Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
Applied Microbiology and Biotechnology (v.62, #1)
Insect cell culture for industrial production of recombinant proteins by L. Ikonomou; Y.-J. Schneider; S. N. Agathos (pp. 1-20).
Insect cells used in conjunction with the baculovirus expression vector system (BEVS) are gaining ground rapidly as a platform for recombinant protein production. Insect cells present several comparative advantages to mammalian cells, such as ease of culture, higher tolerance to osmolality and by-product concentration and higher expression levels when infected with a recombinant baculovirus. Here we review some of the recent developments in protein expression by insect cells and their potential application in large-scale culture. Our current knowledge of insect cell metabolism is summarised and emphasis is placed on elements useful in the rational design of serum-free media. The culture of insect cells in the absence of serum is reaching maturity, and promising serum substitutes (hydrolysates, new growth and production-enhancing factors) are being evaluated. Proteolysis is a problem of the BEVS system due to its lytic nature, and can, therefore, be a critical issue in insect cell bioprocessing. Several cell- or baculovirus proteases are involved in degradation events during protein production by insect cells. Methods for proteolysis control, the optimal inhibitors and culture and storage conditions which affect proteolysis are discussed. Finally, engineering issues related to high-density culture (new bioreactor types, gas exchange, feeding strategies) are addressed in view of their relevance to large-scale culture.
ε-Poly-l-lysine: microbial production, biodegradation and application potential by T. Yoshida; T. Nagasawa (pp. 21-26).
ε-Poly-l-lysine (ε-PL) is a homo-poly-amino acid characterized by the peptide bond between the carboxyl and ε-amino groups of l-lysine. ε-PL shows a wide range of antimicrobial activity and is stable at high temperatures and under both acidic and alkaline conditions. The mechanism of the inhibitory effect of ε-PL on microbial growth is the electrostatic adsorption to the cell surface of microorganisms on the basis of its poly-cationic property. Due to this antimicrobial activity, ε-PL is now industrially produced in Japan as a food additive by a fermentation process using Streptomyces albulus. In spite of the practical application of ε-PL, the biosynthetic mechanisms of ε-PL have not been clarified at all. ε-PL producers commonly possess membrane-bound ε-PL-degrading aminopeptidase, which might play a role in self-protection.
Non-viral gene therapy: polycation-mediated DNA delivery by M. Thomas; A. M. Klibanov (pp. 27-34).
Gene therapy, i.e., the expression in cells of genetic material with therapeutic activity, holds great promise for the treatment of human diseases. A delivery vehicle (vector), of either viral or non-viral origin, must be used to carry the foreign gene into a cell. Viral vectors take advantage of the facile integration of the gene of interest into the host and high probability of its long-term expression but are plagued by safety concerns. Non-viral vectors, although less efficient at introducing and maintaining foreign gene expression, have the profound advantage of being non-pathogenic and non-immunogenic; they are the subject of this review. Polycation–DNA complexes are particularly attractive for non-viral gene therapy. To perform, they have to attach to the target cell surface, be internalized, escape from endosomes, find a way to the nucleus, and, finally, be available for transcription. The clinical usefulness of polycationic vectors depends on elucidating the role each of these steps plays in gene transfer. Recent progress in consequent rational vector improvement is highlighted by our finding of polyethylenimine derivatives more potent and yet less cytotoxic than the 25-kDa polyethylenimine (one of the most effective non-viral vectors). Such vectors could be further modified with cell-targeting ligands to enhance their utility for in vivo applications.
Kinetics of l-lysine fermentation: a continuous culture model incorporating oxygen uptake rate by S. Ensari; H. C. Lim (pp. 35-40).
For process design and optimization, it is essential to have a mathematical model that represents the system well. Many past studies do not go beyond empirically fitting experimental data. In the present study, an unstructured model incorporating oxygen uptake and dissolved oxygen concentration was developed for a continuous culture of l-lysine. Specific rate expressions of cell growth, substrate consumption, product formation, and oxygen uptake were developed and incorporated in the model. The model predicts very well the effects of operational parameters, such as the dilution rate and the feed substrate concentration. It is also able to predict the unsteady-state dynamics of continuous l-lysine fermentation.
Immobilization of Candida krusei cells producing phytase in alginate gel beads: an application of the preparation of myo-inositol phosphates by C. S. Quan; S. D. Fan; Y. Ohta (pp. 41-47).
Cells of Candida krusei capable of producing phytase were immobilized in Ca-alginate gel beads and used for the preparation of myo-inositol phosphates. The immobilization yield was increased about 5-fold after the beads were treated for 96 h at pH 4.0, 4 °C. The increased yield was retained, even after 1 month, when the cells were kept at this temperature and pH. No shift in the pH optima of phytase of the immobilized cells was observed, compared with that of free cells. However, the optimum temperature for the enzyme of the immobilized cells was 55 °C, which was 15 °C higher than that of free cells. The degradation characteristics of the phytate in immobilized cells packed in a glass column (i.d. 1.2 cm, length 20 cm) were investigated. The variation in the composition of the products results from a change in the flow rate of phytate solution (5 mM). At a flow rate of 1.30 ml/min, a mixture of myo-inositol-2-monophosphate, myo-inositol-1,2,5-triphosphate and myo-inositol-1,2,5,6-tetrakisphosphate was produced, in which the latter two were physiologically active. Also, it was found by NMR analysis that the enzyme of this strain produced only one isomer of each of the inositol phosphates, with the exception of myo-inositol pentakisphosphate. Therefore, the pure isomers were easily isolated using ion-exchange chromatography.
Studies on Bacillus stearothermophilus. Part III. Transformation of testosterone by S. Al-Awadi; M. Afzal; S. Oommen (pp. 48-52).
Bacillus stearothermophilus, a thermophilic bacterium isolated from the Kuwaiti desert, produced a variety of monohydroxy androstene derivatives and an oxidized product when incubated with exogenous testosterone for 24 h at 65°C. The major metabolite was identified as androst-4-en-3,17-dione while minor metabolites included 6α-hydroxyandrost-4-en-3,17-dione, 6β-hydroxyandrost-4-en-3,17-dione, 6α-hydroxytestosterone, and 6β-hydroxytestosterone. These metabolites were purified by TLC and HPLC followed by their identification using 1H- and 13C-NMR and other spectroscopic data.
A novel enzyme, d-3-hydroxyaspartate aldolase from Paracoccus denitrificans IFO 13301: purification, characterization, and gene cloning by J. Q. Liu; T. Dairi; N. Itoh; M. Kataoka; S. Shimizu (pp. 53-60).
A novel enzyme, d-3-hydroxyaspartate aldolase (d-HAA), catalyzing the conversion of d-3-hydroxyaspartate to glyoxylate plus glycine, was purified to homogeneity from Paracoccus denitrificans IFO 13301. d-HAA is strictly d-specific as to the α-position, whereas the enzyme does not distinguish between threo and erythro forms at the β-position. In addition to d-3-hydroxyaspartate, the enzyme also acts on d-threonine, d-3-3,4-dihydroxyphenylserine, d-3-3,4-methylenedioxyphenylserine, and d-3-phenylserine. The d-HAA gene was cloned and sequenced. The gene contains an open reading frame consisting of 1,161 nucleotides corresponding to 387 amino acid residues. The predicted amino acid sequence displayed 35% and 22% identity with that of the d-threonine aldolase of Arthrobacer sp. DK-38 and Alcaligenes xylosoxidan IFO 12669, respectively. This is the first paper reporting both a purified enzyme with d-3-hydroxyaspartate aldolase activity and also its gene cloning.
A small cryptic plasmid from Rhodococcus erythropolis: characterization and utility for gene expression by K. Kostichka; L. Tao; M. Bramucci; J.-F. Tomb; V. Nagarajan; Q. Cheng (pp. 61-68).
Exploration of metabolically diverse rhodococci is generally hampered by the lack of genetic tools. A small cryptic plasmid (pAN12) isolated from Rhodococcus erythropolis strain AN12 was sequenced. Plasmid pAN12 encodes proteins that share homology to replication proteins and putative cell division proteins. Based on in vitro transposon mutagenesis, we determined that the Rep protein of pAN12 is essential for plasmid replication in Rhodococcus spp., and the putative cell division protein Div is important for plasmid stability. The pAN12 replicon is able to replicate in R. erythropolis strains AN12 and CW23 (ATCC 47072) and is compatible with the nocardiophage Q4 replicon present on a Rhodococcus shuttle plasmid pDA71. pAN12 appears to belong to the pIJ101/pJV1 family of rolling circle replication plasmids. Expression of an isoprenoid pathway gene (dxs) on the pAN12-derived multicopy shuttle vector increased production of carotenoid pigments in R. erythropolis ATCC 47072.
Efficient 40°C fermentation of l-lysine by a new Corynebacterium glutamicum mutant developed by genome breeding by J. Ohnishi; M. Hayashi; S. Mitsuhashi; M. Ikeda (pp. 69-75).
We have recently developed a new l-lysine-producing mutant of Corynebacterium glutamicum by "genome breeding" consisting of characterization and reconstitution of a mutation set essential for high-level production. The strain AHP-3 was examined for l-lysine fermentation on glucose at temperatures above 35°C, at which no examples of efficient l-lysine production have been reported for this organism. We found that the strain had inherited the thermotolerance that the original coryneform bacteria was endowed with, and thereby grew and produced l-lysine efficiently up to 41°C. A final titer of 85 g/l after only 28 h was achieved at temperatures around 40°C, indicating the superior performance of the strain developed by genome breeding. When compared with the traditional 30°C fermentation, the 40°C fermentation allowed an increase in yield of about 20% with a concomitant decrease in final growth level, suggesting a significant transition of carbon flux distribution in glucose metabolism. DNA array analysis of metabolic changes between the 30°C and 40°C fermentations identified several differentially expressed genes in central carbon metabolism although we could not find stringent control-like global induction of amino-acid-biosynthetic genes in the 40°C fermentation. Among these changes, two candidates were picked out as the potential causes of the increased production at 40°C; decreased expression of the citrate synthase gene gltA and increased expression of malE, the product of which involves regeneration of pyruvate and NADPH.
The effect of acetate pathway mutations on the production of pyruvate in Escherichia coli by A. Tomar; M. A. Eiteman; E. Altman (pp. 76-82).
We compared pyruvate accumulation in six strains of Escherichia coli and their corresponding ppc mutants. Each strain contained a mutation of a gene involved in the pathway to acetate synthesis. Strains with mutations in genes encoding the pyruvate dehydrogenase complex generally exhibited the greatest pyruvate accumulation of which CGSC6162 (an aceF mutant) and CGSC6162 Δppc were studied in greater detail in controlled fermenters. Both CGSC6162 and CGSC6162 Δppc accumulated greater than 35 g/l pyruvate in a medium supplemented with acetate. We observed pyruvate mass yields from glucose of 0.72 in CGSC6162, with volumetric productivities above 1.5 g l−1 h−1. For CGSC6162 Δppc, we observed pyruvate yields of 0.78 and volumetric productivities above 1.2 g l−1 h−1. CGSC6162 consumed all initially supplied acetate, while CGSC6162 Δppc first consumed and then generated acetate during the course of a 36 h fermentation. Acetate generation and pyruvate oxidase activity was pH- and temperature-dependent, with a pH of 7.0 and the lowest temperature studied (32°C) favoring the greatest pyruvate generation. Lactate was an unexpected by-product even though measured lactate dehydrogenase (LDH) activity was very low.
Characterization and identification of genes essential for dimethyl sulfide utilization in Pseudomonas putida strain DS1 by T. Endoh; K. Kasuga; M. Horinouchi; T. Yoshida; H. Habe; H. Nojiri; T. Omori (pp. 83-91).
Microbial dimethyl sulfide (DMS) conversion is thought to be involved in the global sulfur cycle. We isolated Pseudomonas putida strain DS1 from soil as a bacterium utilizing DMS as a sole sulfur source, and tried to elucidate the DMS conversion mechanism of strain DS1 at biochemical and genetic level. Strain DS1 oxidized DMS to dimethyl sulfone (DMSO2) via dimethyl sulfoxide, whereas the oxidation was repressed in the presence of sulfate, suggesting that a sulfate starvation response is involved in DMS utilization by strain DS1. Two of the five DMS-utilization-defective mutants isolated by transposon5 (Tn5) mutagenesis had a Tn5 insertion in the ssuEADCBF operon, which has been reported to encode a two-component monooxygenase system (SsuED), an ABC-type transporter (SsuABC), and a small protein (SsuF), and also to play a key role in utilization of sulfonates and sulfate esters in another bacterium, P. putida strain S-313. Disruption of ssuD and SsuD enzymatic activity demonstrated that methanesulfonate is a metabolic intermediate of DMS and desulfonated by SsuD. Disruption of ssuC or ssuF also led to a DMS-utilization-defective phenotype. Another two mutants had a defect in a gene homologous to pa2354 from P. aeruginosa PAO1, which encodes a putative transcriptional regulator, while the remaining mutant had a defect in cysM encoding O-acetylserine (thiol)-lyase B.
Glucose oxidation by Gluconobacter oxydans: characterization in shaking-flasks, scale-up and optimization of the pH profile by M. Silberbach; B. Maier; M. Zimmermann; J. Büchs (pp. 92-98).
In this study, the advantage of a novel measuring device for the online determination of oxygen and carbon dioxide transfer rates in shaking-flasks is reported for glucose oxidation by Gluconobacter oxydans. In this fermentation process, this device was used for the characterization of the oxidation pattern of different strains. G. oxydans NCIMB 8084 forms 2,5-diketogluconate from d-glucose in a multi-stage process via three different membrane-bound dehydrogenases. This strain was chosen for a scale-up of the process from shaking-flasks to a 2-l stirred vessel. An enhancement of 2,5-diketogluconate production was realized by controlling the pH at different levels during the fermentation.