3-hydroxypropionic Acid Research Articles

Production of 3-hydroxypropionic acid through malonyl CoA pathway from glucose by recombinant Escherichia coli BL21

Production of 3-hydroxypropionic acid through malonyl CoA pathway from glucose by recombinant Escherichia coli BL21

Identification and characterization of the propanediol utilization protein PduP of Lactobacillus reuteri for 3-hydroxypropionic acid production from glycerol

Although the de novo biosynthetic mechanism of 3-hydroxypropionic acid (3-HP) in glycerol-fermenting microorganisms is still unclear, the propanediol utilization protein (PduP) of Lactobacillus species has been suggested to be a key enzyme in this regard. To verify this hypothesis, a pduP gene from Lactobacillus reuteri was cloned and expressed, and the encoded protein was characterized. Recombinant L. reuteri PduP exhibited broad substrate specificity including 3-hydroxypropionaldehyde and utilized both NAD(+) and NADP(+) as a cofactor. Among various aldehyde substrates tested, the specific activity was highest for propionaldehyde, at pH 7.8 and 37 °C. The K(m) and V(max) values for propionaldehyde in the presence of NAD(+) were 1.18 mM and 0.35 U mg⁻¹, respectively. When L. reuteri pduP was overexpressed in Klebsiella pneumoniae, 3-HP production remarkably increased as compared to the wild-type strain (from 0.18 g L⁻¹ to 0.72 g L⁻¹) under shake-flask culture conditions, and the highest titer (1.38 g L⁻¹ 3-HP) was produced by the recombinant strain under batch fermentation conditions in a bioreactor. This is the first report stating the enzymatic properties of PduP protein and the probable role in biosynthesis of 3-HP in glycerol fermentation.

Preparation and Application of Hybrid SBS Bipolar Membrane in Electro-Generation of HPAc and Succinic Acid

The hybrid SBS-g-(DMAEMA-SiO2)/SBS-g-(AA-StSO3Na-SiO2) bipolar membrane, SBS BPM was prepared by SBS (styrene-butadiene-styrene triblock copolymer) grafted with acrylic acid (AA), sodium sulfonic styene(StSO3Na) through free radical grafting reaction, then the grafting copolymer and γ-mercaptopropyltrimethoxysilane (KH-590) were exposed to UV illumination, and the SiO2 doped anode membrane was obtained through sol–gel reaction of trimethoxysilane group, as similar process the cathode membrane was prepared by SBS grafted with dimethyl amino ethyl methacrylate (DMAEMA) and KH-570. The SBS BPM exhibited good mechanical strength (tensile strength, TS is 1.55MPa) and high temperature tolerance (thermal degradation temperature in N2 is in the range of 230–250°C). The ion exchange capacities (IECs) of SBS-g-(DMAEMA-SiO2) and SBS-g-(AA-StSO3 Na-SiO2) were in the region of 1.42–2.82 mmol/g. The SEM images show that the membrane cross- sections are very smooth where no silica particles could be observed. SBS-g-(DMAEMA-SiO2)/ SBS- g-(AA-StSO3Na-SiO2) bipolar membrane was used as the separator in the electrolysis cell for electro- generation of 2,2-dimethyl-3- hydroxypropionic acid(HPAc) and succinic acid in pairs. The yield of HPAc and the corresponding average current efficiency in the anode chamber were 28.2% and 53.8％, respectively. The yield of succinic acid and the average current efficiency in the cathode chamber were 74.1% and 62.5%, respectively.

Stimulation of reductive glycerol metabolism by overexpression of an aldehyde dehydrogenase in a recombinant Klebsiella pneumoniae strain defective in the oxidative pathway

Previously, we constructed a glycerol oxidative pathway-deficient mutant strain of Klebsiella pneumoniae by inactivation of glycerol dehydrogenase (dhaD) to eliminate by-product synthesis during production of 1,3-propanediol (1,3-PD) from glycerol. Although by-product formation was successfully blocked in the resultant strain, the yield of 1,3-PD was not enhanced, probably because dhaD disruption resulted in insufficient regeneration of the cofactor NADH essential for the activity of 1,3-PD oxidoreductase (DhaT). To improve cofactor regeneration, in the present study we overexpressed an NAD(+)-dependent aldehyde dehydrogenase in the recombinant strain. To this end, an aldehyde dehydrogenase AldHk homologous to E. coli AldH but with NAD(+)-dependent propionaldehyde dehydrogenase activity was identified in K. pneumoniae. Functional analysis revealed that the substrate specificity of AldHk embraced various aldehydes including propionaldehyde, and that NAD(+) was preferred over NADP(+) as a cofactor. Overexpression of AldHk in the glycerol oxidative pathway-deficient mutant AK/pVOTHk resulted in a 3.6-fold increase (0.57 g l(-1) to 2.07 g l(-1)) in the production of 3-hydroxypropionic acid (3-HP), and a 1.1-fold enhancement (8.43 g l(-1) to 9.65 g l(-1)) of 1,3-PD synthesis, when glycerol was provided as the carbon source, compared to the levels synthesized by the control strain (AK/pVOT). Batch fermentation using AK/pVOTHk showed a significant increase (to 70%, w/w) in conversion of glycerol to the reductive metabolites, 1,3-PD and 3-HP, with no production of by-products except acetate.

Ergometría y cambio climático

Introduction and objectives In the short history of ergonomics (approximately 50 years) there have been notable changes in the atmosphere that we breathe, such as large, medium and small particles, as well as the gas composition, with increases in carbon dioxide (CO 2) of about 125%. This situation becomes worse within the buildings where the physiology exercise laboratories are located. The objective of this study was to determine how these atmospheric changes affect humans during exercise. Methods A comparative study was conducted by means of 2 paired ergometric bicycle tests on 13 subjects (12 males and 1 female). One was carried out in the normal laboratory situation (indoor), and the repeat was done in the same laboratory, with a bubble with a system that filtered large, medium and small particles, breathing the air outside the laboratory (outdoor). The parameters that were controlled were: the maximum power achieved on the ergometric bicycle expressed in watts (W), the ergospirometer parameters (VO 2max, VCO 2max, VE max), cardiological parameters: heart beats per minute and 2-hydroxypropanoic acid (La ++) levels and arterialised capillary blood glucose. Results The ergospirometer and cardiac parameters, or those associated with the power achieved on the ergometric bicycle did not change statistically, when we compared the two situations studied. However, the subjects did have higher levels of arterialised capillary lactate (+117%) 3 min after finishing the indoor situation test (7.55±1.81 vs 6.44±1.76 mMol/dl, P<0.016; n=13). We observed identical behaviour in the capillary blood glucose levels, which showed an increase of 112% in the usual situation (indoor) compared to those in the purified (outdoor) air bubble (blood glucose: 90.0±12.2 mg/dl vs 82.15±6.94 mg/dl; P>0.054 (not significant, n=13). Discussion The blood gas analysers for metabolic studies can be calibrated in different atmospheres and correctly determine the capacities and potential energy of these subjects, despite the atmospheric changes. The metabolic changes were sufficient to compensate for the different atmospheres compared, and enabled a similar level of physical performance to be expressed in the effort test and also in the cardiac behaviour during the same, considering the levels of contamination in a laboratory near Barcelona. Conclusions The subjects were able to adapt to the atmospheric changes owing to the gradual contamination. No differences were seen in the two situations established in the metabolic gas analyses under effort, and neither were there any changes in cardiac behaviour. The maximum potential obtained in the laboratory did not change. But, metabolically, a price was paid for atmospheric contamination, as shown by the higher mobilisation of glucose in capillary blood, and also in the higher production of capillary lactate under the conditions of the study.

The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems

The Rut pathway is composed of seven proteins, all of which are required by Escherichia coli K-12 to grow on uracil as the sole nitrogen source. The RutA and RutB proteins are central: no spontaneous suppressors arise in strains lacking them. RutA works in conjunction with a flavin reductase (RutF or a substitute) to catalyze a novel reaction. It directly cleaves the uracil ring between N-3 and C-4 to yield ureidoacrylate, as established by both nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Although ureidoacrylate appears to arise by hydrolysis, the requirements for the reaction and the incorporation of (18)O at C-4 from molecular oxygen indicate otherwise. Mass spectrometry revealed the presence of a small amount of product with the mass of ureidoacrylate peracid in reaction mixtures, and we infer that this is the direct product of RutA. In vitro RutB cleaves ureidoacrylate hydrolytically to release 2 mol of ammonium, malonic semialdehyde, and carbon dioxide. Presumably the direct products are aminoacrylate and carbamate, both of which hydrolyze spontaneously. Together with bioinformatic predictions and published crystal structures, genetic and physiological studies allow us to predict functions for RutC, -D, and -E. In vivo we postulate that RutB hydrolyzes the peracid of ureidoacrylate to yield the peracid of aminoacrylate. We speculate that RutC reduces aminoacrylate peracid to aminoacrylate and RutD increases the rate of spontaneous hydrolysis of aminoacrylate. The function of RutE appears to be the same as that of YdfG, which reduces malonic semialdehyde to 3-hydroxypropionic acid. RutG appears to be a uracil transporter.

A Novel NAD+-dependent aldehyde dehydrogenase encoded by the puuC gene of Klebsiella pneumoniae DSM 2026 that utilizes 3-hydroxypropionaldehyde as a substrate

3-Hydroxypropionic acid (3-HP), a versatile and valuable platform chemical, has diverse industrial applications; but its biological production from glycerol is often limited by the capability of the enzyme aldehyde dehydrogenase (ALDH) to convert an intermediary compound, 3-hydroxypropionaldehyde (3-HPA), to 3-HP. In this study, we report a new ALDH, PuuC, from Klebsiella pneumoniae DSM 2026, that efficiently converts 3-HPA to 3-HP. The identified gene puuC was cloned, expressed in Escherichia coli, purified, and characterized for its properties. The recombinant enzyme with a molecular weight of 53.8 kDa exhibited broad substrate specificity for various aliphatic aldehydes, especially C2–C5 aldehydes. NAD+ was the preferred coenzyme for the oxidation of most aliphatic and aromatic aldehydes tested. The optimum pH and temperature for PuuC activity were pH 8.0 and 45°C. The K m values for 3-HPA and NAD+ were 0.48 and 0.09 mM, respectively. The activity of PuuC was enhanced in the presence of reducing agents such as 2-mercaptoethanol or dithiothreitol, while several metal ions, particularly Hg2+, Ag+, and Cu2+ inhibited its activity. The predicted structure of PuuC indicated the presence of K191 and E194 in close proximity to the glycine motif, suggesting that PuuC belongs to class 2 ALDHs.

Rapid dissection of a complex phenotype through genomic-scale mapping of fitness altering genes

The understanding and engineering of complex phenotypes is a critical issue in biotechnology. Conventional approaches for engineering such phenotypes are often resource intensive, marginally effective, and unable to generate the level of biological understanding desired. Here, we report a new approach for rapidly dissecting a complex phenotype that is based upon the combination of genome-scale growth phenotype data, precisely targeted growth selections, and informatic strategies for abstracting and summarizing data onto coherent biological processes. We measured at high resolution (125 NT) and for the entire genome the effect of increased gene copy number on overall biological fitness corresponding to the expression of a complex phenotype (tolerance to 3-hydroxypropionic acid (3-HP) in Escherichia coli). Genetic level fitness data were then mapped according to various definitions of gene–gene interaction in order to generate network-level fitness data. When metabolic pathways were used to define interactions, we observed that genes within the chorismate and threonine super-pathways were disproportionately enriched throughout selections for 3-HP tolerance. Biochemical and genetic studies demonstrated that alleviation of inhibition of either of these super-pathways was sufficient to mitigate 3-HP toxicity. These data enabled the design of combinatorial modifications that almost completely offset 3-HP toxicity in minimal medium resulting in a 20 g/L and 25-fold increase in tolerance and specific growth, respectively.

Solubilization and structural determination of a glycoconjugate which is assembled into the sheath of Leptothrix cholodnii

The sheath of Leptothrix cholodnii is constructed from a structural glycoconjugate, a straight-chained amphoteric heteropolysaccharide modified with glycine and cysteine. Though the structure of the glycan core is already determined, its modifications with amino acids and other molecules are not fully resolved. In this study, we aimed to determine the chemical structure of the glycoconjugate as a whole. Enantiomeric determination of cysteine in the sheath was performed and as a result, l-cysteine was detected. NMR spectroscopy was endeavored to determine overall structure of the glycoconjugate. Prior to NMR analysis, solubilization of the glycoconjugate was attempted by adding denaturing reagents or by derivatization. As far as tested, sulfonation by performic acid oxidation was suitable for solubilization, but further improvement was achieved by N-acetylation. The approximate molecular weight of the derivative was estimated to be 4.5 × 10 4 by size-exclusion chromatography. The NMR studies for the sulfonated glycoconjugate and its N-acetylated derivative revealed that the sheath glycoconjugate is a glycosaminoglycan consisting of a pentasaccharide repeating unit which is substoichiometrically esterified with 3-hydroxypropionic acid and stoichiometrically amidated with acetic acid and glycyl- l-cysteine.

Production of 3-hydroxypropionic acid by recombinant Klebsiella pneumoniae based on aeration and ORP controlled strategy

A biosynthetic pathway for the production of 3-hydroxypropionic acid (3-HP) from glycerol was estab- lished in recombinant Klebsiella pneumoniae by introducing the aldehyde dehydrogenase gene from Escherichia coli. The activity of aldehyde dehydrogenase, which oxidized 3-hydroxypropionaldehyde (3-HPA) to 3-HP, was detected and 3-HP was produced by the recombinant strains. Three different oxygen supply strategies, associated with measuring the oxidoreduction potential (ORP) during the fermentation under these conditions, were adopted for higher production of 3-HP by the recombinant cells. About 0.8 g/l 3-HP and more 1,3-propanediol production by the recombinant Klebsiella pneumoniae were obtained under completely aerobic conditions. Under micro-aerobic conditions, 3-HP production could be increased to 2.2 g/l and 1,3-propanediol production was almost the same as in the original strain. Under the anaerobic conditions, 1,3-propanediol was the main product and about 1.3 g/l 3-HP was produced. Finally, 3-HP produc- tion of the recombinant strain was increased to 2.8 g/l under micro-aerobic condition with a further two-stage ORP controlled strategy.

Development and evaluation of efficient recombinant Escherichia coli strains for the production of 3‐hydroxypropionic acid from glycerol

3-Hydroxypropionic acid (3-HP) is a commercially valuable chemical with the potential to be a key building block for deriving many industrially important chemicals. However, its biological production has not been well documented. Our previous study demonstrated the feasibility of producing 3-HP from glycerol using the recombinant Escherichia coli SH254 expressing glycerol dehydratase (DhaB) and aldehyde dehydrogenase (AldH), and reported that an "imbalance between the two enzymes" and the "instability of the first enzyme DhaB" were the major factors limiting 3-HP production. In this study, the efficiency of the recombinant strain(s) was improved by expressing DhaB and AldH in two compatible isopropyl-thio-beta-galactoside (IPTG) inducible plasmids along with glycerol dehydratase reactivase (GDR). The expression levels of the two proteins were measured. It was found that the changes in protein expression were associated with their enzymatic activity and balance. While cloning an alternate aldehyde dehydrogenase (ALDH), alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH), instead of AldH, the recombinant E. coli SH-BGK1 showed the highest level of 3-HP production (2.8 g/L) under shake-flask conditions. When an aerobic fed-batch process was carried out under bioreactor conditions at pH 7.0, the recombinant SH-BGK1 produced 38.7 g 3-HP/L with an average yield of 35%. This article reports the highest level of 3-HP production from glycerol thus far.

Combined Success for Efficient Catalysis

Catalysis is the key method to provide mankind with a reliable supply of all sorts of chemicals, while at the same time providing resource-saving, efficient, sustainable, and atom-economic processes. Hence, scientists worldwide have worked hard to develop new methods and routes for better synthetic strategies, with a strong emphasis on catalytic reactions. The main approaches used are heterogeneous and homogenous catalysis using purely chemical reagents, and biocatalysis using isolated enzymes or whole cell systems, such as microorganisms. Indeed, biocatalysis has emerged in the past two decades—boosted by tremendous developments in molecular biology, bioinformatics and protein engineering—from a rather limited discipline, restricted to a few 'useful' and commercially available enzymes, into a mature field with numerous applications already performed on an industrial scale. The most important products are without doubt chiral compounds, which serve as building blocks for the synthesis of pharmaceuticals and agrochemicals, but other areas of biocatalysis are also becoming increasingly important; for instance in the bulk production of 1,3-propanediol or 3-hydroxypropionic acid for polymer synthesis and in biofuels. Thus, mother nature’s ability to fashion a plethora of compounds by enzyme catalysis has been increasingly transferred to challenges in modern chemistry. However, biocatalysis need not be seen as “in competition with” heterogeneous or homogeneous chemical catalysis. The recent past has seen an increasing number of publications in which 'traditional' organic chemists have combined transition metal catalysis with enzymes to achieve, for instance, efficient dynamic kinetic resolutions or multistep one-pot syntheses. I am convinced that the future will see even more examples for the combination of chemocatalysis with enzyme catalysis, and that both areas will go further hand-in-hand towards the integration of different disciplines in multistep synthetic schemes. This, however, requires a platform to provide knowledge about all that is state-of-the-art in neighboring fields of catalysis research, and I feel that ChemCatChem will be best placed to perform this role. This new journal will certainly enable its broad readership the perfect basis for comparison of different approaches for the same goal: highly efficient catalysis. Hence, I am convinced that ChemCatChem will strengthen cross-communication between scientists, and that discoveries in one field of research will fertilize research in other areas to help solve the challenges the next generations of mankind have to face in the future.

Production of 3-Hydroxypropionic Acid from Acrylic Acid by Newly Isolated Rhodococcus erythropolis LG12

A novel microorganism, designated as LG12, was isolated from soil based on its ability to use acrylic acid as the sole carbon source. An electron microscopic analysis of its morphological characteristics and phylogenetic classification by 16S rRNA homology showed that the LG12 strain belongs to Rhodococcus erythropolis. R. erythropolis LG12 was able to metabolize a high concentration of acrylic acid (up to 40 g/l). In addition, R. erythropolis LG12 exhibited the highest acrylic acid-degrading activity among the tested microorganisms, including R. rhodochrous, R. equi, R. rubber, Candida rugosa, and Bacillus cereus. The effect of the culture conditions of R. erythropolis LG12 on the production of 3-hydroxypropionic acid (3HP) from acrylic acid was also examined. To enhance the production of 3HP, acrylic acid-assimilating activity was induced by adding 1 mM acrylic acid to the culture medium when the cell density reached an OD600 of 5. Further cultivation of R. erythropolis LG12 with 40 g/l of acrylic acid resulted in the production of 17.5 g/l of 3HP with a molar conversion yield of 44% and productivity of 0.22 g/I/h at 30 degrees after 72 h.

Effect of process parameters on 3-hydroxypropionic acid production from glycerol using a recombinant Escherichia coli

The top-valued platform chemical, 3-hydroxypropionic acid (3-HP), has a wide range of industrial applications but its biological production is not well established. Previously, the production of 3-HP from glycerol was demonstrated using a recombinant Escherichia coli strain expressing glycerol dehydratase (dhaB) and aldehyde dehydrogenase (aldH). The present investigation focuses on the effect of the culture conditions on the production of 3-HP from glycerol. The physicochemical parameters, such as pH, IPTG concentration, liquid-to-flask volume ratio, and substrate concentration, were examined in flask-scale experiments and obtained the highest titer of 3-HP at 4.4 g l(-1) in 48 h. When a fed-batch process was carried out in a bioreactor under pH-regulated conditions, the recombinant E. coli produced 3-HP at 31 g l(-1) in 72 h with a yield of 0.35 mol mol(-1) glycerol. The maximum specific rate of 3-HP production was estimated to be 3.41 mmol g(-1) cdw h(-1) between 12 and 24 h. Other than 3-HP, propionic acid (3.4 g l(-1)), 1,3-propanediol (2.4 g l(-1)), and lactic acid (1.6 g l(-1)) were produced as the major by-products. This paper reports for the first time a commercially meaningful high titer of 3-HP production.

Biosynthetic pathways for 3-hydroxypropionic acid production

Biobased platform chemicals have attracted growing interest recently. Among them, 3-hydroxypropionic acid receives significant attention due to its applications in the synthesis of novel polymer materials and other derivatives. To establish a biotechnology route instead of the problematic chemical synthesis of 3-hydroxypropionic acid, biosynthetic pathway is required, and the strategies of how to engineer a microbe to produce this product should be considered. In the present review, we summarize and review all known pathways, which could be potentially constructed for 3-hydroxypropionic acid production. Mass and redox balances are discussed in detail. Thermodynamic favorability is evaluated by standard Gibbs free energy. The assembly of pathways and possible solutions are proposed. Several new techniques and future research needs are also covered.

Oxidation of Allyl Alcohol in the Presence of a Gold Catalyst: A Route to 3‐Hydroxypropionic Acid

Paving the route with gold: 3-Hydroxypropionic acid is considered to be a potential building block for both organic synthesis and high-performance polymers. However, there is currently no large-scale chemical process to produce this important molecule. With this aim in mind, a new approach is proposed: aerobic oxidation of allyl alcohol to 3-hydroxypropionic acid using a supported Au/C catalyst.

Cloning, expression, and characterization of an aldehyde dehydrogenase from Escherichia coli K-12 that utilizes 3-Hydroxypropionaldehyde as a substrate

3-Hydroxypropionaldehyde (3-HPA), an intermediary compound of glycerol metabolism in bacteria, serves as a precursor to 3-Hydroxypropionic acid (3-HP), a commercially valuable platform chemical. To achieve the effective conversion of 3-HPA to 3-HP, an aldH gene encoding an aldehyde dehydrogenase in Escherichia coli K-12 (AldH) was cloned, expressed, and characterized for its properties. The recombinant AldH exhibited broad substrate specificity for various aliphatic and aromatic aldehydes. AldH preferred NAD+ over NADP+ as a cofactor for the oxidation of most aliphatic aldehydes tested. Among the aldehydes used, the specific activity was highest (38.1 U mg(-1) protein) for 3-HPA at pH 8.0 and 37 degrees C. The catalytic efficiency (kcat) and the specificity constant (kcat/Km) for 3-HPA in the presence of NAD+ were 28.5 s(-1) and 58.6x10(3) M(-1) s(-1), respectively. The AldH activity was enhanced in the presence of disulfide reductants such as dithiothreitol (DTT) or 2-mercaptoethanol, while several metal ions, particularly Hg2+, Ag+, Cu2+, and Zn2+, inhibited AldH activity. This study illustrates that AldH is a potentially useful enzyme in converting 3-HPA to 3-HP.

Preparation and properties of polymer/zinc oxide nanocomposites using functionalized zinc oxide quantum dots

Poly(methyl methacrylate) (PMMA)/zinc oxide (ZnO) or carbazole polymer (PCEM)/ZnO nanocomposites, which are composed of high molecular weight PMMA or PCEM with narrow molecular weight distributions and ZnO nanoparticles, were successfully prepared by atom transfer radical polymerization (ATRP) initiated by 2-bromo-2-methylpropionyl (BMP) group (ZnBM) introduced onto the ZnO nanoparticle surfaces. Introduction of the BMP group onto the ZnO surfaces was achieved by esterification of OH group of the ZnO surfaces. The chemically attached OH group-having ZnO nanoparticles (ZnHM) were fabricated by sol-gel reaction from zinc acetate dihydrate, followed by treatment of the ZnO nanoparticles with 2-hydroxypropionic acid (HPA). The ZnHM nanoparticles showed one UV absorption and two emission bands: UV emission peak and broad visible emission band, while the ZnBM exhibited broad UV absorption and no emission spectra. The PMMA/ZnO nanocomposites displayed UV absorption and photoluminescent (PL) band with blue emission on the basis of the ZnHM nanoparticles, where the ZnO nanoparticles dispersed homogeneously in the PMMA matrix. The PCEM/ZnO nanocomposites depicted UV emission peak due to the carbazole unit in the UV range, but no visible emission. Thermal properties of the PMMA/ZnO nanocomposites were improved by dispersion of the ZnO nanoparticles into the PMMA, but the PCEM/ZnO nanocomposites showed no improvement of the thermal properties.

A Second Pathway to Degrade Pyrimidine Nucleic Acid Precursors in Eukaryotes

Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. Using a yeast model, Saccharomyces kluyveri, we show that during degradation, uracil is not reduced to dihydrouracil. Six loci, named URC1–6 (for uracil catabolism), are involved in the novel catabolic pathway. Four of them, URC3,5, URC6, and URC2 encode urea amidolyase, uracil phosphoribosyltransferase, and a putative transcription factor, respectively. The gene products of URC1 and URC4 are highly conserved proteins with so far unknown functions and they are present in a variety of prokaryotes and fungi. In bacteria and in some fungi, URC1 and URC4 are linked on the genome together with the gene for uracil phosphoribosyltransferase ( URC6). Urc1p and Urc4p are therefore likely the core components of this novel biochemical pathway. A combination of genetic and analytical chemistry methods demonstrates that uridine monophosphate and urea are intermediates, and 3-hydroxypropionic acid, ammonia and carbon dioxide the final products of degradation. The URC pathway does not require the presence of an active respiratory chain and is therefore different from the oxidative and rut pathways described in prokaryotes, although the latter also gives 3-hydroxypropionic acid as the end product. The genes of the URC pathway are not homologous to any of the eukaryotic or prokaryotic genes involved in pyrimidine degradation described to date.

Production of 3-hydroxypropionic acid from glycerol by a novel recombinant Escherichia coli BL21 strain

3-Hydroxypropionic acid (3-HP) is an important platform chemical from which several commodity and specialty chemicals can be generated. The present investigation focuses on the construction and evaluation of a recombinant strain Escherichia coli SH254 that produces 3-HP from glycerol. The strain was developed by cloning two genes, dhaB of Klebsiella pneumoniae DSM 2026 encoding glycerol dehydratase and aldH of E. coli K-12 MG1655 encoding aldehyde dehydrogenase, respectively. In vitro assays of crude enzyme extract of glycerol dehydratase (DhaB) showed 37.0 U mg −1 protein on glycerol with coenzyme B 12, and partially purified aldehyde dehydrogenase (AldH) exhibited 22.8 U mg −1 protein on 3-hydroxypropionaldehyde (3-HPA) with NAD + as a cofactor. When cultivated aerobically on a glycerol medium containing yeast extract, the recombinant E. coli SH254 produced 3-HP at a maximum of 6.5 mmol l −1 (0.58 g l −1). The highest specific rate and yield of 3-HP production were estimated as 6.6 mmol g −1 cdw h −1 and 0.48 mol mol −1 glycerol, respectively. Although not optimized extensively, this study is encouraging for further development of a bioprocess to produce 3-HP from glycerol.