Cross-induction of the L-fucose system by L-rhamnose in Escherichia coli.

Dissimilation of L-fucose as a carbon and energy source by Escherichia coli involves a permease, an isomerase, a kinase, and an aldolase encoded by the fuc regulon at minute 60.2. Utilization of L-rhamnose involves a similar set of proteins encoded by the rha operon at minute 87.7. Both pathways lead to the formation of L-lactaldehyde and dihydroxyacetone phosphate. A common NAD-linked oxidoreductase encoded by fucO serves to reduce L-lactaldehyde to L-1,2-propanediol under anaerobic growth conditions, irrespective of whether the aldehyde is derived from fucose or rhamnose. In this study it was shown that anaerobic growth on rhamnose induces expression of not only the fucO gene but also the entire fuc regulon. Rhamnose is unable to induce the fuc genes in mutants defective in rhaA (encoding L-rhamnose isomerase), rhaB (encoding L-rhamnulose kinase), rhaD (encoding L-rhamnulose 1-phosphate aldolase), rhaR (encoding the positive regulator for the rha structural genes), or fucR (encoding the positive for the fuc regulon). Thus, cross-induction of the L-fucose enzymes by rhamnose requires formation of L-lactaldehyde; either the aldehyde itself or the L-fuculose 1-phosphate (known to be an effector) formed from it then interacts with the fucR-encoded protein to induce the fuc regulon.     

Fermentation of 6-Deoxyhexoses by Bacillus macerans

Under anaerobic conditions Bacillus macerans ATCC 7068 fermented 6-deoxyhexoses (l-rhamnose, l-fucose, and d-fucose) to a mixture of 1,2-propanediol (PD), acetone, H(2), CO(2), and ethanol. The final PD concentration was proportional to the amount of l-rhamnose fermented ( approximately 0.9 mol of PD per mol of rhamnose). PD was not produced from hexoses (e.g., d-glucose or l-mannose), despite active fermentation of these substrates. Relative to the fermentation of d-glucose, the fermentation of l-rhamnose was accompanied by a twofold reduction in yield of H(2), CO(2), and cell mass. Exposure of cell extracts to l-rhamnose resulted in the transient appearance of an aldehyde intermediate. Cell extracts contained a pyridine nucleotide-linked lactaldehyde reductase activity which converted synthetic d- or l-lactaldehyde to PD. The data suggest an Embden-Meyerhof pathway for 6-deoxyhexose catabolism, with the formation of lactaldehyde by a conventional aldolase cleavage reaction and subsequent reduction to PD.     

Biochemical and molecular characterization of thermo-alkali tolerant xylanase producing bacteria from thermal springs of Manikaran

1,2-Propanediol (propylene glycol) is an existing commodity chemical and can be produced from renewable resources using microbes. By virtue of being a natural product, relevant biochemical pathways can be harnessed into fermentation processes to produce 1,2-propanediol. In the present review, the chemical process and different biological strategies for the production of 1,2-propanediol are reviewed and compared with the potentials and limitations of all processes. For the successful commercial production of this diol, it is necessary to establish the metabolic pathways and production hosts (microorganisms), which are capable of delivering final product with high yields and volumetric productivity. Three pathways which have been recognized for 1,2-propanediol production are discussed here. In the first, de-oxy sugars like fucose and rhamnose are used as the carbon sources, while in the other route, the glycolytic intermediate-dihydroxyacetonephosphate (DHAP) is used to produce 1,2-propanediol via the formation of methylglyoxal. A new pathway of 1,2-propanediol production by lactic acid degradation under anoxic conditions and the enzymes involved is also discussed. The production of this diol has gained attention because of their newer applications in industries such as polymers, food, pharmaceuticals, textiles, etc. Furthermore, improvement in fermentation technology will permit its uses in other applications. Future prospect in the light of the current research and its potential as a major bulk chemical are discussed.     

NAD-linked aldehyde dehydrogenase for aerobic utilization of L-fucose and L-rhamnose by Escherichia coli.

Mutant analysis revealed that complete utilization of L-fucose and L-rhamnose by Escherichia coli requires the activity of a common NAD-linked aldehyde dehydrogenase which converts L-lactaldehyde to L-lactate. Mutations affecting this activity mapped to the ald locus at min 31, well apart from the fuc genes (min 60) encoding the trunk pathway for L-fucose dissimilation (as well as L-1,2-propanediol oxidoreductase) and the rha genes (min 88) encoding the trunk pathway for L-rhamnose dissimilation. Mutants that grow on L-1,2-propanediol as a carbon and energy source also depend on the ald gene product for the conversion of L-lactaldehyde to L-lactate.     

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.     

Anaerobic metabolism of the L-rhamnose fermentation product 1,2-propanediol in Salmonella typhimurium.

When grown anaerobically on L-rhamnose, Salmonella typhimurium excreted 1,2-propanediol as a fermentation product. Upon exhaustion of the methyl pentose, 1,2-propanediol was recaptured and further metabolized, provided the culture was kept under anaerobic conditions. n-Propanol and propionate were found in the medium as end products of this process at concentrations one-half that of 1,2-propanediol. As in Klebsiella pneumoniae (T. Toraya, S. Honda, and S. Fukui, J. Bacteriol. 139:39-47, 1979), a diol dehydratase which transforms 1,2-propanediol to propionaldehyde and the enzymes involved in a dismutation that converts propionaldehyde to n-propanol and propionate were induced in S. typhimurium cultures able to transform 1,2-propanediol anaerobically.     

Structural and biochemical characterisation of a NAD+-dependent alcohol dehydrogenase from Oenococcus oeni as a new model molecule for industrial biotechnology applications

Alcohol dehydrogenases are highly diverse enzymes catalysing the interconversion of alcohols and aldehydes or ketones. Due to their versatile specificities, these biocatalysts are of great interest for industrial applications. The adh3-gene encoding a group III alcohol dehydrogenase was isolated from the gram-positive bacterium Oenococcus oeni and was characterised after expression in the heterologous host Escherichia coli. Adh3 has been identified by genome BLASTP analyses using the amino acid sequence of 1,3-propanediol dehydrogenase DhaT from Klebsiella pneumoniae and group III alcohol dehydrogenases with known activity towards 1,3-propanediol as target sequences. The recombinant protein was purified in a two-step column chromatography approach. Crystal structure determination and biochemical characterisation confirmed that Adh3 forms a Ni²⁺-containing homodimer in its active form. Adh3 catalyses the interconversion of ethanol and its corresponding aldehyde acetaldyhyde and is also capable of using other alcoholic compounds as substrates, such as 1,3-propanediol, 1,2-propanediol and 1-propanol. In the presence of Ni²⁺, activity increases towards 1,3-propanediol and 1,2-propanediol. Adh3 is strictly dependent on NAD⁺/NADH, whereas no activity has been observed with NADP⁺/NADPH as co-factor. The enzyme exhibits a specific activity of 1.1 U/mg using EtOH as substrate with an optimal pH value of 9.0 for ethanol oxidation and 8.0 for aldehyde reduction. Moreover, Adh3 exhibits tolerance to several metal ions and organic solvents, but is completely inhibited in the presence of Zn²⁺. The present study demonstrates that O. oeni is a group III alcohol dehydrogenase with versatile substrate specificity, including Ni²⁺-dependent activity towards 1,3-propanediol.     

Metabolism of L-fucose and L-rhamnose in Escherichia coli: aerobic-anaerobic regulation of L-lactaldehyde dissimilation.

L-Lactaldehyde is a branching point in the metabolic pathway of L-fucose and L-rhamnose utilization. Under aerobic conditions, L-lactaldehyde is oxidized to L-lactate by the enzyme lactaldehyde dehydrogenase, while under anaerobic conditions, L-lactaldehyde is reduced to L-1,2-propanediol by the enzyme propanediol oxidoreductase. Aerobic growth on either of the methyl pentoses induces a lactaldehyde dehydrogenase enzyme which is inhibited by NADH and is very stable under anaerobic conditions. In the absence of oxygen, the cell shifts from the oxidation of L-lactaldehyde to its reduction, owing to both the induction of propanediol oxidoreductase activity and the decrease in the NAD/NADH ratio. The oxidation of L-lactaldehyde to L-lactate is again restored upon a change to aerobic conditions. In this case, only the NAD/NADH ratio may be invoked as a regulatory mechanism, since both enzymes remain active after this change. Experimental evidence in the presence of rhamnose with mutants unable to produce L-lactaldehyde and mutants capable of producing but not further metabolizing it points toward L-lactaldehyde as the effector molecule in the induction of lactaldehyde dehydrogenase. Analysis of a temperature-sensitive mutation affecting the synthesis of lactaldehyde dehydrogenase permitted us to locate an apparently single regulator gene linked to the ald locus at 31 min and probably acting as a positive control element on the expression of the structural gene.     

Bouquet-type dendrimerlike poly(ethylene oxide)s with a focal aldehyde and peripheral hydroxyls

Doubly functionalized dendrimerlike poly(ethylene oxide)s (PEOs) carrying 16 hydroxyl groups at their periphery and one aldehyde group at their focal point were synthesized up to the fourth generation through an iterative divergent approach. First, a protected aldehyde dihydroxyl compound, namely, 3,3-diethoxy-1,2-propanediol, was used as initiator for the anionic ring-opening polymerization (AROP) of ethylene oxide after partial deprotonation (30%) in dimethyl sulfoxide. The two hydroxyls carried by the PEO chain ends of the first generation were subsequently derivatized so as to generate four hydroxyls via a two-step reaction (allylation and osmylation). The next generations of such dendrimerlike PEOs were grown upon repeating the same cycle of AROP and chain-end modification. At the completion of these reactions, the acetal group present at the core was deprotected under acidic conditions to afford the targeted dendrimerlike PEO of fourth generation with a central aldehyde group. The reactivity and accessibility of the latter function was demonstrated upon its conjugation with aniline used as a model compound.     

PduA is a shell protein of polyhedral organelles involved in coenzyme B(12)-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2

Salmonella enterica forms polyhedral organelles involved in coenzyme B(12)-dependent 1,2-propanediol degradation. These organelles are thought to consist of a proteinaceous shell that encases coenzyme B(12)-dependent diol dehydratase and perhaps other enzymes involved in 1,2-propanediol degradation. The function of these organelles is unknown, and no detailed studies of their structure have been reported. Genes needed for organelle formation and for 1,2-propanediol degradation are located at the 1,2-propanediol utilization (pdu) locus, but the specific genes involved in organelle formation have not been identified. Here, we show that the pduA gene encodes a shell protein required for the formation of polyhedral organelles involved in coenzyme B(12)-dependent 1,2-propanediol degradation. A His(6)-PduA fusion protein was purified from a recombinant Escherichia coli strain and used for the preparation of polyclonal antibodies. The anti-PduA antibodies obtained were partially purified by a subtraction procedure and used to demonstrate that the PduA protein localized to the shell of the polyhedral organelles. In addition, electron microscopy studies established that strains with nonpolar pduA mutations were unable to form organelles. These results show that the pduA gene is essential for organelle formation and indicate that the PduA protein is a structural component of the shell of these organelles. Physiological studies of nonpolar pduA mutants were also conducted. Such mutants grew similarly to the wild-type strain at low concentrations of 1,2-propanediol but exhibited a period of interrupted growth in the presence of higher concentrations of this growth substrate. Growth tests also showed that a nonpolar pduA deletion mutant grew faster than the wild-type strain at low vitamin B(12) concentrations. These results suggest that the polyhedral organelles formed by S. enterica during growth on 1,2-propanediol are not involved in the concentration of 1,2-propanediol or coenzyme B(12), but are consistent with the hypothesis that these organelles moderate aldehyde production to minimize toxicity.     

Rheological measurements of the influence of 1,2-propanediol on actin/alpha-actinin gel structure: the effects of temperature and protein concentrations.

In previous studies, we demonstrated that 1,2-propanediol induces shortening and bundling of actin filaments, both in vitro and in vivo, and that it enhances actin/alpha-actinin interaction, especially at low temperature. 1,2-Propanediol also promotes homogeneous microporous networks which can be vitrified by rapid cooling. In the present study, dynamical rheological measurements were performed under various sets of experimental conditions including temperature (4 or 20 degrees C), protein concentrations (actin and alpha-actinin), and 1,2-propanediol presence or absence. Gelation kinetics were monitored, and the resulting actin mechanical properties investigated, in order to untangle the respective effects of the experimental parameters. Whether in the presence or absence of solvent, low temperature brings about a rigidification of the sample, as does high protein concentration, as expected. However, 1,2-propanediol itself involves either softening of the sample (at high temperature and low protein concentration or at low temperature and high protein concentration) or rigidification in the case of low temperature and low protein concentration. These effects result from the competition between actin/alpha-actinin affinity (enhanced by both low temperature and 1,2-propanediol), bundling of filaments (fostered by alpha-actinin for alpha-actinin/actin ratios used), rate of actin polymerization (higher at high temperature), shortening effect of 1,2-propanediol on actin filaments, and chain mobility (lower at high protein concentration). As discussed, only the combination of low temperature and low protein concentration induces full crosslinking of the system into a viscoelastic solid under the influence of 1,2-propanediol.     

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.     

The formation of unhydrated propionaldehyde by dioldehydrase.

When dl-1,2-propanediol is converted to propionaldehyde by dioldehydrase, an enzyme which requires B₁₂-coenzyme, the product is unhydrated propionaldehyde. Its formation was demonstrated by measuring the rate of reduction of the enzymically formed aldehyde by NADH, catalyzed by yeast alcohol dehydrogenase.     

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.     

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.     

Structural determinants for alcohol substrates to be oxidized to formaldehyde by rat liver microsomes.

Glycerol can be oxidized to formaldehyde by rat liver microsomes and by cytochrome P450. The ability of other alcohols to be oxidized to formaldehyde was determined to evaluate the structural determinants of the alcohol which eventually lead to this production of formaldehyde. Monohydroxylated alcohols such as 1- or 2-propanol did not produce formaldehyde when incubated with NADPH and microsomes. Geminal diols such as 1,3-propanediol, 1,3-butanediol, or 1,4-butanediol also did not yield formaldehyde. However, vicinal diols such as 1,2-propanediol or 1,2-butanediol produced formaldehyde. With 1,2-propanediol, the residual two-carbon fragment was found to be acetaldehyde, while with 1,2-butanediol, the residual three-carbon fragment was propionaldehyde. Oxidation of 1,2-propanediol to formaldehyde plus acetaldehyde involved interaction with an oxidant derived from H2O2 plus nonheme iron, since production of the two aldehydic products was completely prevented by catalase or glutathione plus glutathione peroxidase and by chelators such as desferrioxamine or EDTA. The oxidant was not superoxide or hydroxyl radical. Product formation was fivefold lower when NADH replaced NADPH, and was inhibited by substrates, ligands, and inhibitors of cytochrome P450. A charged glycol such as alpha-glycerophosphate (but not the geminal beta-glycerophosphate) was readily oxidized to formaldehyde, suggesting that interaction of the glycol with the oxidant was occurring in solution and not in a hydrophobic environment. These results indicate that the carbon-carbon bond between 1,2-glycols can be cleaved by an oxidant derived from microsomal generated H2O2 and reduction of non-heme iron, with the subsequent production of formaldehyde plus an aldehyde with one less carbon than the initial glycol substrate.     

Reconstitution of the Z-Disk

Methanol-utilizing bacteria, Klebsiella sp. No. 101 and Microcyclus eburneus could grow aerobically and statically on 1,2-propanediol. The authors examined the presence of enzyme activity of adenosyl-B₁₂ dependent diol dehydratase as well as NAD dependent diol dehydroagenase. Adenosyl-B₁₂ dependent diol dehydratase activity was not detected in these organisms, even if these grown statically.

The dehydrogenase activity was found in the extract from these methanol-utilizing bacteria cells grown on various carbon sources. The partially purified enzyme preparation from the cells of Mic. eburneus grown aerobically on 1,2-propanediol dehydrogenated 1,2-propanediol, 1,2-butanediol and 2,3-butanediol. The enzyme activity was separated into two fractions, namely enzyme I and II on DEAE-Sephadex A-25 column chromatography. The enzyme I was different from the enzyme II in the ratio of enzyme activity to 1,2-propanediol and 2,3-butanediol, heat stability, pH stability and pH optimum, and effect of 2-mercaptoethanol.     

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.     

Loss of aldehyde dehydrogenase in an Escherichia coli mutant selected for growth on the rare sugar L-galactose.

Escherichia coli K-12 converts L-fucose to dihydroxyacetone phosphate (C-1 to C-3) and L-lactaldehyde (C-4 to C-6) by a pathway specified by the fuc regulon. Aerobically, L-lactaldehyde serves as a carbon and energy source by the action of an aldehyde dehydrogenase of broad specificity; the product, L-lactate, is then converted to pyruvate. Anaerobically, L-lactaldehyde serves as an electron acceptor to regenerate NAD from NADH by the action of an oxidoreductase; the reduced product, L-12-propanediol, is excreted. A strain selected for growth on L-galactose (a structural analog of L-fucose) acquired a broadened inducer specificity because of an altered fucR gene encoding the activator protein for the fuc regulon (Y. Zhu and E. C. C. Lin, J. Mol. Evol. 23:259-266, 1986). In this study, a second mutation that abolished aldehyde dehydrogenase activity was discovered. The L-fucose pathway converts L-galactose to dihydroxyacetone phosphate and L-glyceraldehyde. Aldehyde dehydrogenase then converts L-glyceraldehyde to L-glycerate, which is toxic. Loss of the dehydrogenase averts the toxicity during growth on L-galactose, but reduces by one-half the aerobic growth yield on L-fucose. When mutant cells induced in the L-fucose system were incubated with radioactive L-fucose, accumulation of radioactivity occurred if the substrate was labeled at C-1 but not if it was labeled C-6. Complete aerobic utilization of carbons 4 through 6 of L-fucose depends not only on an adequate activity of aldehyde dehydrogenase to trap L-lactaldehyde as its anionic acid but also on the lack of L-1,2-propanediol oxidoreductase activity, which converts L-lactaldehyde to a readily excreted alcohol.