Search

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.

Carbon monoxide (CO) is highly toxic but is an abundant carbon source that can be utilized for the production of hydrogen (H 2 ). CO-dependent H 2 production is catalyzed by a unique enzyme complex composed of carbon monoxide dehydrogenase (CODH) and CO-dependent hydrogenase (CO–H 2 ase), both of which contain metal cluster(s). In this study, CODH and the required maturation proteins from the novel facultative anaerobic bacterium Citrobacter amalonaticus Y19 were cloned and heterologously expressed in Escherichia coli . For functional expression of CODH in E. coli , only CooF (ferredoxin-like protein) and CooS (CODH), not the maturation proteins, were needed. The recombinant E. coli BL21(DE3)- cooFS showed a 3.5-fold higher specific CODH activity (4.9 U mg protein −1 ) compared to C. amalonaticus Y19 (Y19) (1.4 U mg protein −1 ). Purified heterologous CODH from the soluble cell-free extract of the recombinant E. coli showed a specific activity of 170.6 U mg protein −1 . Recombinant E. coli harboring Y19 CODH and maturation proteins did not produce H 2 from CO, suggesting that the native hydrogenases present in E. coli could not substitute the Y19 CO–H 2 ase for CO-dependent H 2 production.

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 (k cat/K m) 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 k cat/K m 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 k cat/K m 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.

The abilities of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus DSM 8903 to ferment switchgrass (SWG), microcrystalline cellulose (MCC) and glucose to hydrogen (H 2 ) in one-step were examined. Hydrogen production from glucose reached the theoretical maximum for dark fermentation of 4 mol H 2 /mol glucose. The H 2 yield on MCC and SWG after 6 days of fermentation was 23.2 mmol H 2 /L or 9.4 mmol H 2 /g MCC and 14.3 mmol H 2 /L or 11.2 mmol H 2 /g SWG, respectively. The rate of H 2 formation however was higher on MCC (0.7 mmol/L h) than SWG (0.1 mmol/L h). C. saccharolyticus DSM 8903 was able to produce H 2 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.

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 Δ glpK Δ dhaT ( 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±3 mM 3-HP with more than 50% yield on glycerol in shake flasks and 250±10 mM 3-HP with approximately 30% yield on glycerol in a fed-batch bioreactor.

Background Fermentative hydrogen (H2) production suffers from low carbon-to-H2 yield, to which problem, co-production of ethanol and H2 has been proposed as a solution. For improved co-production of H2 and ethanol, we developed Escherichia coli BW25113 ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB Δpta-ackA ΔpfkA (SH8*) and overexpressed Zwf and Gnd, the key enzymes in the pentose-phosphate (PP) pathway (SH8*_ZG). However, the amount of accumulated pyruvate, which was significant (typically 0.20 mol mol−1 glucose), reduced the co-production yield. Results In this study, as a means of reducing pyruvate accumulation and improving co-production of H2 and ethanol, we developed and studied E. coli SH9*_ZG with functional acetate production pathway for conversion of acetyl-CoA to acetate (pta-ackA+). Our results indicated that the presence of the acetate pathway completely eliminated pyruvate accumulation and substantially improved the co-production of H2 and ethanol, enabling yields of 1.88 and 1.40 mol, respectively, from 1 mol glucose. These yields, significantly, are close to the theoretical maximums of 1.67 mol H2 and 1.67 mol ethanol. To better understand the glycolytic flux distribution, glycolytic flux prediction and RT-PCR analyses were performed. Conclusion The presence of the acetate pathway along with activation of the PP pathway eliminated pyruvate accumulation, thereby significantly improving co-production of H2 and ethanol. Our strategy is applicable to anaerobic production of biofuels and biochemicals, both of which processes demand high NAD(P)H. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0510-5) contains supplementary material, which is available to authorized users.

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.

Escherichia fergusonii KLU01, a propitious bacterial strain isolated from oil contaminated soil was identified to be hydrocarbon degrading, heavy metal tolerant and a potent producer of biosurfactant using diesel oil as the sole carbon and energy source. The biosurfactant produced by the strain was characterized to be a lipopeptide. The minimum active dose and critical micelle concentration of the biosurfactant were found as 0.165 ± 0.08 μg and 36 mg/L, respectively. In spite of being an excellent emulsifier, the biosurfactant showed an incredible stability at extremes of temperature, pH and at various concentrations of NaCl, CaCl2 and MgCl2. Also the bacterium manifested tolerance towards Manganese, Iron, Lead, Nickel, Copper and Zinc. The strain emerges as a new class of biosurfactant producer with potential environmental and industrial applications, especially in hydrocarbon degradation and heavy metal bioremediation.

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.

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.

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.

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.

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 Hg(2+), 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 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.

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.

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 3.6 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 fed-batch culture was carried out under microaerobic conditions at pH 7.0 in a 5-l bioreactor, the recombinant K. pneumoniae ∆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 1,3-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.

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.

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.

not Available.