Lactate production as representative of the fermentation potential of Corynebacterium glutamicum 2262 in a one-step process.

The fermentative properties of thermo-sensitive strain Corynebacterium glutamicum 2262 were investigated in processes coupling aerobic cell growth and the anaerobic fermentation phase. In particular, the influence of two modes of fermentation on the production of lactate, the fermentation product model, was studied. In both processes, lactate was produced in significant amount, 27 g/L in batch culture, and up to 55.

Biodegradation of Phenanthrene by Pseudomonas putida and a Bacterial Consortium in the Presence and in the Absence of a Surfactant.

This study describes the biodegradation of phenanthrene in aqueous media in the presence and in the absence of a surfactant, Brij 30. Biodegradations were performed using either Pseudomonas putida DSMZ 8368 or a bacterial consortium Pyr01 isolated from one PAHs-polluted site. P.

Polyelectrolyte microcapsules with entrapped multicellular tumor spheroids as a novel tool to study the effects of photodynamic therapy.

In the current study, semi-permeable alginate-oligochitosan microcapsules for multicellular tumor spheroids (MTS) generation were elaborated and tested, to estimate a response of the microencapsulated MTS (MMTS) to photodynamic therapy (PDT). The microcapsules (mean diameter 600 μm) with entrapped human breast adenocarcinoma MCF-7 cells were obtained using an electrostatic bead generator, and MMTS were generated by in vitro long-term cell cultivation. The formed MMTS were incubated in Chlorin e6 photosensitizer solution and then irradiated using 650-nm laser light.

Effect of surfactant pluronic F-68 on CHO cell growth, metabolism, production, and glycosylation of human recombinant IFN-γ in mild operating conditions.

The control of glycosylation to satisfy regulatory requirements and quality consistency of recombinant proteins produced by different processes has become an important issue. With two N-glycosylation sites, γ-interferon (IFN-γ) can be seen as a prototype of a recombinant therapeutic glycoprotein for this purpose. The effect of the nonionic surfactant Pluronic F-68 (PF-68) on cell growth and death was investigated, as well as production and glycosylation of recombinant IFN-γ produced by a CHO cell line that was maintained in a rich protein-free medium in the absence or presence of low agitation.

OdhI dephosphorylation kinetics during different glutamate production processes involving Corynebacterium glutamicum.

In Corynebacterium glutamicum, the activity of the 2-oxoglutarate dehydrogenase complex was shown to be controlled by the phosphorylation of a 15-kDa protein OdhI by different serine/threonine protein kinases. In this paper, the phosphorylation status and kinetics of OdhI dephosphorylation were assessed during glutamate producing processes triggered by either a biotin limitation or a temperature upshock from 33 degrees C to 39 degrees C. A dephosphorylation of OdhI in C.

Related effects of cell adaptation to serum-free conditions on murine EPO production and glycosylation by CHO cells.

The necessity to perform serum-free cultures to produce recombinant glycoproteins generally requires an adaptation procedure of the cell line to new environmental conditions, which may therefore induce quantitative and qualitative effects on the product, particularly on its glycosylation. In previous studies, desialylation of EPO produced by CHO cells was shown to be dependent on the presence of serum in the medium. In this paper, to discriminate between the effects of the adaptation procedure to serum-free medium and the effects of the absence of serum on EPO production and glycosylation, adapted and non-adapted CHO cells were grown in serum-free and serum-containing media.

Cell envelope fluidity modification for an effective glutamate excretion in Corynebacterium glutamicum 2262.

1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was used to assess the cell envelope fluidity of Corynebacterium glutamicum 2262 during a temperature-triggered glutamate producing process. Because the fluorescence lifetime of TMA-DPH was shown to be constant all over the process, fluorescence anisotropy can be considered as a good index of cell envelope fluidity. When the temperature of the fed-batch culture was increased from 33 to 39 degrees C to induce glutamate excretion, the fluorescence anisotropy values decreased from 0.

Characterization of a Corynebacterium glutamicum lactate utilization operon induced during temperature-triggered glutamate production.

Gene expression changes of glutamate-producing Corynebacterium glutamicum were identified in transcriptome comparisons by DNA microarray analysis. During glutamate production induced by a temperature shift, C. glutamicum strain 2262 showed significantly higher mRNA levels of the NCgl2816 and NCgl2817 genes than its non-glutamate-producing derivative 2262NP.

Feedback-resistant acetohydroxy acid synthase increases valine production in Corynebacterium glutamicum.

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.

Dynamics of glutamate synthesis and excretion fluxes in batch and continuous cultures of temperature-triggered Corynebacterium glutamicum.

Corynebacterium glutamicum 2262 strain, when triggered for glutamate excretion, experiences a rapid decrease in growth rate and increase in glutamate efflux. In order to gain a better quantitative understanding of the factors controlling the metabolic transition, the fermentation dynamics was investigated for a temperature-sensitive strain cultivated in batch and glucose-limited continuous cultures. For non-excreting cells at 33 degrees C, increasing the growth rate resulted in strong increases in the central metabolic fluxes, but the intracellular glutamate level, the oxoglutarate dehydrogenase complex (ODHC) activity and the flux distribution at the oxoglutarate node remained essentially constant.

Generation of high-recombinant- protein-producing Chinese hamster ovary (CHO) cells.

The purpose of this chapter is to propose a practical procedure for the generation and selection of Chinese hamster ovary cells producing high levels of recombinant protein by combining in vitro and in vivo amplification of the foreign gene. A detailed description of the expression and amplification plasmids utilized for the in vitro generation of long DNA concatenamers, as well as the cell transfection and selection protocols are given. The procedure required for in vivo gene amplification using the dihydrofolate reductase/methotrexate system is also described.

HPCE monitoring of the N-glycosylation pattern and sialylation of murine erythropoietin produced by CHO cells in batch processes.

Using capillary electrophoresis coupled to laser-induced fluorescence (HPCE-LIF), it was possible to profile N-linked oligosaccharides from EPO, including species containing sialic acid, during the course of batch cultures performed either in serum-free or serum-containing medium. Although an unusual high heterogeneity of the N-linked oligosaccharides was observed by both SDS-PAGE and HPCE analysis, the patterns of mEPO glycans after desialylation by mild acid hydrolysis were found to be quite constant over the course of the cultures either with or without serum supplementation. In contrast, when the protein was analyzed by HPCE without acidic desialylation, fingerprints of N-linked oligosaccharides changed with time in serum-containing conditions.

Glutamate as an inhibitor of phosphoenolpyruvate carboxylase activity in Corynebacterium glutamicum.

The glutamate-producing bacterium, Corynebacterium glutamicum is known to possess two anaplerotic enzymes: pyruvate carboxylase (Pc) and phosphoenolpyruvate carboxylase (PEPc). In vitro, this latter enzyme appeared to be inhibited by different glutamic acid salts, whereas ammonium-glutamate had no influence on Pc activity. To investigate the in vivo relevance of PEPc activity inhibition, the intracellular concentration of glutamate was determined throughout the glutamate-producing process.

Cinnamic acid 4-hydroxylase mechanism-based inactivation by psoralen derivatives: cloning and characterization of a C4H from a psoralen producing plant-Ruta graveolens-exhibiting low sensitivity to psoralen inactivation.

Cinnamate 4-hydroxylase (C4H, EC 1.14.13.

Instability of glutamate production by Corynebacterium glutamicum 2262 in continuous culture using the temperature-triggered process.

Kinetics and physiology of Corynebacterium glutamicum 2262 cultured for extended periods in continuous mode were investigated at 33, 39 and 41 degrees C. At 33 degrees C no glutamate production occurred whatever the dilution rates tested (ranging between 0.05 and 0.

Reduction of CMP-N-acetylneuraminic acid hydroxylase activity in engineered Chinese hamster ovary cells using an antisense-RNA strategy.

Rodent cells, widely used for the industrial production of recombinant human glycoproteins, possess CMP-N-acetylneuraminic acid hydroxylase (CMP-Neu5Ac hydroxylase; EC 1.14.13.