Subramanian Mohan Raj

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 (2.8 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. 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.

Effect of puuC overexpression and nitrate addition on glycerol metabolism and anaerobic 3-hydroxypropionic acid production in recombinant Klebsiella pneumoniae ΔglpKΔdhaT

3-Hydroxypropionic acid (3-HP), an industrially important platform chemical, is used as a precursor during the production of many commercially important chemicals. Recently, recombinant strains of K. pneumoniae overexpressing an NAD(+)-dependent γ-glutamyl-γ-aminobutyraldehyde dehydrogenase (PuuC) enzyme of K. pneumoniae DSM 2026 were shown to produce 3-HP from glycerol without the addition coenzyme B(12), which is expensive. However, 3-HP production in K. pneumoniae is accompanied with NADH generation, and this always results in large accumulation of 1,3-propanediol (1,3-PDO) and lactic acid. In this study, we investigated the potential use of nitrate as an electron acceptor both to regenerate NAD(+) and to prevent the formation of byproducts during anaerobic production of 3-HP from glycerol. Nitrate addition could improve NAD(+) regeneration, but decreased glycerol flux towards 3-HP production. To divert more glycerol towards 3-HP, a novel recombinant strain K. pneumoniae ΔglpKΔdhaT (puuC) was developed by disrupting the glpK gene, which encodes glycerol kinase, and the dhaT gene, which encodes 1,3-propanediol oxidoreductase. This strain showed improved cellular NAD(+) concentrations and a high carbon flux towards 3-HP production. Through anaerobic cultivation in the presence of nitrate, this recombinant strain produced more than 40±3mM 3-HP with more than 50% yield on glycerol in shake flasks and 250±10mM 3-HP with approximately 30% yield on glycerol in a fed-batch bioreactor.

Consolidated bioprocessing of untreated switchgrass to hydrogen by the extreme thermophile Caldicellulosiruptor saccharolyticus DSM 8903.

The abilities of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus DSM 8903 to ferment switchgrass (SWG), microcrystalline cellulose (MCC) and glucose to hydrogen (H2) in one-step were examined. Hydrogen production from glucose reached the theoretical maximum for dark fermentation of 4 mol H2/mol glucose. The H2 yield on MCC and SWG after 6 days of fermentation was 23.2 mmol H2/L or 9.4 mmol H2/g MCC and 14.3 mmol H2/L or 11.2 mmol H2/g SWG, respectively. The rate of H2 formation however was higher on MCC (0.7 mmol/Lh) than SWG (0.1 mmol/Lh). C. saccharolyticus DSM 8903 was able to produce H2 directly from mechanically-comminuted SWG without any physicochemical or biological pretreatment. Combining four processing steps (pretreatment, enzyme production, saccharification and fermentation) into a single biorefinery operation makes C. saccharolyticus DSM 8903 a promising candidate for consolidated bioprocessing (CBP) of lignocellulosic biomass.

Co-production of hydrogen and ethanol from glucose by modification of glycolytic pathways in Escherichia coli - from Embden-Meyerhof-Parnas pathway to pentose phosphate pathway.

Hydrogen (H2) production from glucose by dark fermentation suffers from the low yield. As a solution to this problem, co‐production of H2 and ethanol, both of which are good biofuels, has been suggested. To this end, using Escherichia coli, activation of pentose phosphate (PP) pathway, which can generate more NADPH than the Embden‐Meyhof‐Parnas (EMP) pathway, was attempted. Overexpression of two key enzymes in the branch nodes of the glycolytic pathway, Zwf and Gnd, significantly improved the co‐production of H2 and ethanol with concomitant reduction of pyruvate secretion. Gene expression analysis and metabolic flux analysis (MFA) showed that, upon overexpression of Zwf and Gnd, glucose assimilation through the PP pathway, compared with that of the EMP or Entner‐Doudoroff (ED) pathway, was greatly enhanced. The maximum co‐production yields were 1.32 mol H2 mol−1 glucose and 1.38 mol ethanol mol−1 glucose, respectively. It is noteworthy that the glycolysis and the amount of NAD(P)H formed under anaerobic conditions could be altered by modifying (the activity of) several key enzymes. Our strategy could be applied for the development of industrial strains for biological production of reduced chemicals and biofuels which suffers from lack of reduced co‐factors.

Thermophilic hydrogen production from renewable resources: current status and future perspectives.

Hydrogen (H2) is considered an alternative fuel of the future due to its high energy density and non-polluting nature. H2 energy provides many advantages over fossil fuels in that it is renewable, eco-friendly, and efficient. The global demand for H2 is increasing significantly; however, matching the supply of cost-competitive H2 to meet the current demand is a major technological barrier. H2 can be produced from lignocellulosic biomass and serve as a raw material for the synthesis of many industrially important chemicals. The use of thermophilic bacteria for biological production of H2 appears to be a promising alternative route to the current H2 production technologies. However, the carbon and H2 production metabolisms in most thermophilic bacteria have not yet been completely understood. This paper summarizes the recent research progress made toward understanding the carbon utilization for H2 production and developing gene manipulation techniques to enhance the H2 production capabilities in thermophilic bacteria. It reviews the current status, future directions and opportunities that thermophiles can offer to enable a cost-competitive and environmentally benign H2 production bioprocess.

3-Hydroxyisobutyrate dehydrogenase-I from Pseudomonas denitrificans ATCC 13867 degrades 3-hydroxypropionic acid

This study examined the role and physiological relevance of 3-hydroxyisobutyrate dehydrogenase-I (3HIBDHI) of Pseudomonas denitrificans ATCC 13867 in the degradation of 3-hydroxypropionic acid (3-HP) during 3-HP production. The gene encoding 3HIBDH-I of P. denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant 3HIBDH-I was then purified on a Ni-NTA-HP column and characterized for its choice of substrates, cofactors, metals, reductants, and the optimal temperature and pH. The recombinant 3HIBDH-I showed a high catalytic constant (kcat/Km) of 604.1 ± 71.1 mM/S on (S)-3-hydroxyisobutyrate, but no detectable activity on (R)-3-hydroxyisobutyrate. 3HIBDH-I preferred NAD+ over NADP+ as a cofactor for its catalytic activity. The kcat/Km determined for 3-HP was 15.40 ± 1.43 mM/S in the presence of NAD+ at 37°C and pH 9.0. In addition to (S)-3-hydroxyisobutyrate and 3-HP, 3HIBDH-I utilized l-serine, methyl-d,l-serine, and methyl-(S)-(+)-3-hydroxy-2-methylpropionate; on the other hand, the kcat/Km values determined for these substrates were less than 5.0mM/S. Ethylenediaminetetraacetic acid, 2-mercaptoethanol, dithiothreitol and Mn2+ increased the activity of 3HIBDHI significantly, whereas the presence of Fe2+, Hg2+ and Ag+ in the reaction mixture at 1.0 mM completely inhibited its activity. This study revealed the characteristics of 3HIBDH-I and its significance in 3-HP degradation.

Cloning, Expression and Characterization of 3-Hydroxyisobutyrate Dehydrogenase from Pseudomonas denitrificans ATCC 13867

The gene encoding an NAD+-dependent, 3-hydroxyisobutyrate dehydrogenase (3HIBDH-IV) from Pseudomonas denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL 21 (DE3) and characterized to understand its physiological relevance in the degradation of 3-hydroxypropionic acid (3-HP). The deduced amino acid sequence showed high similarity to other 3-hydroxyisobutyrate dehydrogenase isozymes (3HIBDHs) of P. denitrificans ATCC 13867. A comparison of 3HIBDH-IV with its relevant enzymes along with molecular docking studies suggested that Lys171, Asn175 and Gly123 are important for its catalytic function on 3-hydroxyacids. The recombinant 3HIBDH-IV was purified to homogeneity utilizing a Ni-NTA-HP resin column in high yield. 3HIBDH-IV was very specific to (S)-3-hydroxyisobutyrate, but also catalyzed the oxidation of 3-HP to malonate semialdehyde. The specific activity and half-saturation constant (K m) for 3-HP at 30°C and pH 9.0 were determined to be 17 U/mg protein and 1.0 mM, respectively. Heavy metals, such as Ag+ and Hg2+, completely inhibited the 3HIBDH-IV activity, whereas dithiothreitol, 2-mercaptoethanol and ethylenediaminetetraacetic acid increased its activity 1.5–1.8-fold. This paper reports the characteristics of 3HIBDH-IV as well as its probable role in 3-HP degradation.

Metabolic Engineering of Microorganisms for Biohydrogen Production

Abstract Metabolic engineering has been studied extensively to improve microbial hydrogen (H2) production. Elimination or modification of carbon metabolic pathways, incorporation of nonnative metabolic pathways leading to H2 production, utilization of various carbon substrates, and improvement of H2-producing enzymes (hydrogenase and nitrogenase) were the major focuses of these studies. This chapter highlights recent developments with metabolic engineering toward improving the yield and rate of microbial biohydrogen production.