Isolation and characterization of a new mucoid-free Klebsiella pneumoniae strain for 2,3-butanediol production

The secretion of mucoid substances by Klebsiella pneumoniae, a natural 2,3-butanediol (2,3-BD) hyper-producer, hinders its application in large-scale fermentation because of pathogenicity, fermentation instability, and downstream difficulty. In this study, 14 K. pneumoniae strains were isolated from a waste water treatment plant and their 2,3-BD production efficiencies were assessed with the strain K. pneumoniae DSM2026. Among various strains isolated, K. pneumoniae GSC010 and GSC112 produced relatively large amounts of 2,3-BD compared to other isolates; and their 2,3-BD production was consistent with DSM2026. Meanwhile, mucoidic characteristics of GSC010 were more or less similar to DSM2026, which was observed by scanning electron microscope (SEM) as a characteristic intercalated thread anchored on the surface of the cells. However, no polysaccharide materials were found in a non-mucoid cell, GSC112. Fed-batch culture of GSC112 with continuous glucose feeding resulted in the production of 2,3-BD at 52.4 g/l with 2,3-BD yield and overall productivity of 0.27 g/g glucose and 0.52 g/l/h, respectively. These results strongly suggest that the newly isolated mucoid-free K. pneumoniae GSC112 has potential for industrial production of 2,3-BD.Keywords: 2,3-Butanediol, Klebsiella pneumoniae, isolation, capsular polysaccharides, scanning electron microscop

Production of 3-Hydroxypropionic Acid From Glycerol by Recombinant Pseudomonas denitrificans

3-Hydroxypropionic acid (3-HP) can be produced from glycerol through two sequential enzymatic reactions that are catalyzed by a coenzyme B-12-dependent glycerol dehydratase and an NAD(P)(+)-dependent aldehyde dehydrogenase (ALDH), respectively. Pseudomonas denitrificans synthesizes coenzyme B-12 under aerobic conditions, where NAD(P)(+) is regenerated efficiently. Hence, it is considered an ideal host for the production of 3-HP from glycerol under aerobic conditions. In this study, recombinant strains of P. denitrificans were developed and their potential for the production of 3-HP from glycerol was evaluated. When the enzymes, glycerol dehydratase (DhaB) and glycerol dehydratase reactivase (GdrAB), of Klebsiella pneumoniae were expressed heterologously, P. denitrificans could produce 3-HP at 37.7mmol/L with 62% (mol/mol) yield on glycerol. Glucose was required as the carbon and energy sources for cell growth. The overexpression of heterologous ALDH was not essential; however, the titer and yield of 3-HP were improved to 54.7mmol/L and 67% (mol/mol), respectively, when an ALDH gene (puuC) from K. pneumoniae was overexpressed. One serious drawback hindering the use of P. denitrificans as a recombinant host for 3-HP production is that it oxidizes 3-HP to malonate and utilizes 3-HP as a carbon source for growth. This is the first report on the development and use of recombinant P. denitrificans for 3-HP production from glycerol. Biotechnol. Bioeng. 2013;110: 3177-3187

Development of recombinant Klebsiella pneumoniae Delta dhaT strain for the co-production of 3-hydroxypropionic acid and 1,3-propanediol 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 (PTT). In this study, an NAD(+)-dependent gamma-glutamyl-gammaaminobutyraldehyde dehydrogenase (PuuC) of K. pneumoniae DSM 2026, which oxidizes 3-hydroxypropionaldehyde to a platform chemical 3-hydroxypropionic acid (3-HP), was cloned and 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 (1,3-PDOR), was deleted from the chromosome for the balanced production of 3-HP and 1,3-PDO. The recombinant K. pneumoniae Delta dhaT, expressing puuC, produced 3.6 g 3-HP and 3.0 g 1,3-PDO per liter with an average yield of 81% on glycerol carbon in shake flask culture under microaerobic conditions. When a fedbatch culture was carried out under microaerobic conditions at pH 7.0 in a 5-1 bioreactor, the recombinant K. pneumoniae Delta dhaT (puuC) strain produced 16.0 g 3-HP and 16.8 g 1,3-PDO per liter with a cumulative yield of 51% on glycerol carbon in 24 h. The production of 1,3PDO in the dhaT-deletion mutant was attributed to the expression of NAD(P) H-dependent hypothetical oxidore-ductase. This study demonstrates the feasibility of obtaining two commercially valuable chemicals, 3-HP and 1,3PDO, at a significant scal

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 isopropylthio-beta-galactoside (1PTG) 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 dehydrogenise (ALDH), alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH), instead of A1dH, 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

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

Production of 3-hydroxypropionic acid via malonyl-CoA pathway using recombinant Escherichia coli strains

Malonyl-CoA is an intermediary compound that is produced during fatty acid metabolism. Our study aimed to produce the commercially important platform chemical 3-hydroxypropionic acid (3-HP) from its immediate precursor malonyl-CoA by recombinant Escherichia coli strains heterologously expressing the mcr gene of Chloroflexus aurantiacus DSM 635, encoding an NADPH-dependent malonyl-CoA reductase (MCR). The recombinant E. coli overexpressing mcr under the 15 promoter showed MCR activity of 0.015 U mg(-1) protein in crude cell extract and produced 0.71 mmol/L of 3-HP in 24 h in shake flask cultivation under aerobic conditions with glucose as the sole source of carbon. When acetyl-CoA carboxylase and biotinilase, encoded by the genes accADBCb (ACC) of E. coli K-12 were overexpressed along with MCR, the final 3-HP titer improved by 2-fold, which is 1.6 mM. Additional expression of the gene pntAB, encoding nicotinamide nucleotide transhydrogenase that converts NADH to NADPH, increased 3-HP production to 2.14 mM. The strain was further developed by deleting the sucAB gene, encoding a-ketoglutarate dehydrogenase complex in tricarboxylic acid (TCA) cycle, or blocking lactate and acetate production pathways, and evaluated for the production of 3-HP. We report on the feasibility of producing 3-HP from glucose through the malonyl-CoA pathway

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 (k (cat)) and the specificity constant (k(cat)/K-m) for 3-HPA in the presence of NAD(+) were 28.5 s(-1) and 58.6 x 10(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

Effects of mutation of 2,3-butanediol formation pathway on glycerol metabolism and 1,3-propanediol production by Klebsiella pneumoniae J2B

The current study investigates the impact of mutation of 2,3-butanediol (BDO) formation pathway on glycerol metabolism and 1,3-propanediol (PDO) production by lactate dehydrogenase deficient mutant of Klebsiella pneumoniae J2B. To this end, BDO pathway genes, budA, budB, budC and budO (whole-bud operon), were deleted from K. pneumoniae J2B Delta ldhA and the mutants were studied for glycerol metabolism and alcohols (PDO, BDO) production. Delta budO-mutant-only could completely abolish BDO production, but with reductions in cell growth and PDO production. By modifying the culture medium, the Delta budO mutant could recover its performance on the flask scale. However, in bioreactor experiments, the Delta budO mutant accumulated a significant amount of pyruvate (>73 mM) in the late phase and PDO production stopped concomitantly. Glycolytic intermediates of glycerol, especially glyceraldehyde-3-phosphate (G3P) was highly inhibitory to glycerol dehydratase (GDHt); its accumulation, followed by pyruvate accumulation, was assumed to be responsible for the Delta budO mutant's low PDO production.ope