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ABSTRACT: Gluconobacter oxydans is famous for its rapid and incomplete oxidation of a wide range of sugars and sugar alcohols. The organism is known for its efficient oxidation of D-glucose to D-gluconate, which can be further oxidized to two different keto-D-gluconates, 2-keto-D-gluconate and 5-keto-D-gluconate, as well as 2,5-di-keto-D-gluconate. For this oxidation chain and for further oxidation reactions, G. oxydans possesses a high number of membrane-bound dehydrogenases. In this review, we focus on the dehydrogenases involved in D-glucose oxidation and the products formed during this process. As some of the involved dehydrogenases contain pyrroloquinoline quinone (PQQ) as a cofactor, also PQQ synthesis is reviewed. Finally, we will give an overview of further PQQ-dependent dehydrogenases and discuss their functions in G. oxydans ATCC 621H (DSM 2343).
Journal of Molecular Microbiology and Biotechnology 02/2009; 16(1-2):6-13. · 1.95 Impact Factor
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ABSTRACT: Gluconobacter oxydans is known for causing rapid and incomplete oxidation of a wide range of sugars, sugar acids and sugar alcohols. Therefore, this microorganism is already employed in several biotechnological processes that involve incomplete oxidation of a substrate, e.g. vitamin C or dihydroxyacetone production. To fully exploit the oxidative potential of G. oxydans, characterization of the biological role of gene products is essential. To take advantage of the genome sequence of G. oxydans DSM 2343, based on pBBR1MCS5, we constructed a new cloning and expression vector. The newly established vector pEXGOX will significantly decrease duration of cloning and increase cloning efficiency. It has the following advantages: (i) small size (5.7 kbp); (ii) complete sequence; (iii) variety of unique restriction sites; (iv) direct cloning of PCR products; (v) strong promoter. The pEXGOX plasmid was successfully used to clone G. oxydans genes and has the potential to facilitate studies of gene function of several G. oxydans open reading frames.
International Journal of Food Microbiology 07/2008; 125(1):91-5. · 3.33 Impact Factor
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Kirsten Schroer,
Ursula Mackfeld,
Ivy Ai Wei Tan,
Christian Wandrey,
Florian Heuser, Stephanie Bringer-Meyer,
Andrea Weckbecker,
Werner Hummel,
Thomas Daussmann,
Rupert Pfaller,
Andreas Liese,
Stephan Lütz
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ABSTRACT: The reduction of methyl acetoacetate was carried out in continuously operated biotransformation processes catalyzed by recombinant Escherichia coli cells expressing an alcohol dehydrogenase from Lactobacillus brevis. Three different cell types were applied as biocatalysts in three different cofactor regeneration approaches. Both processes with enzyme-coupled cofactor regeneration catalyzed by formate dehydrogenase or glucose dehydrogenase are characterized by a rapid deactivation of the biocatalyst. By contrast the processes with substrate-coupled cofactor regeneration by alcohol dehydrogenase catalyzed oxidation of 2-propanol could be run over a period of 7 weeks with exceedingly high substrate and cosubstrate concentrations of up to 2.5 and 2.8 mol L(-1), respectively. Even under these extreme conditions, the applied biocatalyst showed a good stability with only marginal leakage of intracellular cofactors.
Journal of Biotechnology 01/2008; 132(4):438-44. · 3.05 Impact Factor
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ABSTRACT: An in vivo system was developed for the biotransformation of D-fructose into D-mannitol by the expression of the gene mdh encoding mannitol dehydrogenase (MDH) from Leuconostoc pseudomesenteroides ATCC12291 in Bacillus megaterium. The NADH reduction equivalents necessary for MDH activity were regenerated via the oxidation of formate to carbon dioxide by coexpression of the gene fdh encoding Mycobacterium vaccae N10 formate dehydrogenase (FDH). High-level protein production of MDH in B. megaterium required the adaptation of the corresponding ribosome binding site. The fdh gene was adapted to B. megaterium codon usage via complete chemical gene synthesis. Recombinant B. megaterium produced up to 10.60 g/L D-mannitol at the shaking flask scale. Whole cell biotransformation in a fed-batch bioreactor increased D-mannitol concentration to 22.00 g/L at a specific productivity of 0.32 g D-mannitol (gram cell dry weight)(-1) h(-1) and a D-mannitol yield of 0.91 mol/mol. The nicotinamide adenine dinucleotide (NAD(H)) pool of the B. megaterium producing D-mannitol remained stable during biotransformation. Intra- and extracellular pH adjusted itself to a value of 6.5 and remained constant during the process. Data integration revealed that substrate uptake was the limiting factor of the overall biotransformation. The information obtained identified B. megaterium as a useful production host for D-mannitol using a resting cell biotransformation approach.
Biotechnology Journal 12/2007; 2(11):1408-16.
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ABSTRACT: A recombinant oxidation/reduction cycle for the conversion of D-fructose to D-mannitol was established in resting cells of Corynebacterium glutamicum. Whole cells were used as biocatalysts, supplied with 250 mM sodium formate and 500 mM D-fructose at pH 6.5. The mannitol dehydrogenase gene (mdh) from Leuconostoc pseudomesenteroides was overexpressed in strain C. glutamicum ATCC 13032. To ensure sufficient cofactor [nicotinamide adenine dinucleotide (reduced form, NADH)] supply, the fdh gene encoding formate dehydrogenase from Mycobacterium vaccae N10 was coexpressed. The recombinant C. glutamicum cells produced D-mannitol at a constant production rate of 0.22 g (g cdw)(-1) h(-1). Expression of the glucose/fructose facilitator gene glf from Zymomonas mobilis in C. glutamicum led to a 5.5-fold increased productivity of 1.25 g (g cdw)(-1) h(-1), yielding 87 g l(-1) D-mannitol from 93.7 g l(-1) D-fructose. Determination of intracellular NAD(H) concentration during biotransformation showed a constant NAD(H) pool size and a NADH/NAD(+) ratio of approximately 1. In repetitive fed-batch biotransformation, 285 g l(-1) D-mannitol over a time period of 96 h with an average productivity of 1.0 g (g cdw)(-1) h(-1) was formed. These results show that C. glutamicum is a favorable biocatalyst for long-term biotransformation with resting cells.
Applied Microbiology and Biotechnology 10/2007; 76(3):545-52. · 3.42 Impact Factor
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ABSTRACT: The genus Gluconobacter is well known for its rapid and incomplete oxidation of a wide range of substrates. Therefore, Gluconobacter oxydans especially is used for several biotechnological applications, e.g., the efficient oxidation of glycerol to dihydroxyacetone (DHA). For this reaction, G. oxydans is equipped with a membrane-bound glycerol dehydrogenase that is also described to oxidize sorbitol, gluconate, and arabitol. Here, we demonstrated the impact of sldAB overexpression on glycerol oxidation: Beside a beneficial effect on the transcript level of the sldB gene, the growth on glycerol as a carbon source was significantly improved in the overexpression strains (OD 2.8 to 2.9) compared to the control strains (OD 2.8 to 2.9). Furthermore, the DHA formation rate, as well as the final DHA concentration, was affected so that up to 350 mM of DHA was accumulated by the overexpression strains when 550 mM glycerol was supplied (control strain: 200 to 280 mM DHA). Finally, we investigated the effect on sldAB overexpression on the G. oxydans transcriptome and identified two genes involved in glycerol metabolism, as well as a regulator of the LysR family.
Applied Microbiology and Biotechnology 10/2007; 76(3):553-9. · 3.42 Impact Factor
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ABSTRACT: Gluconobacter oxydans DSM 2343 is known to catalyze the oxidation of glucose to gluconic acid, and subsequently, to 2-keto-D-gluconic acid (2-KGA) and 5-keto-D-gluconic acid (5-KGA), by membrane-bound and soluble dehydrogenases. In G. oxydans MF1, in which the membrane-bound gluconate-2-dehydrogenase complex was inactivated, formation of the undesired 2-KGA was absent. This mutant strain uniquely accumulates high amounts of 5-KGA in the culture medium. To increase the production rate of 5-KGA, which can be converted to industrially important L-(+)-tartaric acid, we equipped G. oxydans MF1 with plasmids allowing the overproduction of the soluble and the membrane-bound 5-KGA-forming enzyme. Whereas the overproduction of the soluble gluconate:NADP 5-oxidoreductase resulted in the accumulation of up to 200 mM 5-KGA, the detected 5-KGA accumulation was even higher when the gene coding for the membrane-bound gluconate-5-dehydrogenase was overexpressed (240 to 295 mM 5-KGA). These results provide a basis for designing a biotransformation process for the conversion of glucose to 5-KGA using the membrane-bound as well as the soluble enzyme system.
Applied Microbiology and Biotechnology 12/2006; 73(2):443-51. · 3.42 Impact Factor
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ABSTRACT: L-Ascorbic acid has been industrially produced for around 70 years. Over the past two decades, several innovative bioconversion systems have been proposed in order to simplify the long time market-dominating Reichstein method, a largely chemical synthesis by which still a considerable part of L-ascorbic acid is produced. Here, we describe the current state of biotechnological alternatives using bacteria, yeasts, and microalgae. We also discuss the potential for direct production of l-ascorbic acid exploiting novel bacterial pathways. The advantages of these novel approaches competing with current chemical and biotechnological processes are outlined.
Journal of Biotechnology 07/2006; 124(1):196-205. · 3.05 Impact Factor
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ABSTRACT: Gluconobacter oxydans DSM 2343 (ATCC 621H)catalyzes the oxidation of glucose to gluconic acid and subsequently to 5-keto-D-gluconic acid (5-KGA), a precursor of the industrially important L-(+)-tartaric acid. To further increase 5-KGA production in G. oxydans, the mutant strain MF1 was used. In this strain the membrane-bound gluconate-2-dehydrogenase activity, responsible for formation of the undesired by-product 2-keto-D-gluconic acid, is disrupted. Therefore, high amounts of 5-KGA accumulate in the culture medium. G. oxydans MF1 was equipped with plasmids allowing the overexpression of the membrane-bound enzymes involved in 5-KGA formation. Overexpression was confirmed on the transcript and enzymatic level. Furthermore, the resulting strains overproducing the membrane-bound glucose dehydrogenase showed an increased gluconic acid formation, whereas the overproduction of gluconate-5-dehydrogenase resulted in an increase in 5-KGA of up to 230 mM. Therefore, these newly developed recombinant strains provide a basis for further improving the biotransformation process for 5-KGA production.
Biotechnology Journal 06/2006; 1(5):556-63.
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ABSTRACT: Recently, we reported on the construction of a whole-cell biotransformation system in Escherichia coli for the production of D: -mannitol from D: -fructose. Supplementation of this strain with extracellular glucose isomerase resulted in the formation of 800 mM D: -mannitol from 1,000 mM D: -glucose. Co-expression of the xylA gene of E. coli in the biotransformation strain resulted in a D: -mannitol concentration of 420 mM from 1,000 mM D: -glucose. This is the first example of conversion of D: -glucose to D: -mannitol with direct coupling of a glucose isomerase to the biotransformation system.
Applied Microbiology and Biotechnology 01/2006; 69(4):397-403. · 3.42 Impact Factor
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ABSTRACT: A whole-cell biotransformation system for the reduction of prochiral carbonyl compounds, such as methyl acetoacetate, to chiral hydroxy acid derivatives [methyl (R)-3-hydroxy butanoate] was developed in Escherichia coli by construction of a recombinant oxidation/reduction cycle. Alcohol dehydrogenase from Lactobacillus brevis catalyzes a highly regioselective and enantioselective reduction of several ketones or keto acid derivatives to chiral alcohols or hydroxy acid esters. The adh gene encoding for the alcohol dehydrogenase of L. brevis was expressed in E. coli. As expected, whole cells of the recombinant strain produced only low quantities of methyl (R)-3-hydroxy butanoate from the substrate methyl acetoacetate. Therefore, the fdh gene from Mycobacterium vaccae N10, encoding NAD+-dependent formate dehydrogenase, was functionally coexpressed. The resulting two-fold recombinant strain exhibited an in vitro catalytic alcohol dehydrogenase activity of 6.5 units mg-1 protein in reducing methyl acetoacetate to methyl (R)-3-hydroxy butanoate with NADPH as the cofactor and 0.7 units mg-1 protein with NADH. The in vitro formate dehydrogenase activity was 1.3 units mg-1 protein. Whole resting cells of this strain catalyzed the formation of 40 mM methyl (R)-3-hydroxy butanoate from methyl acetoacetate. The product yield was 100 mol% at a productivity of 200 micromol g-1 (cell dry weight) min-1. In the presence of formate, the intracellular [NADH]/[NAD+] ratio of the cells increased seven-fold. Thus, the functional overexpression of alcohol dehydrogenase in the presence of formate dehydrogenase was sufficient to enable and sustain the desired reduction reaction via the relatively low specific activity of alcohol dehydrogenase with NADH, instead of NADPH, as a cofactor.
Applied Microbiology and Biotechnology 04/2005; 66(6):629-34. · 3.42 Impact Factor
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ABSTRACT: 1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) is the second enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. The structure of the apo-form of this enzyme from Zymomonas mobilis has been solved and refined to 1.9-A resolution, and that of a binary complex with the co-substrate NADPH to 2.7-A resolution. The subunit of DXR consists of three domains. Residues 1-150 form the NADPH binding domain, which is a variant of the typical dinucleotide-binding fold. The second domain comprises a four-stranded mixed beta-sheet, with three helices flanking the sheet. Most of the putative active site residues are located on this domain. The C-terminal domain (residues 300-386) folds into a four-helix bundle. In solution and in the crystal, the enzyme forms a homo-dimer. The interface between the two monomers is formed predominantly by extension of the sheet in the second domain. The adenosine phosphate moiety of NADPH binds to the nucleotide-binding fold in the canonical way. The adenine ring interacts with the loop after beta1 and with the loops between alpha2 and beta2 and alpha5 and beta5. The nicotinamide ring is disordered in crystals of this binary complex. Comparisons to Escherichia coli DXR show that the two enzymes are very similar in structure, and that the active site architecture is highly conserved. However, there are differences in the recognition of the adenine ring of NADPH in the two enzymes.
Biochimica et Biophysica Acta 05/2004; 1698(1):37-44. · 4.66 Impact Factor
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ABSTRACT: In the gram negative, obligately ethanologenic bacterium Zymomonas mobilis a pyruvate dehydrogenase complex was identified and the complex was enriched from cell extracts. This multienzyme complex is responsible for acetyl-CoA biosynthesis from pyruvate. No activities of related multienzyme complexes, 2-ketoglutarate dehydrogenase and branched chain keto acid dehydrogenase, could be detected.
Archives of Microbiology 01/1993; 159(2):197-199. · 1.43 Impact Factor
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ABSTRACT: The physiological basis of the exceptionally high sugar tolerance of Zymomonas mobilis was investigated. Determinations of the internal metabolite concentrations of Z. mobilis showed that an increase in the extracellular glucose concentration was accompanied by a parallel rise in the intracellular glucose concentration, bringing about an almost complete osmotic balance between internal and external space. Studies of glucose transport confirmed that Z. mobilis has a facilitated diffusion system which enables a rapid equilibration between internal and external glucose concentrations. Studies using the non-metabolisable sugars maltose (impermeable) and xylose (permeable) revealed that these sugars were able to alter the osmotic pressure on the cytoplasmic membrane resulting in volume changes.
Applied Microbiology and Biotechnology 12/1990; 34(4):518-523. · 3.42 Impact Factor
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ABSTRACT: The interaction of the membrane-bound glucose dehydrogenase from the anaerobic but aerotolerant bacterium Zymomonas mobilis with components of the electron transport chain has been studied. Cytoplasmic membranes showed reduction of oxygen to water with the substrates glucose or NADH. The effects of the respiratory chain inhibitors piericidin, capsaicin, rotenone, antimycin, myxothiazol, HQNO, and stigmatellin on the oxygen comsumption rates in the presence of NADH or glucose as substrates indicated that a complete and in the most parts identical respiratory chain is participating in the glucose as well as in the NADH oxidation. Furthermore, the presence of coenzyme Q10 (ubiquinone 10) in Z. mobilis was demonstrated. Extraction from and reincorporation of the quinone into the membranes revealed that ubiquinone is essential for the respiratory activity with glucose and NADH. In addition, a membrane-associated tetramethyl-p-phenylene-diamine-oxidase activity could be detected in Z. mobilis.
Archives of Microbiology 10/1990; 154(6):536-543. · 1.43 Impact Factor
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ABSTRACT: The synthesis of oxalacetate and malate in the ethanol-producing bacterium Zymomonas mobilis have been investigated. Cell-free extracts were examined for pyruvate carboxylase, phosphoenolpyruvate (PEP) carboxylase, PEP carboxytransphosphorylase, PEP carboxykinase, and malic enzyme, but only PEP carboxylase and nicotine adenine dinucleotide (NAD)-dependent malic enzyme activities could be detected. The PEP carboxylase, partially purified from extracts, was not affected by acetyl-coenzyme A. Intermediates of the tricarboxylic acid cycle and aspartate inhibited the enzyme competitively with PEP. Of these, citrate and -ketoglutarate were the strongest inhibitors. The physiological roles of PEP carboxylase and malic enzyme in Z. mobilis are discussed.
Applied Microbiology and Biotechnology 09/1989; 31(5):529-536. · 3.42 Impact Factor
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ABSTRACT: The influence of different primary aliphatic alcohols on the activities of two key enzymes in hopanoid biosynthesis of Zymomonas mobilis was investigated. By use of 14C- and 3H-labelled substrates the enzymes 3-hydroxy-3-methylglutaryl-CoA-reductase and squalene-hopenecyclase were detected with activities of 1.6 pmol x (min x mg protein)-1 and 2.3 pmol x- (min x mg protein)-1, respectively. Cells grown in the presence of 6% (v/v) ethanol did not show higher activities of these enzymes than cells grown in the presence of 1% (v/v) ethanol. Furthermore, 3-hydroxy-3-methylglutaryl-CoA-reductase was not activated by ethanol. However, ethanol activated the squalene-hopene-cyclase when added to the enzyme test system. Besides ethanol, propanol also had a positive effect on the squalene-hopene-cyclase: the enzyme's activity increased 1.7-fold in the presence of either alcohol at a concentration of 6% (v/v). This corresponded with a similar increase of hopanoid content of whole cells when grown in the presence of 6% (v/v) added ethanol or propanol. These results indicated that the squalene-hopene-cyclase has a regulatory function in the alcohol dependent hopanoid biosynthesis of Z. mobilis.
Applied Microbiology and Biotechnology 01/1989; 30(2):170-175. · 3.42 Impact Factor
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ABSTRACT: Zymomonas mobilis ATCC 29191 is able to degrade gluconate but cannot use it as a single carbon and energy source. Gluconate is phosphorylated by a gluconate kinase (EC 2.7.1.12) and the resulting 6-phosphogluconate is further catabolized to yield about 0.8 mol ethanol per mol of gluconate, considerable amounts of acetate and acetoin. This product spectrum agrees with the theoretical yield of only one reduction equivalent if gluconate is phosphorylated by a kinase and subsequently metabolized via the Entner-Doudoroff pathway.Furthermore, Z. mobilis contains a membrane-bound enzyme system which is able to oxidize glucose to gluconate. Cell-free extracts were active in an assay system with Wurster's blue as electron acceptor, and various aldoses as well as maltose, mannitol and sorbitol could be oxidized. The affinity for sorbitol was very low (K
m
=330 mM) but reasonable for glucose (K
m
=2.8 mM). The pH optimum for the glucose-oxidizing reaction was 6.5, while that for sorbitol oxidation was 5.5.
Applied Microbiology and Biotechnology 12/1987; 27(4):378-382. · 3.42 Impact Factor
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ABSTRACT: Ethanol production with bacteria. Strains of Saccharomyces cerevisiae have mostly been used for the production of ethanol from sugar by yeasts. Recently it was shown that the bacterium Zymomonas mobilis has some advantages compared to yeast for the production of industrial alcohol. Compared to traditional yeast fermentation, ethanol yield is about 5% higher than with yeast, since less sugar is incorporated into cell material by this bacterium. Like yeast, Zymomonas mobilis has remarkably high ethanol tolerance which enables the bacterium to produce ethanol concentrations of more than 13 vol.-% from sugar solutions of appropriate concentration. Investigations of the spectrum of lipids present have shown that this bacterium contains large quantities of hopanoids which are presumably of significance for the stabilization of cell membranes in the presence of ethanol. Since the cost of the sugar greatly influences the profitability fraction formed in the production of glucose syrup from wheat flour was investigated. It was shown that after enzymatic saccharification of this waste starch the glucose was efficiently fermented to ethanol by Zymomonas mobilis. It is planned to broaden the substrate spectrum of Zymomonas mobilis by gene cloning techniques so that in future pentoses, e. g. xylose or arabinose, can also be fermented to ethanol by this organism.
Chemie Ingenieur Technik 08/1987; 59(9):695 - 700. · 0.59 Impact Factor
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ABSTRACT: The influence of different culture conditions on the hopanoid content of Zymomonas mobilis was investigated in batch cultures. With a gas-liquid chromatographic method it could be shown that the content of 1,2,3,4-tetrahydroxypentane-29-hopane (THBH) reached a maximum value in the stationary phase due to the high level of ethanol accumulated in the medium. The hopanoid content increased sharply with the addition of ethanol to the culture. Ethanol was shown to be the most effective of the alcohols tested in causing an increase of the hopanoid content. Furthermore, an alteration of the incubation temperature from 14 to 37C also caused an increase of the amount of hopanoids.
Applied Microbiology and Biotechnology 09/1986; 25(1):32-36. · 3.42 Impact Factor
