Quantitative analysis on inactivation and reactivation of recombinant glycerol dehydratase from Klebsiella pneumoniae XJPD-Li

The genes encoding glycerol dehydratase were cloned and characterized by genomic DNA from Klebsiella pneumoniae XJPD-Li, and the assigned accession number EF634063 was available from the GenBank database. The DNA sequence analysis showed that the clone included three ORFs (dhaB, dhaC and dhaE, encoding α, β and γ subunit of glycerol dehydratase, respectively). Among three subunits of glycerol dehydratase, amino acid residues H₁₃, S₁₉₃, N₃₅₉, E₄₀₇, and M₅₁₅ of α subunit, N₄₇, L₁₅₀, V₁₈₉ of β subunit are different with what had been reported. Subsequently, the expression vector was constructed and transformed into E. coli BL21, and the colony carried genes of glycerol dehydratase were selected. SDS-PAGE examination showed that the three subunits were well expressed. The specific activity of recombined glycerol dehydratase reached to 0.299 U mg^?1, which was about 3 times comparing with that of the wild strain. The research also displayed that both glycerol and O₂ could inactive the glycerol dehydratase expressed in E. coli quickly in 10 min. The inactivated glycerol dehydratase could be effectively reactivated under the system as follows: the concentration of ATP, Mg²⁺ and coenzyme B₁₂ were 50 mM, 10 mM and 3 μM, respectively, when the ratio (W/W) of glycerol dehydratase to reactivation factor was 4:1. The O₂-inactivated and glycerol-inactivated dehydratase could be reactivated to 97.3% and 98.9% of initial activity in 10 min in above-mentioned conditions, respectively. The reactivation factor together with ATP was considered as the “ON/OFF” reactivating condition.     

Utilization of Glycerol as a Hydrogen Acceptor by Lactobacillus reuteri: Purification of 1,3-Propanediol:NAD+ Oxidoreductase

Lactobacillus reuteri utilizes exogenously added glycerol as a hydrogen acceptor during carbohydrate fermentations, resulting in higher growth rates and cell yields than those obtained during growth on carbohydrates alone. Glycerol is first converted to 3-hydroxypropionaldehyde by a coenzyme B₁₂-dependent glycerol dehydratase and then reduced to 1,3-propanediol by an NAD⁺ -dependent oxidoreductase. The latter enzyme was purified and determined to have a molecular weight of 180,000; it is predicted to exist as a tetramer of identical 42,000-molecular-weight subunits.     

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.     

Cloning, expression and reactivating characterization of glycerol dehydratase reactivation factor from Klebsiella pneumoniae XJPD-Li

Genes dhaF and dhaG encoding the α and β subunits of glycerol dehydratase reactivation factor (GDHtR) were amplified from the genomic DNA of Klebsiella pneumoniae XJPD-Li. The identity of the deduced amino acid sequence of the β subunit was relatively low compared with that of K. pneumoniae (U30903), where the 96th amino acid residue was found to be the more active amino acid histidine instead of glutamine in K. pneumoniae (U30903). A specific GDHtR activity of approximately 30 U/mg was attained in Escherichia coli BL21 (pET-28a (+)-dhaFG). His6-tagged GDHtR was purified by Ni-nitrilotriacetate chromatography, and the enzyme was purified 2.6-fold in a yield of 20.7%. The study showed that both glycerol and O₂-inactivated glycerol dehydratase (GDHt) could be quickly reactivated by GDHtR in the presence of ATP, Mg²⁺ and coenzyme B₁₂. However, the glycerol-inactivated GDHt was more easily reactivated than O₂-inactivated GDHt. In the first 10 min of the reactivation reaction, the average reactivation rate was 0.18 and 0.12 μmol/min for glycerol and O₂-inactivated GDHt, respectively.     

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.     

Enzymes Involved in Anaerobic Polyethylene Glycol Degradation by Pelobacter venetianus and Bacteroides Strain PG1

In extracts of polyethylene glycol (PEG)-grown cells of the strictly anaerobically fermenting bacterium Pelobacter venetianus, two different enzyme activities were detected, a diol dehydratase and a PEG-degrading enzyme which was characterized as a PEG acetaldehyde lyase. Both enzymes were oxygen sensitive and depended on a reductant, such as titanium citrate or sulfhydryl compounds, for optimal activity. The diol dehydratase was inhibited by various corrinoids (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, and methylcobalamin) by up to 37% at a concentration of 100 μM. Changes in ionic strength and the K⁺ ion concentration had only limited effects on this enzyme activity; glycerol inhibited the enzyme by 95%. The PEG-degrading enzyme activity was stimulated by the same corrinoids by up to 80%, exhibited optimal activity in 0.75 M potassium phosphate buffer or in the presence of 4 M KCI, and was only slightly affected by glycerol. Both enzymes were located in the cytoplasmic space. Also, another PEG-degrading bacterium, Bacteroides strain PG1, contained a PEG acetaldehyde lyase activity analogous to the corresponding enzyme of P. venetianus but no diol dehydratase. Our results confirm that corrinoid-influenced PEG degradation analogous to a diol dehydratase reaction is a common strategy among several different strictly anaerobic PEG-degrading bacteria.     

Towards a phylogeny of phototrophic purple sulfur bacteria — the genus Ectothiorhodospira

While most strains of heterofermentative lactobacilli and strains of Leuconostoc species contained only traces of a dehydratase reacting with glycerol or propanediol-1,2, three strains of Lactobacillus brevis and one strain of L. buchneri that metabolized glycerol readily in the presence of glucose, contained propanediol-1,2 dehydratase (EC 4.2.1.28). This cobamide requiring enzyme from L. brevis B 18 was partially purified. It reacts with the substrates propanediol-1,2, glycerol and ethanediol-1,2 with the relative activities of about 3:2:1. This ratio remained unchanged throughout the purification procedure. The substrate affinities were measured: propanediol-1,2 K _m=0.6 mM, glycerol K _m=4 mM, ethanediol-1,2 K _m=5.3 mM coenzyme B₁₂ (substrate glycerol) K _m=0.007 mM. The activity of the dehydratase was promoted by potassium or ammonium ions and inhibited by sodium, lithium, magnesium or specially manganese. The apparent molecular weight of propanediol-1,2 dehydratase was determined as M_r=180,000.Dedicated to Prof. Dr. H. G. Schlegel on behalf of his 60th birthday     

Immobilization of glycerol dehydrogenase on magnetic silica nanoparticles for conversion of glycerol to value-added 1,3-dihydroxyacetone

Abstract

Glycerol dehydrogenase (GlyDH) which oxidizes glycerol to the value-added chemical, 1,3-dihydroxyacetone, is of interest due to the oversupply of glycerol as a by-product of the biodiesel industry. To exploit the enzymatic oxidation of glycerol industrially, silica coated magnetic Fe₃O₄ nanoparticles were prepared and then activated with an amino-silane reagent for covalent immobilization of GlyDH via a glutaraldehyde linkage. At the optimal glutaraldehyde concentration of 0.05% (v/v), an enzyme loading of up to 57.5 mg/g-nanoparticles was achieved with 81.1% of the original activity retained. Reaction kinetic analysis indicated that the immobilized GlyDH had almost the same Michaelis-Menten constants for both NAD⁺ and glycerol as the free GlyDH did. However, after immobilization the turnover number k_cat of the GlyDH decreased from 164 s^?1 to 113 s^?1, and the reaction was 1.3-fold less sensitive to inhibition by DHA, which could compensate the decrease in k_cat. The immobilized GlyDH was also less sensitive to changes in pH and temperature, and showed a 5.3-fold improvement in thermal stability at 50^°C. Furthermore, excellent reusability was observed such that 10 cycles of re-use only led to 9% loss of enzyme activity.     

1,3-Propandiol production by engineered Hansenula polymorpha expressing dha genes from Klebsiella pneumoniae

Currently, 1,3-propanediol (1,3-PD) is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensive chemical synthesis. A methylotrophic yeast Hansenula polymorpha was engineered by expression of dhaB1, dhaB2, dhaB3, dhaB _RA1 and dhaB _RA2 encoding glycerol dehydratase complex and dhaT encoding 1,3-PD oxidoreductase from Klebsiella pneumoniae under direction of promoter of glyceraldehyde-3 phosphate dehydrogenase (GAPDH). The engineered recombinant yeast strain can produce 1,3-PD from glucose (2.4 g L⁻¹) as well as glycerol (0.8 g L⁻¹), which might lead to a safe and cost-effective method for industrial production of 1,3-PD from various biomass resources.     

Enhancement of gene detection frequencies by combining DNA-based stable-isotope probing with the construction of metagenomic DNA libraries

Summary DNA-based stable-isotope probing (SIP) using ¹³C-labeled growth substrates as bait is a powerful tool for the selective DNA isolation from microorganisms that are actively involved in consuming these substrates. To enhance the detection frequency of target genes in screens for new natural products, we have combined for the first time DNA-based SIP with the construction of metagenomic libraries. To isolate genes encoding coenzyme B₁₂-dependent glycerol dehydratases an enrichment of glycerol-fermenting microorganisms from a sediment sample of the Wadden Sea was performed by using glycerol–¹³C₃ as sole carbon source. Subsequently, the ¹³C-labeled DNA was separated from the naturally abundant ¹²C-DNA by density centrifugation, and used for library generation. Screening of the constructed libraries for the target genes revealed that the gene detection frequencies employing DNA-based SIP for enrichment of genomes harboring dehydratase genes were 2.1- to 3.8-fold higher than those recorded by using a traditional step with unlabeled glycerol for enrichment.     

Low-solubility glycerol dehydratase,a chimeric enzyme of coenzyme B<Subscript>12</Subscript>-dependent glycerol and diol dehydratases

Coenzyme B₁₂-dependent diol and glycerol dehydratases are isofunctional enzymes, which catalyze dehydration of 1, 2-diols to produce corresponding aldehydes. Although the two types of dehydratases have high sequence homology, glycerol dehydratase is a soluble cytosolic enzyme, whereas diol dehydratase is a low-solubility enzyme associated with carboxysome-like polyhedral organelles. Since both the N-terminal 20 and 16 amino acid residues of the β and γ subunits, respectively, are indispensable for the low solubility of diol dehydratase, we constructed glycerol dehydratase-based chimeric enzymes which carried N-terminal portions of the β and γ subunits of diol dehydratase in the corresponding subunits of glycerol dehydratase. Addition of the diol dehydratase-specific N-terminal 34 and 33 amino acid residues of the β and γ subunits, respectively, was not enough to lower the solubility of glycerol dehydratase. A chimeric enzyme which carries the low homology region (residues 35–60) of the diol dehydratase β subunit in addition to the diol dehydratase-specific extra-regions of β and γ subunits showed low solubility comparable to diol dehydratase, although its hydropathy plot does not show any prominent hydrophobic peaks in these regions. It was thus concluded that short N-terminal sequences are sufficient to change the solubility of the enzyme.     

Purification and Characterization of Glycerol Dehydratase from Lactobacillus reuteri

A coenzyme B₁₂-dependent glycerol dehydratase from Lactobacillus reuteri has been purified and characterized. The dehydratase has a molecular weight of approximately 200,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single major band with a molecular weight of 52,000. K_m values for substrates and coenzyme B₁₂ were in the millimolar and the submicromolar range, respectively.     

Construction and Characterization of a 1,3-Propanediol Operon

The genes for the production of 1,3-propanediol (1,3-PD) in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, are naturally under the control of two different promoters and are transcribed in different directions. These genes were reconfigured into an operon containing dhaB followed by dhaT under the control of a single promoter. The operon contains unique restriction sites to facilitate replacement of the promoter and other modifications. In a fed-batch cofermentation of glycerol and glucose, Escherichia coli containing the operon consumed 9.3 g of glycerol per liter and produced 6.3 g of 1,3-PD per liter. The fermentation had two distinct phases. In the first phase, significant cell growth occurred and the products were mainly 1,3-PD and acetate. In the second phase, very little growth occurred and the main products were 1,3-PD and pyruvate. The first enzyme in the 1,3-PD pathway, glycerol dehydratase, requires coenzyme B₁₂, which must be provided in E. coli fermentations. However, the amount of coenzyme B₁₂ needed was quite small, with 10 nM sufficient for good 1,3-PD production in batch cofermentations. 1,3-PD is a useful intermediate in the production of polyesters. The 1,3-PD operon was designed so that it can be readily modified for expression in other prokaryotic hosts; therefore, it is useful for metabolic engineering of 1,3-PD pathways from glycerol and other substrates such as glucose.     

3-Hydroxypropionaldehyde production from crude glycerol by <Emphasis Type="Italic">Lactobacillus diolivorans</Emphasis> with enhanced glycerol uptake

Background

In their quest for sustainable development and effective management of greenhouse gas emissions, our societies pursue a shift away from fossil-based resources towards renewable resources. With 95% of our current transportation energy being petroleum based, the application of alternative, carbon-neutral products—among them biodiesel—is inevitable. In order to enhance the cost structure of biodiesel biorefineries, the valorization of the crude glycerol waste stream into high-value platform chemicals is of major importance.

Results

The purpose of this study is the production of 3-hydroxypropionaldehyde (3-HPA) from biodiesel-derived crude glycerol by Lactobacillus diolivorans. Particular focus is given on overcoming potential limitations of glycerol transport into the cell, in order to use the cells’ total glycerol dehydratase capability towards the formation of 3-HPA as the main product. Recombinant overexpression of the endogenous glycerol uptake facilitating protein PduF results in a significant increase of glycerol conversion by a factor of 1.3. Concomitantly, glycerol dehydratase activity increased from initially 1.70 ± 0.03 U/mg protein to 2.23 ± 0.11 U/mg protein. With this approach, an average productivity of 4.8 g_3-HPA/(g_CDM h) yielding up to 35.9 g/L 3-HPA and 0.91 mol_3-HPA/mol_Glycerol have been obtained.

Conclusion

Lactobacillus diolivorans proves to be a valuable cell factory for the utilization of crude glycerol delivering high-value C3 chemicals like 3-HPA, 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP). Enhancing the glycerol influx into the cell by genetic engineering was successful paving the way towards the commercial production of 3-HPA.

     

Characterization of glycerol dehydratase expressed by fusing its α- and β-subunits

The gdh and gdhr genes, encoding B₁₂-dependent glycerol dehydratase (GDH) and glycerol dehydratase reactivase (GDHR), respectively, in Klebsiella pneumoniae, were cloned and expressed in E. coli. Part of the β-subunit was lost during GDH purification when co-expressing α, β and γ subunit. This was overcome by fusing the β-subunit to α- or γ-subunit with/without the insertion of a linker peptide between the fusion moieties. The kinetic properties of the fusion enzymes were characterized and compared with wild type enzyme. The results demonstrated that the fusion protein GDHALB/C, constructed by linking the N-terminal of β-subunit to the C-terminal of α subunit through a (Gly₄Ser)₄ linker peptide, had the greatest catalytic activity. Similar to the wild-type enzyme, GDHALB/C underwent mechanism-based inactivation by glycerol during catalysis and could be reactivated by GDHR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Allosteric interactions in glycerol dehydratase. Purification of enzyme and effects of positive and negative cooperativity for glycerol

An improved method for the purification of the apoenzyme (AB complex) of glycerol dehydratase from Aerobacter aerogenes is presented. One hundredfold purification Was achieved. This purification was possible due to stabilization of the AB complex by glycerol. Using chromatography on Sephadex G-200, the highest degree of association of AB complex was found in glycine buffer in the presence of glycerol.Potassium ions, in contrast to glycerol, seem to weaken the forces which bind subunits A and B together. This may be the action of potassium necessary for the performance of glycerol dehydratase activity, since potassium is required for holoenzyme activity.From kinetic studies it appears that the enzyme exhibits homotrophic effects with regard to glycerol binding sites, which can he extended from positive (in the presence of glycine buffer) to negative (in the presence of ethanolamine buffer) cooperativity. The high cooperativity between glycerol binding sites on the enzyme, in glycine buffer, can be abolished by the addition of phosphate. By decreasing the value of the Hill coefficient and increasing the V of the enzymatic reaction, phosphate seems to act as an allosteric activator.     

Adaptation of acidogenic sludge to increasing glycerol concentrations for biohydrogen production

Hydrogen is a promising alternative as an energetic carrier and its production by dark fermentation from wastewater has been recently proposed, with special attention to crude glycerol as potential substrate. In this study, two different feeding strategies were evaluated for replacing the glucose substrate by glycerol substrate: a one-step strategy (glucose was replaced abruptly by glycerol) and a step-by-step strategy (progressive decrease of glucose concentration and increase of glycerol concentration from 0 to 5 g L^?1), in a continuous stirred tank reactor (12 h of hydraulic retention time (HRT), pH 5.5, 35 °C). While the one-step strategy led to biomass washout and unsuccessful H₂ production, the step-by-step strategy was efficient for biomass adaptation, reaching acceptable hydrogen yields (0.4?±?0.1 mol_H2?mol^?1 _{glycerol consumed}) around 33 % of the theoretical yield independently of the glycerol concentration. Microbial community structure was investigated by single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) fingerprinting techniques, targeting either the total community (16S ribosomal RNA (rRNA) gene) or the functional Clostridium population involved in H₂ production (hydA gene), as well as by 454 pyrosequencing of the total community. Multivariate analysis of fingerprinting and pyrosequencing results revealed the influence of the feeding strategy on the bacterial community structure and suggested the progressive structural adaptation of the community to increasing glycerol concentrations, through the emergence and selection of specific species, highly correlated to environmental parameters. Particularly, this work highlighted an interesting shift of dominant community members (putatively responsible of hydrogen production in the continuous stirred tank reactor (CSTR)) according to the gradient of glycerol proportion in the feed, from the family Veillonellaceae to the genera Prevotella and Clostridium sp., putatively responsible of hydrogen production in the CSTR.     

Production of 1,3-propanediol from Glycerol by Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Using Incompatible Plasmids System

1,3-Propanediol (1,3-PD) has numerous applications in polymers, cosmetics, foods, lubricants, and medicines as a bifunctional organic compound. The genes for the production of 1,3-PD in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, and gdrAB, which encodes glycerol dehydratase reactivating factor, are naturally under the control of different promoters and are transcribed in different directions. These genes were coexpressed in E. coli using two incompatible plasmids (pET28a and pET22b) in the presence of selective pressure. The recombinant E. coli coexpressed the glycerol dehydratase, 1,3-propanediol oxidoreductase and reactivating factor for the glycerol dehydratase at high levels. In a fed-batch fermentation of glycerol and glucose, the recombinant E. coli containing these two incompatible plasmids consumed 14.3 g/l glycerol and produced 8.6 g/l 1,3-propanediol. In the substitution case of yqhD (encoding alcohol dehydrogenase from E. coli) for dhaT, the final 1,3-propanediol concentration of the recombinant E. coli could reach 13.2 g/l.     

Constituents of a Peptidal Antibiotic P168 Produced by Paecilomyces lilacinus (Thom) Samson

Two glycerol dehydrogenases, GD-I and GD-II, were purified from Geotrichum candidum by precipitation of ammonium sulfate, sequential column chromatography on Blue-Sepharose CL-4B and DEAE-Sepharose CL-6B and gel filtration on Cellulofine GC-700. The purified enzyme preparations were homogeneous upon disc gel electrophoresis. The molecular weights were estimated to be 135,000 for GD-I and 130,000 for GD-II, and their isoelectric points were 5.9 and 6.2, respectively. The specific activity of GD-I was twice that of GD-II. However, their other properties were very similar: Their optimum pHs for the oxidation of glycerol were 10.5, and those for the reduction of dihydroxyacetone were 5.5. The enzyme activities were highly activated by Cl-, but inhibited by PO₄^3?, BO₃^3? and 2-mercaptoethanol. The enzymes showed highest activity for glycerol, but also acted on several analogs of glycerol.