Effects of over-expression of glycerol dehydrogenase and 1,3-propanediol oxidoreductase on bioconversion of glycerol into 1,3-propandediol by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> under micro-aerobic conditions

Glycerol dehydrogenase (GDH) and 1,3-propanediol (1,3-PD) oxidoreductase had been proved two key enzymes for 1,3-PD production by Klebsiella pneumoniae. Fed-batch fermentations of the recombinant K. pneumoniae strains, over-expressing the two enzymes individually, were carried out under micro-aerobic conditions, and the behaviors of the recombinants were investigated. Results showed that over-expression of 1,3-PD oxidoreductase did not affect the concentration of 1,3-PD. However, it enhanced the molar yield from 50.6 to 64.0% and reduced the concentration of by-products. Among them, the concentrations of lactic acid, ethanol and succinic acid were decreased by 51.8, 50.6 and 47.4%, respectively. Moreover, in the recombinant the maximal concentration of 3-hydroxypropionaldehyde decreased by 73.6%. Over-expression of GDH decreased the yield of ethanol and 2,3-butanediol, meanwhile it increased the concentration of acetic acid. No significant changes were observed both in 1,3-PD yield and glycerol flux distributed to oxidative branch.     

3-Hydroxypropionaldehyde guided glycerol feeding strategy in aerobic 1,3-propanediol production by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

3-Hydroxypropionaldehyde (3-HPA) is a toxic intermediary metabolite in the biological route of 1,3-propanediol biosynthesis from glycerol. 3-HPA accumulated in culture medium would arouse an irreversible cessation of the fermentation process. The role of substrate (glycerol) on 3-HPA accumulation in aerobic fermentation was investigated in this paper. 1,3-Propanediol oxidoreductase and glycerol dehydratase, two key enzyme catalyzing reactions of 3-HPA formation and consumption, were sensitive to high concentration of 3-HPA. When the concentration of 3-HPA increased to a higher level in medium (ac 10 mmol/L), the activity of 1,3-propanediol oxidoreductase in cell decreased correspondingly, which led to decrease of the 3-HPA conversion rate, then the 3-HPA concentration increasing was accelerated furthermore. 3-HPA accumulation in culture medium was triggered by this positive feedback mechanism. In the cell exponential growth phase, the reaction catalyzed by 1,3-propanediol oxidoreductase was the rate limiting step in 1,3-propanediol production. The level of 3-HPA in culture medium could be controlled by the substrate (glycerol) concentration, and lower level of glycerol could avoid 3-HPA accumulating to a high, lethal concentration. In fed batch fermentation, under the condition of initial glycerol concentration 30 g/L, and keeping glycerol concentration lower than 7–8 g/L in cell exponential growth phase, 3-HPA accumulation could not be incurred. Based on this result, a glycerol feeding strategy was set up in fed batch fermentation. Under the optimized condition, 50.1 g/L of 1,3-propanediol was produced in 24 h, and 73.1 g/L of final 1,3-propanediol concentration was obtained in 54 h.     

Enhanced 1,3-propanediol production in recombinant <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> carrying the gene <Emphasis Type="Italic">yqh</Emphasis>D encoding 1,3-propanediol oxidoreductase isoenzyme

The yqhD gene from Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme (PDORI) and the tetracycline resistant gene (tetR) from plasmid pHY300PLK were amplified by PCR. They were inserted into vector pUC18, yielding the recombinant expression vector pUC18-yqhD-tetR. The recombinant vector was then cloned into Klebsiella pneumoniae ME-308. The overexpression of PDORI in K. pneumoniae surprisingly led to higher 1,3-propanediol production. The final 1,3-propanediol concentration of recombinant K. pneumoniae reached 67.6 g/l, which was 125.33% of that of the original strain. The maximum activity of recombinant PDORI converting 3-HPA to 1,3-PD reached 110 IU/mg after induction by IPTG at 31°C during the fermentation, while it was only 11 IU/mg under the same conditions for the wild type strain. The K _m values of the purified PDORI for 1,3-propanediol and NADP were 12.1 mM and 0.15 mM, respectively. Compared with the original strains, the concentration of the toxic intermediate 3-hydroxypropionaldehyde during the fermentation was also reduced by 22.4%. Both the increased production of 1,3-propanediol and the reduction of toxic intermediate confirmed the significant role of 1,3-propanediol oxidoreductase isoenzyme from E. coli in converting 3-hydroxypropionaldehyde to 1,3-propanediol for 1,3-PD production.     

Metabolic flux and robustness analysis of glycerol metabolism in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The knowledge of the mechanism of flux distribution will benefit understanding cell physiology and regulation of metabolism. In this study, the measured fluxes obtained under steady-state conditions were used to estimate intracellular fluxes and identify the robustness of branch points of the anaerobic glycerol metabolism in Klebsiella pneumoniae for the production of 1,3-propanediol by metabolic flux analysis. The biomass concentration increased as NADH₂/NAD⁺ decreased at low initial concentration and inversed at high initial glycerol concentration. The flux distribution revealed that the branch points of glycerol and dihydroxyacetonephosphate were rigid to the environmental conditions. However, the pyruvate and acetyl coenzyme A metabolisms gave cells the flexibility to regulate the energy and intermediate fluxes under various environmental conditions. Additionly, it was found that the formation rate of ethanol and the ratio of pyruvate dehydrogenase to pyruvate formate lyase appeared visible fluctuations at high glycerol uptake rate.     

Enhancement of 1,3-propanediol Production by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> with Fumarate Addition

Addition of 5 mm fumarate to cultures of Klebsiella pneumoniae enhanced the rate of glycerol consumption and the production of 1,3-propanediol (PDO). Compared to the control, the activity of glycerol dehydrogenase increased by 35, 33 and 46%, the activity of glycerol dehydratase increased by 160, 210 and 115%, and the activity of 1,3-propanediol oxidoreductase increased by 25, 39 and 85% when, respectively, 5, 15 and 25 mm fumarate were provided. At the same time, the ratio of NAD⁺ to NADH decreased by 20, 23 and 29%. Using a 5 l bioreactor with 5 mM fumarate addition, the specific rate of glycerol consumption and the productivity of PDO was 30 mmol/l h and 17 mmol/l h, respectively, both increased by 35% over the control. Revisions requested 15 July 2005; Revisions received 30 August 2005     

Decrease of 3-hydroxypropionaldehyde accumulation in 1,3-propanediol production by over-expressing <Emphasis Type="Italic">dha</Emphasis>T gene in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> TUAC01

Glycerol can be biologically converted to 1,3-propanediol, a key raw material required for the synthesis of polytrimethylene terephthalate and other polyester fibers. In 1,3-propanediol synthesis pathway, 3-hydroxypropionaldehyde (3-HPA) was an inhibitory intermediary metabolite. The accumulation of 3-HPA in broth would cause an irreversible cessation of the fermentation process. With the object of reducing 3-HPA level in the fermentation broth, dhaT gene which encodes 1,3-propanediol oxidoreductase (PDOR) was cloned and over expressed in 1,3-propanediol producing bacterium Klebsiella pneumoniae TUAC01. dhaT gene was linked downstream of the ptac promoter in an expressing vector pDK6 to form plasmid pDK-dhaT. The newly formed pDK-dhaT was transformed to K. pneumoniae TUAC01. Under the inducement of IPTG, PDOR was over-expressed when the constructed strain was cultured on an LB medium or a fermentation medium. A 5 L scale-up fermentation experiment was done to test the 3-HPA accumulation in broth, with the initial substrate glycerol 30 g/L; the peak levels of 3-HPA in broth were 7.55 and 1.49 mmol/L for control host strain and the constructed strain, respectively. In 50 g/L initial glycerol experiment, the peak level of 3-HPA in broth was 12.57 and 2.02 mmol/l for the control host strain and the constructed strain, respectively. Thus the fermentation cessation caused by the toxicity of 3-HPA was alleviated in the constructed strain.     

Multiple growth inhibition of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in 1,3-propanediol fermentation

The inhibition of substrate and product on the growth of Klebsiella pneumoniae in anaerobic and aerobic batch fermentation for the production of 1,3-propanediol was studied. The cells under anaerobic conditions had a higher maximum specific growth rate of 0.19 h^–1 and lower tolerance to 110 g glycerol l^–1, compared to the maximum specific growth rate of 0.17 h^–1 and tolerance to 133 g glycerol l^–1 under aerobic conditions. Acetate was the main inhibitory metabolite during the fermentation under anaerobic conditions, with lactate and ethanol the next most inhibitory. The critical concentrations of acetate, lactate and ethanol were assessed to be 15, 19, 26 g l^–1, respectively. However, cells grown under aerobic conditions were more resistant to acetate and lactate but less resistant to ethanol. The critical concentrations of acetate, lactate and ethanol were assessed to be 24, 26, and 17 g l^–1, respectivelyRevisions requested 8 september; Revisions received 2 November 2004     

Enhanced 2,3-butanediol production by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> SDM

Enhanced 2,3-butanediol (BD) production was carried out by Klebsiella pneumoniae SDM. The nutritional requirements for BD production by K. pneumoniae SDM were optimized statistically in shake flask fermentations. Corn steep liquor powder and (NH₄)₂HPO₄ were identified as the most significant factors by the two-level Plackett–Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform fed-batch fermentations with K. pneumoniae SDM. BD production was then studied in a 5-l bioreactor applying different fed-batch strategies, including pulse fed batch, constant feed rate fed batch, constant residual glucose concentration fed batch, and exponential fed batch. The maximum BD concentration of 150 g/l at 38 h with a diol productivity of 4.21 g/l h was obtained by the constant residual glucose concentration feeding strategy. To the best of our knowledge, these results were new records on BD fermentation. Cuiqing Ma and Ailong Wang contributed equally to this work.     

Optimization of culture conditions for 1,3-propanediol production from crude glycerol by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> using response surface methodology

To produce 1,3-propanediol (1,3-PD) from crude glycerol, cultivation conditions were optimized by response surface methodology (RSM) based on a 2⁵ factorial central composite design (CCD). RSM was adopted to derive a statistical model for the individual and interactive effects of crude glycerol, (NH₄)₂SO₄, pH, cultivation time and temperature on the production of 1,3-PD. Optimal conditions for maximum 1,3-PD production were as follows: crude glycerol, 35 g/L; (NH₄)₂SO₄, 8 g/L; pH, 7.37; cultivation time, 10.8 h; temperature, 36.88°C. Under these optimal conditions, the design expert presented the maximal numerical solution with a predicted 1,3-PD production level of up to 13.74 g/L. The experimental production of 1,3-PD yielded 13.8 g/L, which was in close agreement with the model prediction.     

Production of 1,3-propanediol by Klebsiella pneumoniae from glycerol broth

Broth containing 152 g glycerol l⁻¹ from Candida krusei culture was converted to 1,3-propanediol by Klebsiella pneumoniae. Residual glucose in the broth promoted growth of K. pneumoniae while acetate was inhibitory. After desalination treatment of glycerol broth by electrodialysis, the acetate in the broth was removed. A fed-batch culture with electrodialytically pretreated broth as␣substrate was developed giving 53 g 1,3- propanediol l⁻¹ with a yield of 0.41 g g⁻¹ glycerol and a productivity of 0.94 g l⁻¹ h⁻¹.     

A simple method for extracting C-phycocyanin from <Emphasis Type="Italic">Spirulina platensis</Emphasis> using <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

C-phycocyanin (C-PC) was extracted from fresh Spirulina platensis by deploying a species of non-pathogenic nitrogen-fixing bacteria, namely, Klebsiella pneumoniae. The algal slurry was neither washed nor centrifuged; the bacterial culture was poured into the slurry, the vessel sealed, and crude C-PC extracted after about 24 h. The extraction was clean and efficient, and the purity and concentration of C-PC proved to be of adequate quality.     

Production of 1,3-propanediol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> VPI 3266 using a synthetic medium and raw glycerol

Growth inhibition of Clostridium butyricum VPI 3266 by raw glycerol, obtained from the biodiesel production process, was evaluated. C. butyricum presents the same tolerance to raw and to commercial glycerol, when both are of similar grade, i.e. above 87% (w/v). A 39% increase of growth inhibition was observed in the presence of 100 g l^–1 of a lower grade raw glycerol (65% w/v). Furthermore, 1,3-propanediol production from two raw glycerol types (65% w/v and 92% w/v), without any prior purification, was observed in batch and continuous cultures, on a synthetic medium. No significant differences were found in C. butyricum fermentation patterns on raw and commercial glycerol as the sole carbon source. In every case, 1,3-propanediol yield was around 0.60 mol/mol glycerol consumed.     

Microbial production of 1,3-propanediol from glycerol by Klebsiella pneumoniae under micro-aerobic conditions up to a pilot scale

1,3-Propanediol (1,3-PD) can be produced from glycerol by Klebsiella pneumoniae under micro-aerobic conditions. Recently, this fed-batch fermentation process has been successfully scaled up to 1 m³. The final 1,3-PD concentration, molar yield and volumetric productivity of 72 g l⁻¹, 57% and 2.1 g l⁻¹ h⁻¹, respectively, are close to those of 75 g l⁻¹, 61%, and 2.2 g l⁻¹ h⁻¹ under anaerobic conditions. This process would be suitable for the production of 1,3-PD on a large scale.     

Effect of antibacterials on surface properties and adhesion to uroepithelial cells in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The effect of sub-inhibitory and inhibitory concentrations of antimicrobials including aminoglycosides, third generation cephalosporins and quinolones on the surface properties and adhesion of Klebsiella pneumoniae to uroepithelial cells (UECs) was examined. Antibiotics, ceftazidime and ofloxacin at 1/4, 1/8 × MIC and minimum inhibitory concentration (MIC) induced filament formation in bacteria, however cells treated with amikacin were similar in length to control organisms but showed a rough topology under the scanning electron microscope. An increase in bacterial hydrophobicity and decrease in uronic acid content were noted in the presence of ceftazidime and ofloxacin at MIC and sub-MIC level. However, amikacin at MIC level caused decreased hydrophobicity of the cells and the uronic content remained the same. This study clearly indicates that, although ceftazidime and ofloxacin brought about profound changes in cell surface characteristics, these changes did not result in any advantage to the bacterial cell in terms of adhesion. In contrast, with amikacin, which did not show any appreciable change in cell morphology or surface topology, exposure markedly increased the adherence of bacteria to UECs, indicating that the prophylactic use of this antibiotic not only induces resistance in bacteria but can also promote the colonization of UECs.     

A potential antimicrobial treatment against ESBL-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> using the tellurium compound AS101

Due to the extensive spread of antibiotic-resistant Klebsiella pneumoniae, the non-toxic immunomodulator, ammonium trichloro (dioxoethylene-o, o′) tellurate (AS101), was introduced for the first time in this study. Eleven strains of K. pneumoniae were tested: five were extended spectrum beta lactamase (ESBL)-producing strains and six were non-ESBL-producing strains. The MIC and MBC of ten strains were 9 μg/ml AS101 and 18 μg/ml for one strain. AS101 treatment inhibited bacterial growth in a dose-dependent manner on protein-rich media. No inhibition by AS101 was observed on poorer media. In combination with β-mercaptoethanol (2-ME) or cysteamine, AS101 inhibited bacterial growth in both types of media. Growth inhibition was also shown following AS101 treatment at both lag and log phases. Our data indicate that AS101 enters the bacterium through its porins, causing bacterial destruction. The mechanism of cell death was characterized using several techniques: (a) scanning electron microscopy showed that bacteria treated with AS101 or in combination with cysteamine exhibited evidence of cell-wall damage; (b) X-ray microanalysis demonstrated damage to Na/K pumps; and (c) transmission electron microscopy demonstrated cell lysis. These phenomena suggest that AS101 has antibacterial potential against K. pneumoniae infections. B. Sredni and Y. Nitzan were equal collaborators in this research.     

Regulation of expression of Na<Superscript>+</Superscript>-translocating NADH:quinone oxidoreductase genes in <Emphasis Type="Italic">Vibrio harveyi</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The expression of genes encoding sodium-translocating NADH:quinone oxidoreductase (Na⁺-NQR) was studied in the marine bacterium Vibrio harveyi and in the enterobacterium Klebsiella pneumoniae. It has been shown that such parameters as NaCl concentration, pH value, and presence of an uncoupler in the growth media do not influence significantly the level of nqr expression. However, nqr expression depends on the growth substrates used by these bacteria. Na⁺-NQR is highly repressed in V. harveyi during anaerobic growth, and nqr expression is modulated by electron acceptors and values of their redox potentials. The latter effect was shown to be independent of the ArcAB regulatory system. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Accession number: EF394942 (Vibrio harveyi arcB gene, partial cds).     

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.     

Anaerobic glycerol production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains under hyperosmotic stress

Glycerol formation is vital for reoxidation of nicotinamide adenine dinucleotide (reduced form; NADH) under anaerobic conditions and for the hyperosmotic stress response in the yeast Saccharomyces cerevisiae. However, relatively few studies have been made on hyperosmotic stress under anaerobic conditions. To study the combined effect of salt stress and anaerobic conditions, industrial and laboratory strains of S. cerevisiae were grown anaerobically on glucose in batch-cultures containing 40 g/l NaCl. The time needed for complete glucose conversion increased considerably, and the specific growth rates decreased by 80–90% when the cells were subjected to the hyperosmotic conditions. This was accompanied by an increased yield of glycerol and other by-products and reduced biomass yield in all strains. The slowest fermenting strain doubled its glycerol yield (from 0.072 to 0.148 g/g glucose) and a nearly fivefold increase in acetate formation was seen. In more tolerant strains, a lower increase was seen in the glycerol and in the acetate, succinate and pyruvate yields. Additionally, the NADH-producing pathway from acetaldehyde to acetate was analysed by overexpressing the stress-induced gene ALD3. However, this had no or very marginal effect on the acetate and glycerol yields. In the control experiments, the production of NADH from known sources well matched the glycerol formation. This was not the case for the salt stress experiments in which the production of NADH from known sources was insufficient to explain the formed glycerol.