On the function of the various quinone species in Escherichia coli

The respiratory chain of Escherichia?coli contains three quinones. Menaquinone and demethylmenaquinone have low midpoint potentials and are involved in anaerobic respiration, while ubiquinone, which has a high midpoint potential, is involved in aerobic and nitrate respiration. Here, we report that demethylmenaquinone plays a role not only in trimethylaminooxide-, dimethylsulfoxide- and fumarate-dependent respiration, but also in aerobic respiration. Furthermore, we demonstrate that demethylmenaquinone serves as an electron acceptor for oxidation of succinate to fumarate, and that all three quinol oxidases of E.?coli accept electrons from this naphtoquinone derivative.     

On the mechanism of αC polymer formation in fibrin

Our previous studies revealed that the fibrinogen αC-domains undergo conformational changes and adopt a physiologically active conformation upon their self-association into αC polymers in fibrin. In the present study, we analyzed the mechanism of αC polymer formation and tested our hypothesis that self-association of the αC-domains occurs through the interaction between their N-terminal subdomains and may include β-hairpin swapping. Our binding experiments performed by size-exclusion chromatography and optical trap-based force spectroscopy revealed that the αC-domains self-associate exclusively through their N-terminal subdomains, while their C-terminal subdomains were found to interact with the αC-connectors that tether the αC-domains to the bulk of the molecule. This interaction should reinforce the structure of αC polymers and provide the proper orientation of their reactive residues for efficient cross-linking by factor XIIIa. Molecular modeling of self-association of the N-terminal subdomains confirmed that the hypothesized β-hairpin swapping does not impose any steric hindrance. To "freeze" the conformation of the N-terminal subdomain and prevent the hypothesized β-hairpin swapping, we introduced by site-directed mutagenesis an extra disulfide bond between two β-hairpins of the bovine Aα406-483 fragment corresponding to this subdomain. The experiments performed by circular dichroism revealed that Aα406-483 mutant containing Lys429Cys/Thr463Cys mutations preserved its β-sheet structure. However, in contrast to wild-type Aα406-483, this mutant had lower tendency for oligomerization, and its structure was not stabilized upon oligomerization, in agreement with the above hypothesis. On the basis of the results obtained and our previous findings, we propose a model of fibrin αC polymer structure and molecular mechanism of assembly.     

Cloning of the thermostable α-amylase gene from Pyrococcus woesei in Escherichia coli

Pyrococcus woesei (DSM 3773) α-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)α-amyl and pYTB2α-amyl vectors obtained were used for expression of thermostable α-amylase or fusion of α-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of α-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation—they exhibit only 35% of total cell activity—and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable α-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75°C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95°C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90°C and 110°C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120°C. Maltose was the main end product of starch hydrolysis catalyzed by this α-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.     

Synthesis of α3 phage DNA in replication mutants of Escherichia coli

Host functions for DNA replication of bacteriophage α3, a representative of group A microvirid phages, were studied using dna and rep mutants of Escherichia coli. In dna⁺ cells, conversion of phage α3 single-stranded DNA (SS) into the double-stranded replicative form (RF) was insensitive to 30–150 μg/ml of chloramphenicol, 200 μg/ml of rifampicin, 50 μg/ml of nalidixic acid, or 200 μg/ml of novobiocin. At 43°C, synthesis of the parental RF was inhibited in dnaG and dnaZ mutants, but not in dnaE and rep strains. Replication of phage α3 progeny RF was prevented by 50 μg/ml of mitomycin C (in hcr⁺ bacteria), 50 μg/ml of nalidixic acid or 200 μg/ml of novoviocin, but neither by 30 μg/ml of chloramphenicol nor by 200 μg/ml of rifampicin. Besides dnaG and dnaZ gene products, dnaE and rep functions were essential for the progeny RF synthesis. Host factor dependence of α3 was relatively simple and, in contrast with phages øX174 and G4, α3 did not require dnaB and dnaC(D) activities.     

High-level expression of human αs1-casein in Escherichia coli

Human _s1-casein was expressed efficiently in Escherichia coli. The overproduced recombinant human a _s1-casein was about 25% of the total cell protein. Two different vectors were constructed to express Met-_s1-casein and Met-_s1-casein with a His-affinity tag at the C-terminus. Recombinant Met-_s1-casein with a His-affinity tag was purified to homogeneity using Ni-nitrilotriacetic acid resin. N-terminal sequence of the first 10 amino acid residues of this purified protein was identical to that of mature human _s1-casein with an extra methionine residue at the N-terminus.     

The requirement for the protonated α-amino group for the transport of peptides in Escherichia coli

Many glycine peptides support growth of a glycine auxotroph of Escherichia coli. If the alpha-amino group of these peptides is methylated, the products are still utilized for growth, and also retain comparable ability with the unsubstituted peptides to compete with natural peptides for transport into the cell. In contrast, glycine peptides devoid of an alpha-amino group, or that have the alpha-amino group substituted by one of a number of acyl groups are not utilized, although E. coli possesses intracellular enzymic activity able to release glycine from such compounds; further, these derivatives do not compete with natural peptides for transport into the cell.     

Delayed supplementation of glycine enhances extracellular secretion of the recombinant α-cyclodextrin glycosyltransferase in Escherichia coli

The targeting of recombinant proteins for secretion to the culture medium of Escherichia coli presents significant advantages over cytoplasmic or periplasmic expression. However, a major barrier is inadequate secretion across two cell membranes. In the present study, we attempted to circumvent this secretion problem of the recombinant α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01. It was found that glycine could promote extracellular secretion of the recombinant α-CGTase for which one potential mechanism might be the increase in membrane permeability. However, further analysis indicated that glycine supplementation resulted in impaired cell growth, which adversely affected overall recombinant protein production. Significantly, delayed supplementation of glycine could control cell growth impairment exerted by glycine. As a result, if the supplementation of 1% glycine was optimally carried out at the middle of the exponential growth phase, the α-CGTase activity in the culture medium reached 28.5 U/ml at 44 h of culture, which was 11-fold higher than that of the culture in regular terrific broth medium and 1.2-fold higher than that of the culture supplemented with 1% glycine at the beginning of culture.     

The control by respiration of the uptake of α-methyl glucoside in Escherichia coli K12

The uptake of methyl α-d-glucopyranoside (α-MG) by Escherichia coli K12 was decreased by the addition of substrates which stimulated the rate of oxygen consumption by the cells. The inhibition, which occurred only at non-saturating concentrations of α-MG, was not the result of a stimulation of the rate of exit of intracellular α-MG, and was abolished by the presence of carbonyl cyanide m-chlorophenylhydrazone or sodium azide. Since those drugs inhibit energy conservation at the respiratory chain and did not alter significantly the rate of oxygen consumption under the conditions for the assay of α-MG uptake, it appears that the inhibition of the transport system by respirable substrates is mediated by some form of energy derived from respiration.     

Role of α-helical domains in functioning of ATP-dependent Lon protease of Escherichia coli

Homooligomeric ATP-dependent LonA proteases are bifunctional enzymes belonging to the superfamily of AAA⁺ proteins. Their subunits are formed by five successively connected domains, i.e., N-terminal (N), α-helical (HI(CC)), nucleotide-binding (NB), the second α-helical (H), and proteolytic (P) domains. The presence of the inserted HI(CC) domain determines the uniqueness of LonA proteases among the AAA⁺ proteins. The role of the α-helical domains in the LonA protease functioning was studied with an example of E. coli Lon protease (Ec-Lon). The properties of the intact Ec-Lon and its mutant forms, i.e., Lon-R164A and Lon-R542A bearing the substituted arginine residues at the similar positions in the HI(CC) and H domains, were compared. The H domain was shown to play a crucial role in ATP hydrolysis and enzyme binding to the target protein. The HI(CC) domain is not decisive for the manifestation of the catalytic properties of the enzyme. However, it affects the functioning of Lon ATPase and peptidase sites and is involved in maintaining enzyme stability. The participation of the HI(CC) domain in the formation of three-dimensional structures of LonA proteases and/or their complexes with DNA is suggested.     

Efficient biosynthesis of α-aminoadipic acid via lysine catabolism in Escherichia coli

α-Aminoadipic acid (AAA) is a nonproteinogenic amino acid with potential applications in pharmaceutical, chemical and animal feed industries. Currently, AAA is produced by chemical synthesis, which suffers from high cost and low production efficiency. In this study, we engineered Escherichia coli for high-level AAA production by coupling lysine biosynthesis and degradation pathways. First, the lysine-α-ketoglutarate reductase and saccharopine dehydrogenase from Saccharomyces cerevisiae and α-aminoadipate-δ-semialdehyde dehydrogenase from Rhodococcus erythropolis were selected by in vitro enzyme assays for pathway assembly. Subsequently, lysine supply was enhanced by blocking its degradation pathway, overexpressing key pathway enzymes and improving nicotinamide adenine dineucleotide phosphate (NADPH) regeneration. Finally, a glutamate transporter from Corynebacterium glutamicum was introduced to elevate AAA efflux. The final strain produced 2.94 and 5.64 g/L AAA in shake flasks and bioreactors, respectively. This work provides an efficient and sustainable way for AAA production.     

Metabolic engineering of Escherichia coli for α-farnesene production

Sesquiterpenes are important materials in pharmaceuticals and industry. Metabolic engineering has been successfully used to produce these valuable compounds in microbial hosts. However, the microbial potential of sesquiterpene production is limited by the poor heterologous expression of plant sesquiterpene synthases and the deficient FPP precursor supply. In this study, we engineered E. coli to produce α-farnesene using a codon-optimized α-farnesene synthase and an exogenous MVA pathway. Codon optimization of α-farnesene synthase improved both the synthase expression and α-farnesene production. Augmentation of the metabolic flux for FPP synthesis conferred a 1.6- to 48.0-fold increase in α-farnesene production. An additional increase in α-farnesene production was achieved by the protein fusion of FPP synthase and α-farnesene synthase. The engineered E. coli strain was able to produce 380.0 mg/L of α-farnesene, which is an approximately 317-fold increase over the initial production of 1.2 mg/L.     

Production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli

Most mammalian bioactive peptides possess a C-terminal amino acid amide moiety. The presence of the C-terminal amide is a significant impediment to the recombinant production of α-amidated peptides. α-Amidated peptides are produced in vivo by the enzymatic cleavage of a precursor with a C-terminal glycine residue. Peptidylglycine α-hydroxylating monooxygenase catalyzes the key step in the oxidation of the glycine-extended precursors to the α-amidated peptide. Herein, we detail the production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli possessing a N-terminal fusion to thioredoxin (Trx). Trx was fused to hPHMcc to enhance the yield of the resulting 52 kDa protein as a soluble and catalytically active enzyme. The Trx-hPHMcc-His(6) fusion was purified to homogeneity and exhibited steady-state kinetic parameters that were similar to purified rat PHMcc. The bacterial production of recombinant hPHMcc will foster efforts to generate α-amidated peptides by the co-expression of hPHMcc and the α-amidated peptide precursors in E. coli or the in vitro amidation of recombinantly expressed α-amidated peptide precursors.     

A Functional Raw Starch-Binding Domain of Barley α-Amylase Expressed in Escherichia coli

The mature form of barley seed low-pI α-amylase (BAA1) possesses a raw starch-binding site in addition to the catalytic site. A truncated cDNA encoding the C-terminal region (aa 281–414) and containing the proposed raw starch-binding domain (SBD) but lacking Trp278/Trp279, a previously proposed starch granule-binding site, was synthesized via PCR and expressed in Escherichia coli as an N-terminal His-Tag fusion protein. SBD was produced in the form of insoluble inclusion bodies that were extracted with urea and successfully refolded into a soluble form via dialysis. To determine binding, SBD was purified by affinity chromatography with cycloheptaamylose as ligand cross-linked to Sepharose. This work demonstrates that a SBD is located in the C-terminal region and retains sufficient function in the absence of the N-terminal, catalytic, and Trp278/279 regions.     

Pathway engineering of Escherichia coli for α-ketoglutaric acid production

α-Ketoglutaric acid (α-KG) is a multifunctional dicarboxylic acid in the tricarboxylic acid (TCA) cycle, but microbial engineering for α-KG production is not economically efficient, due to the intrinsic inefficiency of its biosynthetic pathway. In this study, pathway engineering was used to improve pathway efficiency for α-KG production in Escherichia coli. First, the TCA cycle was rewired for α-KG production starting from pyruvate, and the engineered strain E. coli W3110Δ4-PCAI produced 15.66 g/L α-KG. Then, the rewired TCA cycle was optimized by designing various strengths of pyruvate carboxylase and isocitrate dehydrogenase expression cassettes, resulting in a large increase in α-KG production (24.66 g/L). Furthermore, acetyl coenzyme A (acetyl-CoA) availability was improved by overexpressing acetyl-CoA synthetase, leading to α-KG production up to 28.54 g/L. Finally, the engineered strain E. coli W3110Δ4-P_(H)CAI_(H)A was able to produce 32.20 g/L α-KG in a 5-L fed-batch bioreactor. This strategy described here paves the way to the development of an efficient pathway for microbial production of α-KG.     

Surface Expression of ω-Transaminase in Escherichia coli

Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus ω-transaminase in Escherichia coli using a surface display vector based on the autotransporter adhesin involved in diffuse adherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity.     

Induction threshold of the Q - mutant of bacteriophage λ in Escherichia coli

Temperature induction of bacteriophage in Escherichia coli depends on bacterial population density. The lowest rate of viability loss at the temperature threshold results in maximal gene expression of . -Infection causes bacterial cells to lose cell viability and thus decrease temperature induction efficiency. In addition, shifting-up in temperature increases the probability of progeny ; thus, the mortality of bacterial hosts increases and the expression of recombinant proteins by naked significantly decrease.     

What Is the Role of ε in the Escherichia coli ATP Synthase?

The ATP synthase from Escherichia coli is a prototype of the ATP synthases that are found in many bacteria, in the mitochondria of eukaryotes, and in the chloroplasts of plants. It contains eight different types of subunits that have traditionally been divided into F₁, a water-soluble catalytic sector, and F_o, a membrane-bound ion transporting sector. In the current rotary model for ATP synthesis, the subunits can be divided into rotor and stator subunits. Several lines of evidence indicate that is one of the three rotor subunits, which rotate through 360 degrees. The three-dimensional structure of is known and its interactions with other subunits have been explored by several approaches. In light of recent work by our group and that of others, the role of in the ATP synthase from E. coli is discussed.     

Overproduction of fungal ribotoxin α-sarcin in Escherichia coli: generation of an active immunotoxin

α-Sarcin is a ribonucleolytic protein secreted by the mold Aspergillus giganteus. DNA encoding α-sarcin was isolated from the host and cloned into T7 promoter based E. coli expression vectors. Using bacterial outer membrane protein A (OmpA) signal sequence, properly processed recombinant (re-) protein was secreted into the culture medium while in the absence of a signal sequence protein remained insoluble in the bacterial inclusion bodies. The re-α-sarcin was purified to homogeneity by simple chromatographic techniques both from the insoluble and soluble sources with respective yields of 40–50 μg/ml and 2–3 μg/ml. The re-ribotoxin was functionally as active as the native toxin and preserved its specificity. The re-α-sarcin was used in the construction of an active immunotoxin targeted at the human cancer cells overexpressing transferrin receptor (TFR).