage synthesis system for many antibiotics, herbicides, and aromatic amino acids synthesis. In order to reduce energy costs of oxidative fermentation in industry, several thermotolerant acetic acid bacteria that can grow up to 40 degrees C have been isolated. Of such isolated strains, some thermotolerant Acetobacter species were found to be useful for vinegar fermentation at a high temperature such 38-40 degrees C, where mesophilic strains showed no growth. They oxidized higher concentrations of ethanol up to 9% without any appreciable lag time, while alcohol oxidation with mesophilic strains was delayed or became almost impossible under such conditions. Several useful Gluconobacter species of thermotolerant acetic acid bacteria are also found, especially L-erythrulose-producing strains and cyclic alcohol-oxidizing strains. Gluconobacter frateurii CHM 43 is able to rapidly oxidize meso-erythritol at 37 degrees C leading to the accumulation of L-erythrulose, which may replace dihydroxyacetone in cosmetics. G. frateuriiCHM 9 is able to oxidize cyclic alcohols to their corresponding cyclic ketones or aliphatic ketones, which are known to be useful for preparing many different physiologically active compounds such as oxidized steroids or oxidized bicyclic ketones. The enzymes involved in these meso-erythritol and cyclic alcohol oxidations have been purified and shown to be a similar type of membrane-bound quinoproteins, consisting of a high molecular weight single peptide. This is completely different from another quinoprotein, alcohol dehydrogenase of acetic acid bacteria, which consists of three subunits including hemoproteins.     

Cloning of genes coding for L-sorbose and L-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of 2-keto-L-gulonate, a precursor of L-ascorbic acid, in a recombinant G. oxydans strain.

We have purified L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH) from Gluconobacter oxydans T-100 that showed an ability to convert D-sorbitol to 2-keto-L-gulonate (2-KLGA). A genomic library of Gluconobacter oxydans T-100 was screened with a probe, a 180-bp PCR product which was obtained from degenerate oligodeoxyribonucleotides based on the elucidated sequence of the purified SDH (used as primers) and the genomic DNA of G. oxydans T-100 (used as a template). From sequencing of the DNA from a clone positive to the probe, the SNDH and the SDH were estimated to be coded in sequential open reading frames with 1,497 and 1,599 nucleotides, respectively, which was confirmed by expression of the DNA in Escherichia coli that showed both enzymatic activities. The DNA was introduced to a shuttle vector which was prepared from a plasmid of G. oxydans T-100 and pHSG298 to obtain an expression vector designated pSDH155. The production of 2-KLGA by pSDH155 in G. oxydans G624, an L-sorbose-accumulating strain, was improved to 230% compared to that of G. oxydans T-100. Chemical mutation of the host strain to suppress the L-idonate pathway and replacement of the original promoter with that of E. coli tufB resulted in improving the production of 2-KLGA. Consequently, high-level production from D-sorbitol to 2-KLGA (130 mg/ml) was achieved by simple fermentation of the recombinant Gluconobacter.     

山梨酮脱氢酶模块与酮古龙酸杆菌底盘细胞的适配分析

酮古龙酸杆菌Ketogulonigenium vulgare是维生素C二步混菌发酵过程中的产酸菌。山梨酮脱氢酶(L-sorbosone dehydrogenase,缩写为SNDH)作为维生素C直接前体2-酮基-L-古龙酸(2-KGA)合成的关键酶,其作用机制并不十分清楚。借助全基因组测序抽提2个山梨酮脱氢酶基因,分别位于基因组(缩写为sndhg)和质粒(缩写为sndhp)上。通过工程化改造技术在工业产酸菌中构建山梨酮脱氢酶功能模块,比较其对2-KGA产量的影响。研究发现sndhg过表达对菌株产酸影响不明显,sndhp过表达使菌株明显产生副产物。将sndhg和sndhp分别配合辅因子PQQ合成基因pqq A,分别构建sndhg-pqq A和sndhp-pqq A模块,得到的工程菌株产酸情况与之前的结果大致相同。将4株K.vulgare工程菌株分别与内生芽孢杆菌Bacillus endophyticus混合培养传代50 d后,分离菌株进行混菌发酵,其2-KGA的转化率分别提高了15.4%、179%、0.65%和125%。表明混菌适应性进化策略是一种增加功能模块与底盘细胞适配性,进而快速获得优良性状菌种的有效方法。     

Conversion of L-sorbose to L-sorbosone by immobilized cells of Gluconobacter melanogenus IFO 3293.

Gluconobacter melanogenus IFO 3293 cells capable of converting L-sorbose to L-sorbosone were immobilized in polyacrylamide gel. The preferred polymer composition for high activity and stability was determined to contain a total monomer concentration of 7.2% and 16.6% crosslinking agent. No significant differences in optimal conditions for conversion, e.g., pH and temperature, were found in comparison with free cell suspensions. However, in the absence of L-sorbose, the thermal stability of immobilized cells was lower. After the initial loss, the conversion activity of immobilized cells increased, possibly due to lysis, and this increase was related to the polymerization conditions and the incubation temperature for the L-sorbose conversion. The enzymatic activity and stability of the immobilized cells also depended on the physical form of the gel and the aeration levels. Addition of electron acceptors or addition of L-sorbosone to the medium reduced, while addition of neomycin, ampicillin, chloramphenicol, and tetracycline increased the stability of the enzymatic activity.     

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.     

Complete genome sequence of the bacterium Ketogulonicigenium vulgare Y25

Ketogulonicigenium vulgare is characterized by the efficient production of 2KGA from L-sorbose. Ketogulonicigenium vulgare Y25 is known as a 2-keto-L-gulonic acid-producing strain in the vitamin C industry. Here we report the finished, annotated genome sequence of Ketogulonicigenium vulgare Y25.     

Biosynthesis of L-ascorbic acid (vitamin C) by Saccharomyces cerevisiae

Saccharomyces cerevisiae cells incubated with D-glucose (D-Glc), D-galactose or D-mannose (D-Man) synthesised D-erythroascorbic acid (D-EAA) but not L-ascorbic acid (L-AA). Accumulation of D-EAA was observed in cells incubated with D-arabinose (D-Ara) whilst accumulation of L-AA occurred in cells incubated with L-galactose (L-Gal), L-galactono-1,4-lactone and L-gulono-1,4-lactone. When S. cerevisiae cells were incubated with D-[U-(14)C]Glc, D-[U-(14)C]Man or L-[1-(14)C]Gal, incorporation of radioactivity into L-AA was observed only with L-[1-(14)C]Gal. Pre-incubation of yeast cells with D-Ara substantially reduced the incorporation of L-[1-(14)C]Gal into L-AA. Our results indicate that, under appropriate conditions, yeast cells can synthesise L-AA via the pathway naturally used for D-EAA biosynthesis.     

Bacterial conversion of glycerol to beta-hydroxypropionaldehyde

beta-hydroxypropionaldehyde (3-HPA) can be oxidized to acrylic acid, an industrially important chemical used in the manufacture of synthetic plastics and other polymers. Of 19 genera and 55 strains tested, 3 Klebsiella and 2 Enterobacter strains produced 3-HPA. The most efficient strain was Klebsiella pneumoniae NRRL B-4011. Under optimum conditions (28 degrees C; 40 g of semicarbazide hydrochloride per liter, 70 g of glycerol per liter; and pH 6.0), 3.1 g of B-4011 cells per liter accumulated 22 g of 3-HPA per liter at a specific rate of 0.83 g/g per h; however, 14.5 g of cells per liter accumulated 46 g of 3-HPA per liter at a specific rate of 0.41 g/g per h.