Laboratory metabolic evolution improves acetate tolerance and growth on acetate of ethanologenic Escherichia coli under non-aerated conditions in glucose-mineral medium.

In this work, Escherichia coli MG1655 was engineered to produce ethanol and evolved in a laboratory process to obtain an acetate tolerant strain called MS04 (E. coli MG1655: ΔpflB, ΔadhE, ΔfrdA, ΔxylFGH, ΔldhA, PpflB::pdc ( Zm ) -adhB ( Zm ), evolved). The growth and ethanol production kinetics of strain MS04 were determined in mineral medium, mainly under non-aerated conditions, supplemented with glucose in the presence of different concentrations of sodium acetate at pH 7.0 and at different values of acid pH and a constant concentration of sodium acetate (2 g/l). Results revealed an increase in the specific growth rate, cell mass formation, and ethanol volumetric productivity at moderate concentrations of sodium acetate (2-10 g/l), in addition to a high tolerance to acetate because it was able to grow and produce a high yield of ethanol in the presence of up to 40 g/l of sodium acetate. Genomic analysis of the ΔpflB evolved strain identified that a chromosomal deletion of 27.3 kb generates the improved growth and acetate tolerance in MG1655 ΔpflB derivative strains. This deletion comprises genes related to the respiration of nitrate, repair of alkylated DNA and synthesis of the ompC gene coding for porin C, cytochromes C, thiamine, and colonic acid. Strain MS04 is advantageous for the production of ethanol from hemicellulosic hydrolysates that contain acetate.

Comparative analysis of the Salmonella typhi and Escherichia coli ompC genes.

The nucleotide (nt) sequence of the gene encoding the Salmonella typhi OmpC outer membrane protein, and its deduced amino acid (aa) sequence are presented here. The S. typhi ompC gene consists of an open reading frame of 1134 nt, corresponding to a protein of 378 aa; with a 21-aa signal peptide. This protein is 11 aa longer than Escherichia coli OmpC, but it has an identical leader peptide. The mature OmpC sequence shows 79% similarity for both bacteria at the aa level, and 77% similarity at the nt level. Seven main variable regions in the OmpC protein were identified. Five of them correspond to hydrophilic regions and contain aa observed most frequently in turn configurations in soluble proteins. This suggests that these aa stretches could be located on the exterior of the outer membrane. To probe into the genus and species specificity of the main variable regions, we have constructed complementary oligodeoxyribonucleotides. The use of one of them with a small number of DNA samples is illustrated here; no restriction fragment length polymorphism or nt sequence heterogeneity could be found between S. typhi and Salmonella typhimurium.

pBRINT-Ts: a plasmid family with a temperature-sensitive replicon, designed for chromosomal integration into the lacZ gene of Escherichia coli.

A pBRINT-Ts family of integrative vectors for Escherichia coli was constructed by using a temperature-sensitive replicon derived from pSC101, a region of homology to the lacZ gene, and various antibiotic resistance markers (kanamycin, chloramphenicol and gentamycin) for selection of the integrants. The gene or group of genes to be integrated can be inserted into a multiple cloning site, flanked by an antibiotic resistance marker and lacZ sequences. A simple and rapid procedure was developed for the selection of cells where allelic exchange had occurred. With this procedure, the efficiency of integration of around 10-3 was observed, using several E. coli strains. From colonies with an integrated pBRINT-Ts plasmid, we detected an average allelic exchange event frequency of 7.5%. As a test for this system, we integrated the amy gene that codes for the alpha-amylase from B. stearothermophilus, into the lacZ gene of E. coli W3110. Production of alpha-amylase was found to be proportional to copy number; at up to 10 copies per chromosome.

Characterization of the regulatory region of the Escherichia coli penicillin acylase structural gene.

Penicillin acylase is utilized in the enzymatic production of semisynthetic penicillins. The enzyme is composed of two different subunits that originate from a common precursor. The partial nucleotide (nt) sequence of the structural gene has been published. This paper reports the nt sequence of the regulatory region of this gene, the identification of a functional promoter, the transcriptional start point, and the description of possible regulatory regions.

A common precursor for the two subunits of the penicillin acylase from Escherichia coli ATCC11105.

Penicillin acylase (PA) is an industrial enzyme that is used to convert penicillin G into a precursor for semisynthetic penicillins. We have cloned a segment of DNA that codes for the two subunits required for PA activity. We also report the nucleotide sequence of a DNA fragment that codes for (i) the small subunit, (ii) the N-terminal region of the large subunit and (iii) a putative connecting peptide. These results confirm the existence of a common precursor for both peptides.

A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria.

Modifications of microbial genomes often require the use of the antibiotic-resistance (Anb(R))-encoding genes and other easily selectable markers. We have developed a set of such selectable markers (Cm(R), Km(R) and Gm(R)), which could easily be inserted into the genome and subsequently removed by using the Cre/loxP site-specific recombination system of bacteriophage P1. In this manner the same marker could be used more than once in the same background, while the resulting strain could or would remain Anb(R) marker-free. Three plasmids were constructed, each containing a cassette consisting of the Cm(R), Km(R), or Gm(R) gene flanked by two parallel loxP sites and two polylinkers (MCS). To test insertion and excision, cassettes were inserted into the lacZ or galE genes carried on an origamma/pir-dependent suicide plasmid, which contained a dominant Sm(R) gene. The cassettes were crossed into the E. coli genome by homologous recombination (allelic exchange), in a manner analogous to that described by Pósfai et al. [Nucl. Acids Res. 22 (1994) 2392-2398], selecting for the Cm(R), Km(R), or Gm(R), for the LacZ(-) or GalE(-) and for the Sm(S) phenotypes (the latter to assure allelic exchange rather than insertion of the entire plasmid). When required, after selecting the strain with the desired modification, the Cm(R), Km(R), or Gm(R) marker was excised by supplying the Cre function. Cre was provided by the thermosensitive plasmid pJW168, which was transformed into the Anb(R) host at 30 degrees C, and was subsequently eliminated at 42 degrees C. Thus the Anb(R) marker was removed, whereas the lacZ or galE gene remained interrupted by the retained loxP site.

Determination of the nucleotide sequence for the glutamate synthase structural genes of Escherichia coli K-12.

We have determined the complete nucleotide sequence of a 6.3-kb chromosomal HpaI-EcoRI fragment, that contains the structural genes for both the large and small subunits of the Escherichia coli K-12 glutamate synthase (GOGAT) enzyme, as well as the 5'- and 3'-flanking and intercistronic DNA regions. The Mrs of the two subunits, as deduced from the nucleotide (nt) sequence, were estimated as 166,208 and 52,246. Partial amino acid sequence of the GOGAT enzyme revealed that the large subunit starts with a cysteine residue that is probably generated by a proteolytic cleavage. Northern blotting experiments revealed a transcript of approximately 7300 nt, that at least contains the cistrons for both subunits. A transcriptional start point and a functional promoter were identified in the 5' DNA flanking region of the large subunit gene. The messenger RNA nontranslated leader region has 120 nt and shares identity with the leader regions of E. coli ribosomal operons, in particular around the so-called boxA sequence implicated in antitermination. Other possible regulatory sequences are described.

Gastric and intestinal absorption of oxprenolol in humans.

The gastrointestinal absorption of the beta blocker oxprenolol was investigated in four healthy subjects by an intubation technique. Oxprenolol was introduced into the stomach, dissolved in a homogenized meal containing the marker 14C-polyethylene glycol (PEG) 4000. Unlabeled PEG 4000 was perfused during the whole experiment into the duodenum at the ampulla of Vater. Samples of luminal contents were collected at regular intervals over four hours in the stomach, at the angle of Treitz, and 30 cm below this point. Blood was also collected. Oxprenolol was not absorbed in the stomach. About 80% of the drug emptied from the stomach was absorbed in the duodenum, and 80% of that released from the duodenum was absorbed in a 30-cm segment of the jejunum. The amounts absorbed in these two intestinal segments were directly proportional to the amounts delivered. The areas under the plasma concentration-time curves were not related to the amounts absorbed. A single dose of oxprenolol taken with an homogenized meal did not modify the gastric emptying and secretory response.

Chromosomal editing in Escherichia coli. Vectors for DNA integration and excision.

Chromosomal editing constitutes the direct and specific modification of the genetic information present in the chromosome. In the bacterium Escherichia coli, strategies were originally developed for the production of specific proteins, the genotypic improvement of strains, and the analysis of regulation of gene expression. However, with the emerging field of metabolic engineering and genomics, efficient means of targeting specific genetic mutations into the chromosome are most useful. In this review, a summary of the systems currently available to generate insertions and deletions in the chromosome of E. coli are presented, as well as the current knowledge about the genetic mechanisms responsible for these processes.

Production in Escherichia coli of a rat chimeric proinsulin polypeptide carrying human A and B chains and its preparative chromatography.

A pseudohuman proinsulin coding DNA sequence (MMRPI) carrying human A and B chains, was constructed via directed mutagenesis of a previously modified rat proinsulin cDNA (MRPI) and expressed as a tryptophan (Trp)LE-proinsulin fusion protein in Escherichia coli W3110. Expression of the hybrid gene was achieved by depletion of tryptophan from the medium. The heterologous fusion protein, accumulated as insoluble inclusion bodies within the cell, was obtained by differential centrifugation and then solubilized using formic acid. At the junction of the two peptides, a methionine residue allowed proinsulin to be released from the carrier protein by cyanogen bromide treatment. The sulfonated form of this proinsulin polypeptide was easily purified, at a preparative level, using ion exchange chromatography.

Quantitative determination of benazepril and benazeprilat in human plasma by gas chromatography-mass spectrometry using automated 96-well disk plate solid-phase extraction for sample preparation.

An analytical method for the determination of benazepril and its active metabolite, benazeprilat, in human plasma by capillary gas chromatography-mass-selective detection, with their respective labelled internal standard, was developed and validated according to international regulatory requirements. After addition of the internal standards, the compounds were extracted from plasma by solid-phase extraction using automated 96-well plate technology. After elution, the compounds were converted into their methyl ester derivatives by means of a safe and stable diazomethane derivative. The methyl ester derivatives were determined by gas chromatography using a mass-selective detector at m/z 365 for benazepril and benazeprilat and m/z 370 for the internal standards. Intra- and inter-day accuracy and precision were found to be suitable over the range of concentrations between 2.50 and 1000 ng/mL.

Development, application and comparison of an enzyme immunoassay and a high-performance liquid chromatography method for the determination of the aromatase inhibitor CGS 20,267 in biological fluids.

CGS 20,267 is a new potent and selective, nonsteroidal, oral aromatase inhibitor. For its determination in human plasma and urine, an enzyme immunoassay (EIA) and an HPLC method were developed. The EIA showed good precision and accuracy (intra- and interassay variation between 3.0 and 17.7%, recoveries between 81 and 106%) and a quantitation limit of 0.7 nmol/L. A strong cross reactivity of the antibodies with the hydroxy metabolite of CGS 20,267 (CGP 44,645) was observed. The HPLC method showed a quantitation limit in plasma of 28 and 34 nmol/L for CGS 20,267 and CGP 44,645, respectively. For urine, concentrations down to 180 nmol/L (CGS 20,267) and 210 nmol/L (CGP 44,645) could be measured. A cross check between EIA and HPLC on plasma samples from healthy male volunteers or breast cancer patients treated orally with CGS 20,267 revealed an excellent correlation (slope = 0.934, intercept = 26, r = 0.991). However, the EIA measurements of urine samples yielded 3-25 times higher concentrations than those obtained by HPLC. Further, HPLC analysis revealed the presence of CGS 20,267 and cross-reacting metabolites in urine but not in plasma. Therefore, the EIA can only be used for the determination of CGS 20,267 in plasma samples.

Engineering the Escherichia coli fermentative metabolism.

Fermentative metabolism constitutes a fundamental cellular capacity for industrial biocatalysis. Escherichia coli is an important microorganism in the field of metabolic engineering for its well-known molecular characteristics and its rapid growth. It can adapt to different growth conditions and is able to grow in the presence or absence of oxygen. Through the use of metabolic pathway engineering and bioprocessing techniques, it is possible to explore the fundamental cellular properties and to exploit its capacity to be applied as industrial biocatalysts to produce a wide array of chemicals. The objective of this chapter is to review the metabolic engineering efforts carried out with E. coli by manipulating the central carbon metabolism and fermentative pathways to obtain strains that produce metabolites with high titers, such as ethanol, alanine, lactate and succinate.

Signaling by IL-1beta+IFN-gamma and ER stress converge on DP5/Hrk activation: a novel mechanism for pancreatic beta-cell apoptosis.

Chronic inflammation and pro-inflammatory cytokines are important mediators of pancreatic beta-cell destruction in type 1 diabetes (T1D). We presently show that the cytokines IL-1beta+IFN-gamma and different ER stressors activate the Bcl-2 homology 3 (BH3)-only member death protein 5 (DP5)/harakiri (Hrk) resulting in beta-cell apoptosis. Chemical ER stress-induced DP5 upregulation is JNK/c-Jun-dependent. DP5 activation by cytokines also involves JNK/c-Jun phosphorylation and is antagonized by JunB. Interestingly, cytokine-inducted DP5 expression precedes ER stress: mitochondrial release of cytochrome c and ER stress are actually a consequence of enhanced DP5 activation by cytokine-mediated nitric oxide formation. Our findings show that DP5 is central for beta-cell apoptosis after different stimuli, and that it can act up- and downstream of ER stress. These observations contribute to solve two important questions, namely the mechanism by which IL-1beta+IFN-gamma induce beta-cell death and the nature of the downstream signals by which ER stress 'convinces' beta-cells to trigger apoptosis.

Optimum melanin production using recombinant Escherichia coli.

AIMS: A parametric study was conducted to define optimum conditions to achieve high yields in the conversion of tyrosine to eumelanin (EuMel) using recombinant Escherichia coli. METHODS AND RESULTS: Escherichia coli W3110 (pTrcMutmelA) expressing the tyrosinase coding gene from Rhizobium etli and glucose-mineral media were used to transform tyrosine into EuMel. Batch aerobic fermentor cultures were performed to study the effect of temperature, pH and inducer concentration (isopropyl-D-thio-galactopyranoside) on melanin production. Under optimum conditions, 0.1 mmol l(-1) of isopropyl-D-thio-galactopyranoside, temperature of 30 degrees C, and changing pH from 7.0 to 7.5 during the production phase, a 100% conversion of tyrosine into EuMel is obtained. Furthermore, tyrosine feeding allowed us to obtain the highest level (6 g l(-1)) of EuMel produced by recombinant E. coli reported until now. CONCLUSIONS: The most important factors affecting melanin formation and hence influencing the rate and efficiency in the conversion of tyrosine into EuMel in this system, are the temperature and pH. SIGNIFICANCE AND IMPACT OF THE STUDY: Maximum theoretical yield was obtained using a simple culture process and mineral media to convert tyrosine (a medium value compound) into melanin, a high value compound. The process reported here avoids the use of purified tyrosinase, expensive chemical methods or the cumbersome extraction of this polymer from animal or plant tissues.

Determination of oxprenolol in human plasma by high-performance liquid chromatography, in comparison with gas chromatography and gas chromatography-mass spectrometry.

A high-performance liquid chromatographic method for the quantitative assay of oxprenolol in human plasma is described. After addition of alprenolol as internal standard, the compounds are extracted from plasma at alkaline pH into an organic phase and back-extracted into an acidic aqueous phase. Separation of the plasma components and metabolites was achieved on a reversed-phase column. Concentrations down to 66 nmol/l (20 ng/ml) can be determined with UV detection at 222 nm. This technique compares favourably with gas chromatographic and gas chromatographic-mass spectrometric methods.

Determination of 3-deoxy-D-arabino-heptulosonate 7-phosphate productivity and yield from glucose in Escherichia coli devoid of the glucose phosphotransferase transport system.

The effect of inactivation of the glucose phosphotransferase transport system (PTS) on 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) productivity and yield from glucose in Escherichia coli is reported. Strains used in this study were the PTS(+) PB103 and its PTS(-) glucose(+) derivative NF9. Their aroB(-) derivatives PB103B and NF9B were constructed to allow accurate measurement of total carbon flow into the aromatic pathway. The measured specific rates of DAHP synthesis were 0.55 and 0.94 mmol/g-dcw. h and the DAHP molar yields from glucose were 0.43 and 0.71 mol/mol for the PTS(+) aroB(-)and the PTS(-) glucose(+) aroB(-)strains, respectively. For the latter strain, this value represents 83% of the maximum theoretical yield for DAHP synthesis from glucose.

A direct comparison of approaches for increasing carbon flow to aromatic biosynthesis in Escherichia coli.

Different approaches to increasing carbon commitment to aromatic amino acid biosynthesis were compared in isogenic strains of Escherichia coli. In a strain having a wild-type PEP:glucose phosphotransferase (PTS) system, inactivation of the genes encoding pyruvate kinase (pykA and pykF) resulted in a 3.4 fold increase in carbon flow to aromatic biosynthesis. In a strain already having increased carbon flow to aromatics by virtue of overexpression of the tktA gene (encoding transketolase), the pykA and/or pykf mutations had no effect. A PTS- glucose+ mutant showed a 1.6-fold increase in carbon flow to aromatics compared to the PTS+ control strain. In the PTS- glucose+ host background, overexpression of tktA caused a further 3.7-fold increase in carbon flow, while inactivation of pykA and pykF caused a 5.8-fold increase. When all of the variables tested (PTS-glucose+, pykA, pykF, and overexpressed tktA) were combined in a single strain, a 19.9-fold increase in carbon commitment to aromatic biosynthesis was achieved.

Characterization of sugar mixtures utilization by an Escherichia coli mutant devoid of the phosphotransferase system.

Due to catabolite repression in microorganisms, sugar mixtures cannot be metabolized in a rapid and efficient manner. Therefore, the development of mutant strains that avoid this regulatory system is of special interest to fermentation processes. In the present study, the utilization of sugar mixtures by an Escherichia coli mutant strain devoid of the phosphotransferase system (PTS) was characterized. This mutant can transport glucose (PTS- Glucose+ phenotype) by a non-PTS mechanism as rapidly as its wild-type parental strain. In cultures grown in minimal medium supplemented with glucose-xylose or glucose-arabinose mixtures, glucose repressed arabinose- or xylose-utilization in the wild-type strain. However, under the same culture conditions with the PTS- Glucose+ mutant, glucose and arabinose were co-metabolized, but glucose still exerted a partial repressive effect on xylose consumption. In cultures growing with a triple mixture of glucose-arabinose-xylose, the wild-type strain sequentially utilized glucose, arabinose and finally, xylose. In contrast, the PTS- Glucose+ strain co-metabolized glucose and arabinose, whereas xylose was utilized after glucose-arabinose depletion. As a result of glucose-arabinose co-metabolism, the PTS- Glucose+ strain consumed the total amount of sugars contained in the culture medium 16% faster than the wild-type strain. [14C]-Xylose uptake experiments showed that in the PTS- Glucose+ strain, galactose permease increases xylose transport capacity and the observed partial repression of xylose utilization depends on the presence of intracellular glucose.