The metabolism of 1,2-propanediol by the propylene oxide utilizing bacterium Nocardia A60

A bacterium designated Nocardia A60 was isolated for its capacity to utilize propylene oxide (1,2-epoxypropame) aerobically as a carbon and energy source for growth. Extracts of cells grown on the epoxide catalyzed the conversion of propylene oxide to 1,2-propanediol This epoxidase activity was absent in cells grown on 1,2-propanediol or succinate. During growth of the organism on propylene oxide and 1,2-propanediol it contained high levels of diol dehydratase (EC 4.2.1.28). Enhanced levels of propionyl-CoA carboxylase during growth on propylene oxide and 1,2-propanediol suggest that these compounds are metabolized via propionate and succinate.     

Fermentation of 1,2-Propanediol and 1,2-Ethanediol by Some Genera of Enterobacteriaceae, Involving Coenzyme B12-Dependent Diol Dehydratase

Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 was able to grow anaerobically on 1,2-propanediol and 1,2-ethanediol as carbon and energy sources. Whole cells of the bacterium grown anaerobically on 1,2-propanediol or on glycerol catalyzed conversion of 1,2-diols and aldehydes to the corresponding acids and alcohols. Glucose-grown cells also converted aldehydes, but not 1,2-diols, to acids and alcohols. The presence of activities of coenzyme B(12)-dependent diol dehydratase, alcohol dehydrogenase, coenzyme-A-dependent aldehyde dehydrogenase, phosphotransacetylase, and acetate kinase was demonstrated with crude extracts of 1,2-propanediol-grown cells. The dependence of the levels of these enzymes on growth substrates, together with cofactor requirements in in vitro conversion of these substrates, indicates that 1,2-diols are fermented to the corresponding acids and alcohols via aldehydes, acyl-coenzyme A, and acyl phosphates. This metabolic pathway for 1,2-diol fermentation was also suggested in some other genera of Enterobacteriaceae which were able to grow anaerobically on 1,2-propanediol. When the bacteria were cultivated in a 1,2-propanediol medium not supplemented with cobalt ion, the coenzyme B(12)-dependent conversion of 1,2-diols to aldehydes was the rate-limiting step in this fermentation. This was because the intracellular concentration of coenzyme B(12) was very low in the cells grown in cobalt-deficient medium, since the apoprotein of diol dehydratase was markedly induced in the cells grown in the 1,2-propanediol medium. Better cell yields were obtained when the bacteria were grown anaerobically on 1,2-propanediol. Evidence is presented that aerobically grown cells have a different metabolic pathway for utilizing 1,2-propanediol.     

Bacterial 2,3-butanediol dehydrogenases

Enterobacter aerogenes, Aeromonas hydrophila, Serratia marcescens and Staphylococcus aureus possessing L(+)-butanediol dehydrogenase produced mainly meso-butanediol and small amounts of optically active butanediol; Acetobacter suboxydans, Bacillus polymyxa and Erwinia carotovora containing D(-)-butanediol dehydrogenase produced more optically active butanediol than meso-butanediol. Resting and growing cells of these organisms oxidized only one enantiomer of racemic butanediol. The D(-)-butanediol dehydrogenase from Bacillus polymyxa was partially purified (30-fold) with a specific activity of 24.5. Except NAD and NADH no other cofactors were required. Optimum pH-values for oxidation and reduction were pH 9 and pH 7, respectively. The optimum temperature was about 60°C. The molecular weight was 100000 to 107000. The K _m-values were 3.3 mM for D(-)-butanediol, 6.25 mM for meso-butanediol, 0.53 mM for acetoin, 0.2 mM for NAD, 0.1 mM for NADH, 87 mM for diacetyl, 38 mM for 1,2-propanediol; 2,3-pentanedion was not a substrate for this enzyme. The L(+)-butanediol dehydrogenase from Serratia marcescens was purified 57-fold (specific activity 22.3). Besides NAD or NADH no cofactors were required. The optimum value for oxidation was about pH 9 and for reduction pH 4.5. The optimum temperature was 32–36°C. The molecular weight was 100000 to 107000. The K _m-values were 5 mM for meso-butanediol, 10 mM for racemic butanediol, 6.45 for acetoin, 1 mM for NAD, 0.25 mM for NADH, 2.08 mM for diacetyl, 16.7 mM for 2,3-pentanedion and 11.8 mM for 1,2-propanediol.Abbreviations Bud 2,3-butanediol - DH dehydrogenase     

2,3-Butanediol Metabolism in the Acetogen Acetobacterium woodii

The acetogenic bacterium Acetobacterium woodii is able to reduce CO₂ to acetate via the Wood-Ljungdahl pathway. Only recently we demonstrated that degradation of 1,2-propanediol by A. woodii was not dependent on acetogenesis, but that it is disproportionated to propanol and propionate. Here, we analyzed the metabolism of A. woodii on another diol, 2,3-butanediol. Experiments with growing and resting cells, metabolite analysis and enzymatic measurements revealed that 2,3-butanediol is oxidized in an NAD⁺-dependent manner to acetate via the intermediates acetoin, acetaldehyde, and acetyl coenzyme A. Ethanol was not detected as an end product, either in growing cultures or in cell suspensions. Apparently, all reducing equivalents originating from the oxidation of 2,3-butanediol were funneled into the Wood-Ljungdahl pathway to reduce CO₂ to another acetate. Thus, the metabolism of 2,3-butanediol requires the Wood-Ljungdahl pathway.     

Immunochemical evidence for the difference between coenzyme-B12-dependent diol dehydratase and glycerol dehydratase.

Klebsiella pneumoniae ATCC 25955 (formerly named Aerobacter aerogenes PZH 572, Warsaw), which is known to produce coenzyme-B12-dependent glycerol dehydratase when grown anaerobically in a glycerol medium, formed coenzyme-B12-dependent diol dehydratase in a 1,2-propanediol-containing medium. Both the diol dehydratase and the glycerol dehydratase produced by the organism catalyzed the conversion of glycerol, 1,2-propanediol and 1,2-ethanediol to the corresponding aldehydes and underwent concomitant inactivation during the catalysis of glycerol dehydration, as does the diol dehydratase of K. pneumoniae (A. aerogenes) ATCC 8724. However, the two enzymes were distinguishable from each other by the monovalent-cation-selectivity pattern and by substrate specificity; that is, glycerol dehydratase preferred glycerol to 1,2-propanediol as a substrate, whereas diol dehydratase preferred 1,2-propanediol to glycerol, as judged from initial velocity studies. Ouchterlony double-diffusion analysis and immunochemical titration with rabbit antiserum against diol dehydratase of K. pneumoniae ATCC 8724 established clearly that the diol dehydratase of K. pneumoniae ATCC 25955 is immunologically similar to that of K. pneumoniae ATCC 8724, while the glycerol dehydratase of the former is different from the diol dehydratase of both strains. Both the enzymes were found to be distributed in several bacteria of the family Enterobacteriaceae.     

Substrate specificity of coenzyme B12-dependent diol dehydrase: glycerol as both a good substrate and a potent inactivator.

The substrate specificity of adenosylcobalamin-dependent diol dehydrase was further studied in detail using an enzyme preparation that appears homogeneous by ultracentrifugal and gel electrophoretical criteria. Besides 1,2-propanediol and 1,2-ethanediol, glycerol, 1,2- and 2,3-butanediol were found to serve as substrate for the enzyme, whereas 1,3-propanediol was not. Of the substrate analogs tested, glycerol displayed some striking features: it was dehydrated to β-hydroxypropionaldehyde with concomitant inactivation of the enzyme. Although the initial velocity with glycerol was comparable to that with 1,2-propanediol, the dehydration reaction ceased almost completely within 3 min accompanying rapid, irreversible inactivation of the holoenzyme. 1,2- and 2,3-Butanediol were converted to butyraldehyde and methyl ethyl ketone, respectively, at a rate much lower than that with 1,2-propanediol. 2,3-Butanediol is the only compound, other than 1,2-diols, known at present to show a considerable substrate activity.     

Oxidation of primary aliphatic alcohols by Acetobacterium carbinolicum sp. nov., a homoacetogenic anaerobe

Four strains of new homoacetogenic bacteria were enriched and isolated from freshwater sediments and sludge with ethanol, propanol, 1,2-propanediol, or 1,2-butanediol as substrates. All strains were Gram-positive nonsporeforming rods and grew well in carbonate-buffered defined media under obligately anaerobic conditions. Optimal growth occurred at 27° C around pH 7.0. H₂/CO₂, primary aliphatic alcohols C₃–C₅, glucose, fructose, lactate, pyruvate, ethylene glycol, 1,2-propanediol, 2,3-butanediol, acetoin, glycerol, and methyl groups of methoxylated benzoate derivates and betaine were fermented to acetate or, in case of primary alcohols C₃–C₅ and 1,2-propanediol, to acetate and the respective fatty acid. In coculture with methanogens methane was formed, probably due to interspecies hydrogen transfer. Strain WoProp 1 is described as a new species, Acetobacterium carbinolicum. It had a DNA base composition of 38.5±1.0% guanine plus cytosine, and contained murein of crosslinkage type B similar to A. woodii.     

Distribution of coenzyme B12-dependent diol dehydratase and glycerol dehydratase in selected genera of Enterobacteriaceae and Propionibacteriaceae.

The presence of diol dehydratase and glycerol dehydratase was shown in several bacteria of Enterobacteriaceae grown anaerobically on 1,2-propanediol and on glycerol, respectively. Diol dehydratases of Enterobacteriaceae were immunologically similar, but distinct from that of Propionibacterium freudenreichii.     

PduP is a coenzyme-a-acylating propionaldehyde dehydrogenase associated with the polyhedral bodies involved in B<Subscript>12</Subscript>-dependent 1,2-propanediol degradation by<Emphasis Type="Italic"> Salmonella enterica</Emphasis> serovar Typhimurium LT2

Salmonella enterica forms polyhedral bodies involved in coenzyme-B₁₂-dependent 1,2-propanediol degradation. Prior studies showed that these bodies consist of a proteinaceous shell partly composed of the PduA protein, coenzyme-B₁₂-dependent diol dehydratase, and additional unidentified proteins. In this report, we show that the PduP protein is a polyhedral-body-associated CoA-acylating aldehyde dehydrogenase important for 1,2-propanediol degradation by S. enterica. A PCR-based method was used to construct a precise nonpolar deletion of the gene pduP. The resulting pduP deletion strain grew poorly on 1,2-propanediol minimal medium and expressed 105-fold less propionaldehyde dehydrogenase activity (0.011 mol min^–1 mg^–1) than did wild-type S. enterica grown under similar conditions (1.15 mol min^–1 mg^–1). An Escherichia coli strain was constructed for high-level production of His₈-PduP, which was purified by nickel-affinity chromatography and shown to have 15.2 mol min^–1 mg^–1 propionaldehyde dehydrogenase activity. Analysis of assay mixtures by reverse-phase HPLC and mass spectrometry established that propionyl-CoA was the product of the PduP reaction. For subcellular localization, purified His₈-PduP was used as antigen for the preparation of polyclonal antiserum. The antiserum obtained was shown to have high specificity for the PduP protein and was used in immunogold electron microscopy studies, which indicated that PduP was associated with the polyhedral bodies involved in 1,2-propanediol degradation. Further evidence for the localization of the PduP enzyme was obtained by showing that propionaldehyde dehydrogenase activity co-purified with the polyhedral bodies. The fact that both Ado-B₁₂-dependent diol dehydratase and propionaldehyde dehydrogenase are associated with the polyhedral bodies is consistent with the proposal that these structures function to minimize propionaldehyde toxicity during the growth of S. enterica on 1,2-propanediol.     

Cloning, expression, and characterization of coenzyme-B12-dependent diol dehydratase from Lactobacillus diolivorans

The three gldCDE genes from Lactobacillus diolivorans, that encode the three subunits of the glycerol dehydratase, were cloned and the proteins were co-expressed in soluble form in Escherichia coli with added sorbitol and betaine hydrochloride. The purified enzyme exists as a heterohexamer (α₂β₂γ₂) structure with a native molecular mass of 210 kDa. It requires coenzyme B₁₂ for catalytic activity and is subject to suicide inactivation by glycerol during catalysis. The enzyme had maximum activity at pH 8.6 and 37 °C. The apparent K _m values for coenzyme B₁₂, 1,2-ethanediol, 1,2-propanediol, and glycerol were 1.5 μM, 10.5 mM, 1.3 mM, and 5.8 mM, respectively. Together, these results indicated that the three genes gldCDE encoding the proteins make up a coenzyme B₁₂-dependent diol dehydratase and not a glycerol dehydratase.     

Coenzyme B12-dependent diol dehydratase: regulation of apoenzyme synthesis in Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724.

Immunochemical studies demonstrated that Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 produces only a single diol dehydratase whether grown on glycerol or on 1,2-propanediol. The enzyme was subject to induction by 1,2-diols and to catabolite repression reversed by cyclic AMP.     

Utilization of Alcohols by Hansenula miso

The ability of Hansenula miso IFO 0146 to utilize various alcohols and acidic salts as sole sources of carbon and the ability of resting cells to oxidize various alcohols and glucose were studied. Growing cells could utilize only ethanol, glycerol, acetate and lactate, while resting cells grown on ethanol medium could oxidize various alcohols such as 1,2-ethanediol, DL-1,2-propanediol, 1,3-propanediol, meso-2,3-butanediol, DL-1,3-butane-diol, and 1,4-butanediol. From 2 g of 1,2-ethanediol and DL-l,3-butanediol, 1.3 g of glycolic acid and 0.5 g of β-hydroxybutyric acid respectively were produced. The organism formed d-arabinitol from glycerol and glucose, respectively. From 100 ml of culture in medium containing 6 ml of ethanol and 3.0 g of (NH₄)₂HPO₄ as carbon and nitrogen sources 3.40 g of dried cells were obtained.     

Glycerol Dehydrogenase from Cellulomonas sp. NT3060: Purification and Characterization

NAD⁺-dependent glycerol dehydrogenase from Cellulomonas sp. NT3060 was purified by a procedure of 10 steps involving crystallization. Dihydroxyacetone was identified as the oxidation product of glycerol with the enzyme. The purified enzyme did not lose activity on heating below 60°C. The enzyme oxidized other alcohols such as 1,2-propanediol, 2,3-butanediol and glycerol-α-monochlorohydrin, beside glycerol. The enzyme activity was inhibited by p-chloromercuribenzoate, Zn²⁺, Cu²⁺ and Cd²⁺. Oxidation of glyberol was activated by Na⁺ and reduction of dihydroxyacetone was activated by K⁺ at pH 7.5.     

Inactivation of <Emphasis Type="Italic">dhaD</Emphasis> and <Emphasis Type="Italic">dhaK</Emphasis> abolishes by-product accumulation during 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose P_BAD promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.     

Hydrolytic enzyme substrates. I. Chemical synthesis and characterization

The synthesis and characterization of a number of new phosphate, sulfate and acetate esters of 3-(p-nitrophenoxy)-1,2-propanediol (PNG); 3-(2,4-dinitrophenoxy)-1,2-propanediol (DNG); 4-(p-nitrophenoxy)-1,2-butanediol (PNB) and 4-(2,4-dinitrophenoxy)-1,2-butanediol (DNB) are described. These esters were prepared to serve as substrates for their corresponding hydrolytic enzymes. The assay system used to measure enzyme hydrolysis requires periodate oxidation of the diol formed after hydrolysis of the ester. Base treatment of the resulting aldehyde yields either p-nitrophenolate ion or the 2,4-dinitrophenolate ion depending upon the substrate. In the presence of high concentrations of methylamine and excess periodate the oxidation and elimination reactions can be carried out simultaneously at pH 7.5. The reactions leading to these results are described.     

Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides.

The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a alpha2beta2gamma2 composition. The Km for the three main substrates were 1.6 mm for 1,2-propanediol, 5.5 mm for 1,2-ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 micro m. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 degrees C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2-propanediol metabolism pathway.     

Fermentation of glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella pneumoniae

Klebsiella pneumoniae was shown to convert glycerol to 1,3-propanediol, 2,3-butanediol and ethanol under conditions of uncontrolled pH. Formation of 2,3-butanediol starts with some hours' delay and is accompanied by a reuse of the acetate that was formed in the first period. The fermentation was demonstrated in the type strain of K. pneumoniae, but growth was better with the more acid-tolerant strain GT1, which was isolated from nature. In continuous cultures in which the pH was lowered stepwise from 7.3 to 5.4, 2,3-butanediol formation started at pH 6.6 and reached a maximum yield at pH 5.5, whereas formation of acetate and ethanol declined in this pH range. 2,3-Butanediol and acetoin were also found among the products in chemostat cultures grown at pH 7 under conditions of glycerol excess but only with low yields. At any of the pH values tested, excess glycerol in the culture enhanced the butanediol yield. Both effects are seen as a consequence of product inhibition, the undissociated acid being a stronger trigger than the less toxic diols and acid anions. The possibilities for using the fermentation type described to produce 1,3-propanediol and 2,3-butanediol almost without by-products are discussed. Received: 4 February 1998 / Received revision: 30 March 1998 / Accepted: 13 April 1998     

Microbial production of diols as platform chemicals: recent progresses

Diols are chemicals with two hydroxyl groups which have a wide range of appealing applications as chemicals and fuels. In particular, four diol compounds, namely 1,3-propanediol (1,3-PDO), 1,2-propanediol (1,2-PDO), 2,3-butanediol (2,3-BDO) and 1,4-butanediol (1,4-BDO) can be biotechnologically produced by direct microbial bioconversion of renewable materials. These diols are considered as platform green chemicals. We review and discuss here the recent development in the microbial production of these diols, especially regarding the engineering of production strains and optimization of the fermentation processes.     

Identification and Characterization of Coenzyme B12-Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures

To isolate genes encoding coenzyme B₁₂-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.     

Recombinant glycerol dehydratase from <Emphasis Type="Italic">Klebsiella pneumonia</Emphasis> XJPD-Li: induction optimization,purification and characterization

Glycerol dehydratase (GDHt) is the rate limiting enzyme in the biosynthesis of 1,3-propanediol from glycerol. The optimization of inducting process for recombinant GDHt from Klebsiella pneumoniae XJPD-Li carried out to increase specific activity and ratio of soluble form. The optimum condition was inducing under the isopropyl-β-D-thiogalactoside concentration of 0.8 mM and the temperature of 20°C for 3 h. Homogeneity of GDHt then was obtained by affinity chromatography, resulted in 2.11-fold purification and an overall yield of 47.5%. The optimum pH and reaction temperature of GDHt were pH 8.0 and 45°C, respectively. The K _m for glycerol, 1,2-propanediol, 1,2-ethanediol and coenzyme B12 were 0.48 mM, 1.43 mM, 3.07 mM, and 10.03 nM, respectively. The GDHt showed relatively stable even under temperature of 40°C and a bit blunt to oxygen. The thermo-inactivation kinetic models were fit linear under different temperatures.