Earticle

An aldehyde dehydrogenase (AldH), from Klebsiella pneumoniae was cloned, expressed, and characterized for its properties in E. coli BL21(DE3). The enzyme effectively converts 3-hydroxypropionaldehyde (3-HPA), an intermediary compound of glycerol metabolism in bacteria, to 3-hydroxypropionic acid (3-HP), a commercially valuable platform chemical. It also exhibited broad substrate specificity for various aliphatic and aromatic aldehydes. The enzyme showed preference towards NAD+ over NADP+ as a cofactor for the oxidation of most aliphatic aldehydes. When several aldehydes tested, the specific activity of 27.7 U mg-1 protein was recorded for 3-HPA at pH 8.0 and 45°C. The halfsaturation constant (Km) and the specificity constant (kcat) for 3-HPA in the presence of NAD+ were 0.48 mM and 41.44ⅹ103 M-1 S-1, respectively. The AldH activity was enhanced in the presence of disulfide reductants such as dithiothreitol, 2-mercaptoethanol, or semicarbazide hydrochloride. Reduction in the AldH activity was noted in the presence of certain metal ions like Hg2+, Ag+, Cu2+, Fe2+ and Zn2+. This study demonstrated that AldH of K. pneumoniae was very specific towards NAD+ and highly active in converting 3-HPA to 3-HP.

The co-production of 3-hydroxypropionic acid (3HP) and 1,3-propanediol (PDO) from glycerol was studied using the resting cells of a recombinant Klebsiella pneumoniae J2B strain [1] that overexpresses an aldehyde dehydrogenase. Active biomass was produced in M9 under a range of aeration conditions, and shifted to potassium phosphate buffer containing glycerol for bioconversion. The microaerobic or anaerobic conditions were favorable for both the production of active biomass and subsequent bioconversion. At flask level, the recombinant strain grown under microaerobic conditions produced 43.2 mM 3HP and 59.0 mM PDO from glycerol (117 mM) in 30 min with a cumulative yield of 0.87 (mol/mol). The fed-batch bioconversion performed in a 1.5 L bioreactor at a constant pH 7.0 under anaerobic conditions, resulted in 125.6 mM 3HP and 209.5 mM PDO in 12 h with a cumulative overall productivity, yield and maximum specific production rate of 27.9 mmol/L/h, 0.71 (mol/mol) and 128.5 mmol/g CDW/h, respectively.

Currently, a bulk amount of raw glycerol is formed as the main by-product during biodiesel production, which is as high as 10% (w/w) of biodiesel generated. The surplus of raw glycerol has not only greatly disturbed the market of traditional glycerol in the preparation and price, but also brought a significant environmental problem since it cannot be discharged direct into the environment without any treatment. As a result of these issues, a large research effort has been devoted to developing methods to refine glycerol as a low-cost feedstock into industrially valuable materials such as fuels, building blocks, and bioactive substances. 1,3-Propanediol (1,3-PD) is a valuable chemical that is used mainly for the synthesis of polymethylene terephalates by polymerization with terephthalates. Use of this polymer in the manufacturing of textile fiber, film, plastic, etc. is rapidly expanding. Klebsiella pneumoniae is a typical microbial strain capable of producing 1,3-PD, in which the metabolic pathway responsible for the production of 1,3-PD has been well studied. The microbial production of 1,3-PD by K. pneumoniae involves the formation of various by-products. 2,3-Butanediol, one of the main by-products, may serve as an obstacle for obtaining a high purity of 1,3-PD in downstream processes because of its similar boiling point. To eliminate the by-products synthesis, glycerol metabolic pathway was engineered by deleting from the chromosomal DNA the genes involved in the synthesis of by-products. The by-product formation except for acetate was eliminated in the resultant strains. Additionally, 3-hydroxypropionic acid (3-HP) that is one of the most interest among alternative chemicals derived from biomass as precursor of acryl derivatives, could be produced from glycerol by the native and engineered strains of K. pneumoniae. The recombinant strains would be valuable as a platform strain for development of industrial process of production of 1,3-PD or 3-HP from glycerol.

Klebsiella pneumoniae converts glycerol to the specialty chemical 1,3- propanediol (1,3-PDO), which is used for the production of polytrimethylene terepthalate. Here, an NAD+-dependent gamma-glutamyl-gammaaminobutyraldehyde dehydrogenase (PuuC) of K. pneumoniae, which oxidizes 3-hydroxypropionaldehyde to a platform chemical 3-hydroxypropionic acid (3-HP), was overexpressed in K. pneumoniae DSM 2026 for the co-production of 3-HP and 1,3-PDO from glycerol. In addition, the gene dhaT, encoding NADH-dependent 1,3-propanediol oxidoreductase, was deleted from the chromosome for the balanced production of 3-HP and 1,3-PDO. The recombinant K. pneumoniae ΔdhaT, expressing PuuC, produced 3.6 g/L 3-HP and 3.0 g/L 1,3-PDO with an average yield of 81% on glycerol carbon in shake flask culture under microaerobic conditions. When a fed-batch culture was carried out under microaerobic conditions at pH 7.0 in a 5 L bioreactor, the recombinant K. pneumoniae ΔdhaT (puuC) strain produced 16.0 g/L 3-HP and 16.8 g/L 1,3-PDO with a cumulative yield of 51% on glycerol carbon in 24 h. The production of 1,3-PDO in the dhaT-deletion mutant was attributed to the expression of NAD(P)H-dependent hypothetical oxidoreductase. This study demonstrates the feasibility of obtaining two commercially valuable chemicals, 3-HP and 1,3-PDO, at a significant scale.

Although the propanediol utilization: CoA dependent propionaldehyde dehydrogenase (PduP) has been suggested to be involved in the de novo biosynthetic mechanism of 3-hydroxypropionic acid (3-HP) in glycerol- fermenting process, it is still poorly understood. Thus in here, we disrupted the pduP gene on the chromosomal DNA of K. pneumoniae for investigating the enzyme effect on 3-HP production. The mutant strain still produce 3-HP but accumulates 3-hydroxypropionaldehyde (3-HPA) after consumed glycerol completely that resulted in cell death as well as decreased final titer of 3-HP production. To analyze the enzyme properties, pduP was cloned and expressed in E. coli. Recombinant K. pneumoniae PduP exhibited broad substrate specificity including 3-HPA and utilized both NAD+ and NADP+ as cofactors. Among various aldehyde substrates tested, the specific activity was highest for propionaldehyde (PA), at pH 8.0 and 37°C. The Km and Vmax values for PA in the presence of NAD+ were 0.22 mM and 0.09 U mg-1, respectively. When pduP was overexpressed in K. pneumoniae, 3-HP production remarkably increased from 1.0g L-1 to 1.8 g L-1. The presentation demonstrated firstly in the role of PduP in cellular biosynthesis of 3-HP from glycerol.

3-Hydroxypropionic acid (3-HP) is a potential feedstock for producing many industrially important chemicals. Previously, we reported the production of 3-HP from glycerol by recombinant E. coli SH-BGK1 with a highest titer of 2.8 g/L in shake-flask experiments and 38.7 g /L in bioreactor experiments. However, the 3-HP yield on glycerol was too low (35%). To improve the 3-HP yield on glycerol the gene encoding glycerol kinase (glpK), which converts glycerol to glycerol-3-phosphate, was disrupted. When the resting cells of resulting recombinant strain, harboring glycerol dehydrate and aldehyde dehydrogenase, was subjected to shake flask cultivation the theoretical yield of 3-HP on glycerol was improved to >95%. This study demonstrates the highest yield of 3-HP on glycerol.

Production of 3-Hydroxypropionic acid (3-HP), often limited by the capability of the enzyme, aldehyde dehydrogenase (ALDH), which converts an intermediate, 3-hydroxypropionaldehyde (3-HPA) to 3-HP. Here we report a new ALDH, PuuC, from K. pneumoniae. The purified PuuC exhibited specificity towards 3-HPA and NAD+ was the preferred coenzyme. The optimum pH and temperature for PuuC activity were pH 8.0 and 45°C, respectively. The Km of 3-HPA and NAD+ were 0.48 and 0.09 mM, respectively. Under aerobic fermentation of glycerol by the recombinant E.coli BL21 carrying the genes (dhaB, gdrAB and puuC) in shake flask produced 52.77 mmol l-1 3-HP with the maximum yield of 0.58 mol mol-1 of glycerol at 24 h.

3-Hydroxypropionic acid (3-HP) is a potential feedstock for producing many industrially important chemicals. Previously, we reported the production of 3-HP from glycerol by recombinant E. coli SH-BGK1 with a highest titer of 2.8 g/L in shake-flask experiments and 38.7 g /L in bioreactor experiments. The yield was appeared to be about 35%. Most carbons in glycerol were diverted to growth and the production of other metabolites like lactic acid, acetic acid etc. To improve the yield, glycerol kinase (glpFKX) gene cluster which includes glycerol transport protein were disrupted. Then, for the glycerol transport, glpF gene was cloned and overexpressed under a constitutive tac promoter in a low copy pACYCDuet vector. The resulting construct was grown in a rich medium and the cells were resuspended and used for resting cell experiment for the production of 3-HP. With the new construct, the yield on glycerol improved to the theoretical maximum of 100%. However, it was necessary to supplement glucose as a carbon source for maintaining cell viability and enzymatic activities for the 3-HP synthesis from glycerol.

3-Hydroxypropionic acid (3-HP), an important platform chemical for several commodity and speciality chemicals was produced in E. coli by over expressing dhaB of Klebsiella pneumoniae DSM 2026 encoding glycerol dehydratase and aldH of E. coli K-12 MG1655 encoding aldehyde dehydrogenase. Adenosylcobalamin, an essential and expensive coenzyme should be provided extrinsically for the activity of DhaB in production of 3-HP in E. coli. To overcome the limitation of coenzyme B12 in E. coli, the K. pneumoniae strain, which naturally synthesizes 3-hydroxypropionaldehyde (3-HPA), an intermediary compound of glycerol metabolism and serves as the precursor for 3-HP production, was studied. An efficient production of 3-HP in K. pneumoniae required expression of aldH encoding aldehyde dehydrogenase which converts 3-HPA to 3-HP. By over expressing an aldH genes in K. pneumoniae under various cultural conditions a reasonable titer of 3-HP was achieved. Under microaerobic condition, a maximum titre of 1.44 ± 0.2 gram per litre was successfully produced.

3-Hydroxypropionic acid (3-HP) is an important platform chemical having diverse industrial applications, but its biological production has not been well studied. We have developed various recombinant Escherichia coli biocatalysts for the production of 3-HP from glycerol, and demonstrated that the strain E. coli SH254, expressing glycerol dehydratase (dhaB1, dhaB2, and dhaB3) and aldehyde dehydrogenase (aldH), could effectively produce 3-HP at 6.5 mmol 1  . The recombinant E. coli SH254 strain was further improved by changing the expression vectors for glycerol dehydratase and aldehyde dehydrogenase and cloning the additional genes (gdrAB) which encode dehydratase reactivase. The resultant recombinant SH-BGA1 exhibited improved activities of the two relevant enzymes in vivo and a higher 3-HP titer of 51 mmol 3-HP 1  in shake-flask conditions. When a pH-stat, fed-batch culture was conducted in 5-L bioreactor, the recombinant E. coli SH-BGA1 produced 3-HP at 346±20 mmol or 31±1.8g 3-HP per liter in 72h. The final yield of 3-HP was 0.35 mol mol  glycerol. The maximum specific rate of 3-HP production was estimated at 1.43 mmol g  cdw h   between 8 and 72 h. Here, for the first time, we report the high titer of 3-HP production using the recombinant E.coli SH-BGA1.

3-hydroxypropionic acid (3-HP), also called as hydracrylic acid, is one of the valuable platform chemicals and widely used in industrial application. As chemical synthesis of 3-HP requires expensive starting materials and high energy consuming process, its production through microorganism based bio-process has been developed. Although one of the recent reports showed commercially available 3-HP production in Escherichia coli, with heterologous enzyme expression and process optimization, there is still room for improvement on its productivity and titer. In this research, glycerol which is a waste from bio-fuel production process was used as a substrate as well as sole carbon source and novel pathway was introduced and reconstructed in Escherichia coli for increased 3-HP production. 6 genes encoding pathway enzymes and the enzyme reactivase were expressed under constitutive strong promoters and maximized 5’-untranslated regions (5’-UTR). The engineered strain was able to convert glycerol to 3-HP successfully.

3-hydroxypropionic acid (3-HP) is expected to build up huge market in biomass derived valuable platform chemicals recently and its commercial mass production will led to bio-refinery industrial development. In glycerol metabolism of bacteria, 3-HP is generally known as converting from 3-hydroxypropionaldehyde (3-HPA) by aldehyde dehydrogenase. To find out the appropriate enzyme for achieving the effective catalysis, 32 kinds of gene encoding each of the dehydrogenase from Lactobacillus reuteri were cloned with dhaT which encoding 1,3-propandiol oxidoreductase, and expressed in Klebsiella pneumoniae AK strain (1). Among 32 recombinants, a half of them illustrated enhancement aspects comparing with the control while eight showed lower 3-HP production. The highest 3-HP production was investigated in the recombinant strain which containing a gene encoding propanediol utilization protein. The enzyme will be useful for improvement of the 3-HP production.

3-hydroxypropionic acid (3-HP), an industrially important platform chemical is used as a precursor for many commercially important chemicals. Recently, the production of 3-HP from glycerol was demonstrated in recombinant K. pneumoniae without the addition of expensive coenzyme B12 [1]. 3-HP production in K. pneumoniae is always associated with the production of 1,3-propanediol and lactic acid due to the generation of large amounts of NADH [2]. To enhance 3-HP yield on glycerol by regenerating NAD+, nitrate was supplemented in the culture medium. But this resulted in significant decrease in 3-HP production and majority of carbon was utilized for cell biomass generation, due to the presence of undesirable anaerobic respiration pathway. To overcome this limitation, a novel recombinant strain, Klebsiella pneumoniae DdhaTDglpK (puuC) was developed. This strain significantly increased 3-HP production at a maximum of 22.2 g/L in the presence of nitrate under anaerobic condition with a yield on glycerol at 0.35 mol/mol.

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 cofactors. Among various aldehyde substrates tested, the specific activity was highest for propionaldehyde, at pH 7.8 and 37°C. The Km and Vmax 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 (2.51 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.

To produce an important platform chemical 3-hydroxypropionic acid (3-HP) from glycerol, we developed a recombinant E. coli BL21 strain by cloning two sets of genes; dhaB and gdrAB under T7 promoters with pCDFDuet vector; and the gene KGSADH under pRSFDuet vector: In which the gene dhaB encodes an enzyme glycerol dehydratase of Klebsiella pneumoniae, capable of converting glycerol to 3-hydroxypropionaldehyde (3-HPA); and the genes gdrAB of K. pneumoniae which encode the reactivation factor of glycerol dehydratase, whilst the gene KGSADH of Azospirillum brasilense encodes γ-ketoglutaric semialdehyde dehydrogenase, that is capable of converting 3-HPA to 3-HP. When analyzing the crude-cell extracts of recombinant proteins, DhaB exhibited 95 U/mg protein on glycerol with coenzyme B12, while the enzyme KGSADH exhibited 2.9 U/mg protein on 3-HPA in the presence of NAD+ as hydrogen acceptor. Upon cultivating the recombinant on a glycerol medium under aerobic condition, it produced a maximum of 19.6 mmol 3-HP or 1.76 g per liter at 0.5 mM IPTG induction. The rate and yield of 3-HP production were estimated as 1.1 mmol g-1 cdw h-1 and 0.28 mol mol-1 glycerol, respectively. Although the final titer of 3-HP is too low for commercial applications, our results in the present investigation demonstrate the utility of the recombinant for further development of a bioprocess to improve 3-HP from glycerol.

Co-production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO) was suggested as an innovative strategy to overcome several limitations occurring in the single production of 3-HP from glycerol. In this study, two new isolates of Klebsiella pneumoniae, which produce less lipopolysaccharide (LPS) thus considered less pathogenic than K. pneumoniae DSM 2026, were compared and evaluated for their potential for the co-production of 3-HP and 1,3-PDO. The newly isolated strains showed significantly faster sedimentation rate than DSM, which should be beneficial for downstream processing. Analysis of genome sequences of the isolates confirmed the presence of all genes necessary for glycerol assimilation, 1,3-PDO production and biosynthesis of coenzyme $B_{12}$. Co-production yield was highest under anaerobic condition while cell growth was highest under aerobic condition. Both strains showed similarly good performance for the co-production although J2B gave the slightly higher co-production yield of 0.80 mol/mol than GSC021 (0.75 mol/mol). The evaluation of the newly developed strains presented here should be useful in designing similar evaluation experiments for other microorganisms.