Growth rate of a non-fermentative <Emphasis Type="Italic">Escherichia coli</Emphasis> strain is influenced by NAD<Superscript>+</Superscript> regeneration

By complementing a non-fermentative Escherichia coli (ldhA ⁻ pflB ⁻) strain with the recombinant Zymomonas mobilis ethanol pathway (pdc, adhB), we evaluated the effect of different levels of enzymatic activity on growth rate demonstrating that there is a direct relationship between anaerobic growth rate and the total specific activity of pyruvate decarboxylase, which is the limiting enzyme of this specific fermentative NAD⁺ regenerating pathway. This relationship was proved to be useful to establish a selection strategy based on growth rate for the analysis of lctE libraries, which encode lactate dehydrogenase from Bacillus subtilis.     

Regulation of NAD<Superscript>+</Superscript>-dependent isocitrate dehydrogenase in the citrate producing yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

The mechanism of the increased accumulation (overproduction) of citric acids in the yeast Yarrowia lipolytica while growing in the presence of glucose under nitrogen deficiency was investigated. The limitation of the yeast growth by the source of nitrogen decreases the total content of nucleotides and increases the ratios of ATP/AMP and NADH/NAD⁺. NAD⁺-Dependent isocitrate dehydrogenase, an enzyme of the tricarboxylic acid cycle playing a key role in the regulation of biosynthesis of citric and isocitric acids, was isolated from Y. lipolytica. The molecular weights of the native enzyme and its subunits were found to be 412 and 52 kD, respectively. It is concluded that the enzyme is a homooligomer consisting of eight subunits. Investigation of the effect of some intermediates of the tricarboxylic acid cycle on the activity of this enzyme suggests that the enhanced excretion of citric acids can be caused by the inhibition of NAD⁺-dependent isocitrate dehydrogenase due to the decrease in the content of AMP and increase in the NADH/NAD⁺ ratio in the cells of Y. lipolytica under depletion of nitrogen.Translated from Biokhimiya, Vol. 69, No. 12, 2004, pp. 1706–1714.Original Russian Text Copyright © 2004 by Morgunov, Solodovnikova, Sharyshev, Kamzolova, Finogenova.     

Expression of protein engineered NADP<Superscript>+</Superscript>-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD⁺-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP⁺. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP⁺-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP⁺-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.     

Thymidine production by overexpressing NAD<Superscript>+</Superscript> kinase in an <Emphasis Type="Italic">Escherichia coli</Emphasis> recombinant strain

Intracellular NADPH/NADP⁺ ratio in cells grown on various production media with different carbon and nitrogen sources had a positive correlation with the thymidine production. To improve thymidine production in a previously engineered E. coli strain, NAD⁺ kinase was overexpressed in it resulting in the NADPH/NADP⁺ ratio shifting from 0.184 to 0.267. The [NADH + NADP⁺]/[NAD⁺ + NADPH] ratio was, however, not significantly altered. In jar fermentation, 740 mg thymidine l⁻¹ was produced in parental strain, while 940 mg l⁻¹ of thymidine was produced in NAD⁺ kinase-expressing strain.     

Kinetic Modeling of Acetophenone Reduction Catalyzed by Alcohol Dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter</Emphasis> sp.

NADPH-dependent alcohol dehydrogenase (ADH) from Thermoanaerobacter sp. was kinetically characterized using reduction of acetophenone as a model. To achieve 98% conversion of acetophenone, cofactor regeneration by oxidation of 2-propanol with the same enzyme was used. The enzyme was stable in the batch reactor. It was enantioselective towards (S)-1-phenylethanol (ee>99.5%). Due to its high deactivation in continuously operated stirred tank reactor (k_d=0.0141 min⁻¹) there was no way to keep high conversion of acetophenone at 98%. The deactivation occurred in the repetitive batch as well. A mathematical model for the acetophenone reduction with cofactor regeneration describing the behaviour in a batch, repetitive-batch and continuously stirred tank reactor was developed.     

<Emphasis Type="Italic">Haloquadratum walsbyi</Emphasis> Yields a Versatile,NAD<Superscript>+</Superscript>/NADP<Superscript>+</Superscript> Dual Affinity,Thermostable, Alcohol Dehydrogenase (<Emphasis Type="Italic">Hw</Emphasis>ADH)

This study presents the first example of an alcohol dehydrogenase (ADH) from the halophilic archaeum Haloquadratum walsbyi (HwADH). A hexahistidine-tagged recombinant HwADH was heterologously overexpressed in Haloferax volcanii. HwADH was purified in one step and was found to be thermophilic with optimal activity at 65 °C. HwADH was active in the presence of 10% (v/v) organic solvent. The enzyme displayed dual cofactor specificity and a broad substrate scope, and maximum activity was detected with benzyl alcohol and 2-phenyl-1-propanol. HwADH accepted aromatic ketones, acetophenone and phenylacetone as substrates. The enzyme also accepted cyclohexanol and aromatic secondary alcohols, 1-phenylethanol and 4-phenyl-2-butanol. H. walsbyi may offer an excellent alternative to other archaeal sources to expand the toolbox of halophilic biocatalysts.     

Anaerobic synthesis of succinic acid by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> strains with activated NAD<Superscript>+</Superscript>-reducing pyruvate dehydrogenase complex

Effect of constitutive expression of the aceEF-lpdA operon genes coding for the enzymes of NAD⁺-reducing pyruvate dehydrogenase complex on the anaerobic production of succinic acid from glucose by recombinant Escherichia coli strains was studied. Basic producer strains were obtained by inactivation of the main pathways for synthesis of acetic and lactic acids through deletion of the genes ackA, pta, poxB, and ldhA (SGM0.1) in E. coli MG1655 strain and by additional introduction of the Bacillus subtilis pyruvate carboxylase (SGM0.1 [pPYC]). A constitutive expression of the genes aceEF-lpdA in derivatives of the basic strains SGM0.1 P_L-aceEF-lpdA and SGM0.1 P_L-aceEF-lpdA [pPYC] was provided by replacing the native regulatory region of the operon with the lambda phage PL promoter. Molar yields of succinic acid in anaerobic glucose fermentation by strains SGM0.1 P_L-aceEF-lpdA and SGM0.1 P_L-aceEF-lpdA [pPYC] exceeded the corresponding yields of control strains by 2 and 33% in the absence and by 9 and 26% in the presence in media of HCO₃⁻ ion. It is concluded that an increase in the succinic acid production by strain SGM0.1 P_L-aceEF-lpdA [pPYC] as compared with the strains SGM0.1 and SGM0.1 [pPYC], which synthesize this substance in the reductive branch of the tricarboxylic acid cycle, is caused by activation of the glyoxylate shunt.     

Transport of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript> by an antiporter-related subunit from the <Emphasis Type="Italic">Escherichia coli </Emphasis>NADH dehydrogenase I produced in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The NADH dehydrogenase I from Escherichia coli is a bacterial homolog of the mitochondrial complex I which translocates Na⁺ rather than H⁺. To elucidate the mechanism of Na⁺ transport, the C-terminally truncated NuoL subunit (NuoL_N) which is related to Na⁺/H⁺ antiporters was expressed as a protein A fusion protein (ProtA–NuoL_N) in the yeast Saccharomyces cerevisiae which lacks an endogenous complex I. The fusion protein inserted into membranes from the endoplasmatic reticulum (ER), as confirmed by differential centrifugation and Western analysis. Membrane vesicles containing ProtA–NuoL_N catalyzed the uptake of Na⁺ and K⁺ at rates which were significantly higher than uptake by the control vesicles under identical conditions, demonstrating that ProtA–NuoL_N translocated Na⁺ and K⁺ independently from other complex I subunits. Na⁺ transport by ProtA–NuoL_N was inhibited by EIPA (5-(N-ethyl-N-isopropyl)-amiloride) which specifically reacts with Na⁺/H⁺ antiporters. The cation selectivity and function of the NuoL subunit as a transporter module of the NADH dehydrogenase complex is discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Design of a secondary alcohol degradation pathway from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> DSM 50106 in an engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

The genes encoding an alcohol dehydrogenase, Baeyer–Villiger monooxygenase and an esterase from P. fluorescens DSM 50106, which seemed to be metabolically connected based on the sequence of the corresponding open reading frames, were cloned into one vector (pABE) and functionally expressed in Escherichia coli. Overall expression levels were quite low, however, using whole cells of E. coli JM109 pABE expressing the three recombinant enzymes, conversion of secondary alcohols (C_n) to the corresponding primary alcohols (C_n−2) and acetic acid via ketone and ester was possible. In this way, 2-decanol was almost completely converted within 20 h at 30°C. Thus, it could be shown that the three enzymes are metabolically connected and that they are most probably involved in alkane degradation via sub-terminal oxidation of the acyclic aliphatic hydrocarbons.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> malate dehydrogenase,a novel solubility enhancer for heterologous proteins synthesized in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Using 2-dimensional gel electrophoresis, the Escherichia coli proteome response to a heat-shock stress was analyzed and a 1.6-fold increase of malate dehydrogenase was observed even under the heat-shock condition where the total number of soluble proteins decreased by about 5%. We subsequently demonstrated that, as an N-terminus fusion expression partner, malate dehydrogenase facilitated the folding of, and dramatically increased the solubility of, many aggregation-prone heterologous proteins in E. coli cytoplasm. Therefore, malate dehydrogenase is well suited for production of a biologically active fusion mutant of cutinase (Pseudomonas putida origin) that is currently of considerable to biotechnology and commercial industries.     

Expression of NAD<Superscript>+</Superscript>-dependent formate dehydrogenase in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis> and its involvement in anaerobic metabolism and H<Subscript>2</Subscript> production

An expression system for NAD⁺-dependent formate dehydrogenase gene (fdh1), from Candida boidinii, was constructed and cloned into Enterobacter aerogenes IAM1183. With the fdh1 expression, the total H₂ yield was attributed to a decrease in activity of the lactate pathway and an increase of the formate pathway flux due to the NADH regeneration. Analysis of the redox state balance and ethanol-to-acetate ratio in the fdhl-expressed strain showed that increased reducing power arose from the reconstruction of NADH regeneration pathway from formate thereby contributing to the improved H₂ production.     

Discovery of an acidic,thermostable and highly NADP<Superscript>+</Superscript> dependent formate dehydrogenase from <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> NRRL B-30929

Objectives

To identify a robust NADP⁺ dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929 (LbFDH) with unique biochemical properties.

Results

A new NADP⁺ dependent formate dehydrogenase gene (fdh) was cloned from genomic DNA of L. buchneri NRRL B-30929. The recombinant construct was expressed in Escherichia coli BL21(DE3) with 6?×?histidine at the C-terminus and the purified protein obtained as a single band of approx. 44 kDa on SDS-PAGE and 90 kDa on native-PAGE. The LbFDH was highly active at acidic conditions (pH 4.8–6.2). Its optimum temperature was 60 °C and 50 °C with NADP⁺ and NAD⁺, respectively and its T_m value was 78 °C. Its activity did not decrease after incubation in a solution containing 20% of DMSO and acetonitrile for 6 h. The K_M constants were 49.8, 0.12 and 1.68 mM for formate (with NADP⁺), NADP⁺ and NAD⁺, respectively.

Conclusions

An NADP⁺ dependent FDH from L. buchneri NRRL B-30929 was cloned, expressed and identified with its unusual characteristics. The LbFDH can be a promising candidate for NADPH regeneration through biocatalysis requiring acidic conditions and high temperatures.

     

Kinetic model of functioning and regulation of <Emphasis Type="Italic">Escherichia coli</Emphasis> isocitrate dehydrogenase

To describe published experimental data on the functioning of E. coli isocitrate dehydrogenase (IDH), a Rapid Equilibrium Random Bi Ter mechanism involving the formation of two dead-end enzyme complexes is proposed and a kinetic model of enzyme functioning is constructed. The enzyme is shown to be regulated through reversible phosphorylation by IDH kinase/phosphatase; the latter, in its turn, is controlled by IDH substrates and also by a number of central metabolites—pyruvate, 3-phosphoglycerate, and AMP—reflecting the energy demand of the cell. Using the model, it is shown that an increase in the concentration of the above effectors raises the fraction of active IDH and thus enhances the Krebs cycle flux. The ratio between the free and the phosphorylated forms of IDH is more sensitive to AMP, NADP, and isocitrate than to pyruvate and 3-phosphoglycerate. The model also predicts changes in the ratio between phosphorylated and active forms of IDH in the Krebs cycle that occur with a change in the energy and biosynthetic loads on E. coli cells.     

Characterization of NADP<Superscript>+</Superscript>-specific <Emphasis Type="SmallCaps">l</Emphasis>-rhamnose dehydrogenase from the thermoacidophilic Archaeon <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis>

Thermoplasma acidophilum utilizes l-rhamnose as a sole carbon source. To determine the metabolic pathway of l-rhamnose in Archaea, we identified and characterized l-rhamnose dehydrogenase (RhaD) in T. acidophilum. Ta0747P gene, which encodes the putative T. acidophilum RhaD (Ta_RhaD) enzyme belonging to the short-chain dehydrogenase/reductase family, was expressed in E. coli as an active enzyme catalyzing the oxidation of l-rhamnose to l-rhamnono-1,4-lactone. Analysis of catalytic properties revealed that Ta_RhaD oxidized l-rhamnose, l-lyxose, and l-mannose using only NADP⁺ as a cofactor, which is different from NAD⁺/NADP⁺-specific bacterial RhaDs and NAD⁺-specific eukaryal RhaDs. Ta_RhaD showed the highest activity toward l-rhamnose at 60 °C and pH 7. The K _m and k _cat values were 0.46 mM, 1,341.3 min⁻¹ for l-rhamnose and 0.1 mM, 1,027.2 min⁻¹ for NADP⁺, respectively. Phylogenetic analysis indicated that branched lineages of archaeal RhaD are quite distinct from those of Bacteria and Eukarya. This is the first report on the identification and characterization of NADP⁺-specific RhaD.     

Production of succinate by a <Emphasis Type="Italic">pfl</Emphasis>B <Emphasis Type="Italic">ldh</Emphasis>A double mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing malate dehydrogenase

The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO₃), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO₃ also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose.     

Periplasmic domain of CusA in an <Emphasis Type="Italic">Escherichia coli</Emphasis> Cu<Superscript>+</Superscript>/Ag<Superscript>+</Superscript> transporter has metal binding sites

The resistance nodulation division (RND)-type efflux systems are utilized in Gram-negative bacteria to export a variety of substrates. The CusCFBA system is the Cu⁺ and Ag⁺ efflux system in Escherichia coli, conferring resistance to lethal concentrations of Cu⁺ and Ag⁺. The periplasmic component, CusB, which is essential for the assembly of the protein complex, has Cu⁺ or Ag⁺ binding sites. The twelve-span membrane protein CusA is a homotrimeric transporter, and has a relatively large periplasmic domain. Here, we constructed the periplasmic domain of CusA by joining two DNA segments and then successfully expressed and purified the protein. Isothermal titration calorimetry experiments revealed Ag⁺ binding sites with Kds of 10⁻⁶–10⁻⁵ M. Our findings suggest that the metal binding in the periplasmic domain of CusA might play an important role in the function of the efflux pump.     

Crystal structures of <Emphasis Type="Italic">Leptospira interrogans</Emphasis> FAD-containing ferredoxin-NADP<Superscript>+</Superscript> reductase and its complex with NADP<Superscript>+</Superscript>

Background  

Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs.     

Characterization of alcohol dehydrogenase 1 and 3 from <Emphasis Type="Italic">Neurospora crassa</Emphasis> FGSC2489

Alcohol dehydrogenase (ADH) is a key enzyme in the production and utilization of alcohols. Some also catalyze the formation of carboxylate esters from alcohols and aldehydes. The ADH1 and ADH3 genes of Neurospora crassa FGSC2489 were cloned and expressed in recombinant Escherichia coli to investigate their alcohol dehydrogenation and carboxylate ester formation abilities. Homology analysis and sequence alignment of amino acid sequence indicated that ADH1 and ADH3 of N. crassa contained a zinc-binding consensus sequence and a NAD⁺-binding motif and showed 54–75% identity with fungi ADHs. N. crassa ADH1 was expressed in E. coli to give a specific activity of 289 ± 9 mU/mg using ethanol and NAD⁺ as substrate and cofactor, respectively. Corresponding experiments on the expression and activity of ADH3 gave 4 mU/mg of specific activity. N. crassa ADH1 preferred primary alcohols containing C3–C8 carbons to secondary alcohols such as 2-propanol and 2-butanol. N. crassa ADH1 possessed 5.3 mU/mg of specific carboxylate ester-forming activity accumulating 0.4 mM of ethyl acetate in 18 h. Substrate specificity of various linear alcohols and aldehydes indicated that short chain-length alcohols and aldehydes were good substrates for carboxylate ester production. N. crassa ADH1 was a primary alcohol dehydrogenase using cofactor NAD⁺ preferably and possessed carboxylate ester-forming activity with short chain alcohols and aldehydes.     

Virtual screening of specific chemical compounds by exploring <Emphasis Type="Italic">E.coli</Emphasis> NAD<Superscript>+</Superscript>-dependent DNA ligase as a target for antibacterial drug discovery

Unique substrate specificity compared with ATP-dependent human DNA ligases recommends E.coli NAD⁺-ligases as potential targets. A plausible strategy is to identify the structural components of bacterial DNA ligase that interact with NAD⁺ and then to isolate small molecules that recognize these components and thereby block the binding of NAD⁺ to the ligase. This work describes a molecular modeling approach to detect the 3D structure of NAD⁺-dependent DNA ligase in E. coli whose partial structure was determined by wet lab experiments and rest structure was left as such on the road for repairment. We applied protein-drug docking approach to detect the binding affinity of this enzyme with Quinacrine and some of its virtual derivatives. In silico docking results predict that the virtual derivative of Quinacrine (C21H26ClN3O2) has greater binding affinity than Quinacrine. Drug likeness value of 0.833 was observed for this derivative without showing any toxicity risk.     

Overexpression in a non-native halophilic host and biotechnological potential of NAD<Superscript>+</Superscript>-dependent glutamate dehydrogenase from <Emphasis Type="Italic">Halobacterium salinarum</Emphasis> strain NRC-36014

Enzymes produced by halophilic archaea are generally heat resistant and organic solvent tolerant, and accordingly important for biocatalytic applications in ‘green chemistry’, frequently requiring a low-water environment. NAD⁺-dependent glutamate dehydrogenase from an extremely halophilic archaeon Halobacterium salinarum strain NRC-36014 was selected to explore the biotechnological potential of this enzyme and genetically engineered derivatives. Over-expression in a halophilic host Haloferax volcanii provided a soluble, active recombinant enzyme, not achievable in mesophilic Escherichia coli, and an efficient purification procedure was developed. pH and salt dependence, thermostability, organic solvent stability and kinetic parameters were explored. The enzyme is active up to 90 °C and fully stable up to 70 °C. It shows good tolerance of various miscible organic solvents. High concentrations of salt may be substituted with 30 % DMSO or betaine with good stability and activity. The robustness of this enzyme under a wide range of conditions offers a promising scaffold for protein engineering.