Sunghoon Park

Recent advances in biological production of 3-hydroxypropionic acid

3-Hydroxypropionic acid (3-HP) is a valuable platform chemical that can be produced biologically from glucose or glycerol. This review article provides an overview and the current status of microbial 3-HP production. The constraints of microbial 3-HP production and possible solutions are also described. Finally, future prospects of biological 3-HP production are discussed.

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‐β‐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), α‐ketoglutaric semialdehyde dehydrogenase (KGSADH), instead of AldH, the recombinant E. coli SH‐BGK1 showed the highest level of 3‐HP production (2.8u2009g/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 38.7u2009g 3‐HP/L with an average yield of 35%. This article reports the highest level of 3‐HP production from glycerol thus far. Biotechnol. Bioeng. 2009; 104: 729–739

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 T5 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 24h 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 α-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.

Current status of the metabolic engineering of microorganisms for biohydrogen production.

The improvement of H2 production capabilities of hydrogen (H2)-producing microorganisms is a challenging issue. Microorganisms have evolved for fast growth and substrate utilization rather than H2 production. To develop good H2-producing biocatalysts, many studies have focused on the redirection and/or reconstruction of cellular metabolisms. These studies included the elimination of enzymes and carbon pathways interfering or competing with H2 production, the incorporation of non-native metabolic pathways leading to H2 production, the utilization of various carbon substrates, the rectification of H2-producting enzymes (nitrogenase and hydrogenase) and photophosphorylation systems, and in silico pathway flux analysis, among others. Owing to these studies, significant improvements in the yield and rate of H2 production, and in the stability of H2 production activity, were reached. This review presents and discusses the recent developments in biohydrogen production, with a focus on metabolic pathway engineering.

Development of recombinant Klebsiella pneumoniae ∆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-gamma-aminobutyraldehyde 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 ∆dhaT, expressing puuC, produced 3.6xa0g 3-HP and 3.0xa0g 1,3-PDO per liter 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 pHxa07.0 in a 5-l bioreactor, the recombinant K. pneumoniae ∆dhaT (puuC) strain produced 16.0xa0g 3-HP and 16.8xa0g 1,3-PDO per liter with a cumulative yield of 51% on glycerol carbon in 24xa0h. 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.

Co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol using resting cells of recombinant Klebsiella pneumoniae J2B strain overexpressing aldehyde dehydrogenase

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 that overexpresses an aldehyde dehydrogenase (KGSADH). Active biomass was produced in a mineral salt medium containing yeast extract and glycerol 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 the flask level, the recombinant strain (2.0xa0gu2009CDW/L) grown under microaerobic conditions produced 43.2xa0mM 3HP and 59.0xa0mM PDO from glycerol (117xa0mM) in 30xa0min with a cumulative yield of 0.87u2009(mol/mol). The fed-batch bioconversion, which was performed in a 1.5-L bioreactor with 1.0xa0gu2009CDW/L at a constant pHxa07.0 under anaerobic conditions, resulted in 125.6xa0mM 3HP and 209.5xa0mM PDO in 12xa0h with a cumulative overall productivity, yield, and maximum specific production rate of 27.9xa0mmol/L/h, 0.71 (mol/mol), and 128.5xa0mmol/g CDW/h, respectively. Lactate, succinate and 2,3-butanediol were the major by-products, whereas the production of acetate and ethanol was marginal. This is the first report of the simultaneous production of 3HP and PDO from glycerol using a resting cell system.

Production of 3-hydroxypropionic acid from glycerol by acid tolerant Escherichia coli

The biological production of 3-hydroxypropionic acid (3-HP) has attracted significant attention because of its industrial importance. The low titer, yield and productivity, all of which are related directly or indirectly to the toxicity of 3-HP, have limited the commercial production of 3-HP. The aim of this study was to identify and select a 3-HP tolerant Escherichia coli strain among nine strains reported to produce various organic acids efficiently at high titer. When transformed with heterologous glycerol dehydratase, reactivase and aldehyde dehydrogenase, all nine E. coli strains produced 3-HP from glycerol but the level of 3-HP production, protein expression and activities of the important enzymes differed significantly according to the strain. Two E. coli strains, W3110 and W, showed higher levels of growth than the others in the presence of 25xa0g/L 3-HP. In the glycerol fed-batch bioreactor experiments, the recombinant E. coli W produced a high level of 3-HP at 460xa0±xa010xa0mM (41.5xa0±xa01.1xa0g/L) in 48xa0h with a yield of 31xa0% and a productivity of 0.86xa0±xa00.05xa0g/L h. In contrast, the recombinant E. coli W3110 produced only 180xa0±xa08.5xa0mM 3-HP (15.3xa0±xa00.8xa0g/L) in 48xa0h with a yield and productivity of 26xa0% and 0.36xa0±xa00.02xa0g/L h, respectively. This shows that the tolerance to and the production of 3-HP differ significantly among the well-known, similar strains of E. coli. The titer and productivity obtained with E. coli W were the highest reported thus far for the biological production of 3-HP from glycerol by E. coli.

Production of 1,3-propanediol from glycerol using the newly isolated Klebsiella pneumoniae J2B.

Recently, novel Klebsiella pneumoniae J2B, which grows rapidly on glycerol as the carbon source without forming pathogenic and sticky lipopolysaccharides, was isolated. Current study examined the ability of K. pneumoniae J2B to produce 1,3-propanediol (PDO) from glycerol. To this end, a deletion mutant for lactate formation was constructed. The ldhA mutant strain produced negligible lactate but more 2,3-butanediol (BDO). When K. pneumoniae ΔldhA was cultivated in glycerol fed-batch mode, the PDO titer of 58.0 g/L with a yield of 0.35 g/g and an overall volumetric productivity of 1.3g/L/h were obtained. BDO was the main byproduct (26.6g/L). Less than 10 g/L of the other metabolites was produced. As PDO and other metabolites accumulated, the rate of PDO production decreased significantly due mainly to the toxic effects of these metabolites. This study highlights the potential of newly isolated K. pneumoniae J2B for the production of PDO from glycerol.

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 B12‐dependent glycerol dehydratase and an NAD(P)+‐dependent aldehyde dehydrogenase (ALDH), respectively. Pseudomonas denitrificans synthesizes coenzyme B12 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 37.7u2009mmol/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 54.7u2009mmol/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.

Evidence of percolation related power law behavior in the thermal conductivity of nanotube/polymer composites

A power law relation for the thermal conductivity, indicative of percolation, is reported through measurements on carbon nanotube/polymer composites. Our results contradict earlier assertions and indicate that synthesis methodologies may be adapted to facilitate such behavior. Consistent modeling of the experimentally determined electrical and thermal conductivity anisotropy, in addition to the incorporation of interfacial resistance, was used to understand the underlying mechanisms and variations.