Jean-Louis Goergen

The collagen receptor DDR2 regulates proliferation and its elimination leads to dwarfism.

The discoidin domain receptor 2 (DDR2) is a member of a subfamily of receptor tyrosine kinases whose ligands are fibrillar collagens, and is widely expressed in postnatal tissues. We have generated DDR2‐deficient mice to establish the in vivo functions of this receptor, which have remained obscure. These mice exhibit dwarfism and shortening of long bones. This phenotype appears to be caused by reduced chondrocyte proliferation, rather than aberrant differentiation or function. In a skin wound healing model, DDR2−/− mice exhibit a reduced proliferative response compared with wild‐type littermates. In vitro, fibroblasts derived from DDR2−/− mutants proliferate more slowly than wild‐type fibroblasts, a defect that is rescued by introduction of wild‐type but not kinase‐dead DDR2 receptor. Together our results suggest that DDR2 acts as an extracellular matrix sensor to modulate cell proliferation.

Characterization of a Corynebacterium glutamicum Lactate Utilization Operon Induced during Temperature-Triggered Glutamate Production

ABSTRACT 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. Reverse transcription-PCR analysis showed that the two genes together constitute an operon. NCgl2816 putatively codes for a lactate permease, while NCgl2817 was demonstrated to encode quinone-dependent l-lactate dehydrogenase, which was named LldD. C. glutamicum LldD displayed Michaelis-Menten kinetics for the substrate l-lactate with a Km of about 0.51 mM. The specific activity of LldD was about 10-fold higher during growth on l-lactate or on an l-lactate-glucose mixture than during growth on glucose, d-lactate, or pyruvate, while the specific activity of quinone-dependent d-lactate dehydrogenase differed little with the carbon source. RNA levels of NCgl2816 and lldD were about 18-fold higher during growth on l-lactate than on pyruvate. Disruption of the NCgl2816-lldD operon resulted in loss of the ability to utilize l-lactate as the sole carbon source. Expression of lldD restored l-lactate utilization, indicating that the function of the permease gene NCgl2816 is dispensable, while LldD is essential, for growth of C. glutamicum on l-lactate.

Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum

ABSTRACT 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 (5.4 kb, Kmr). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum ΔilvA ΔpanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.

Characterisation of the enzyme activities involved in the valine biosynthetic pathway in a valine-producing strain of Corynebacterium glutamicum

The enzyme activities of the valine biosynthetic pathway and their regulation have been studied in the valine-producing strain, Corynebacterium glutamicum 13032DeltailvApJC1ilvBNCD. In this micro-organism, this pathway might involve up to five enzyme activities: acetohydroxy acid synthase (AHAS), acetohydroxy acid isomeroreductase (AHAIR), dihydroxyacid dehydratase and transaminases B and C. For each enzyme, kinetic parameters (optimal temperature, optimal pH and affinity for substrates) were determined. The first enzyme of the pathway, AHAS, was shown to exhibit a weak affinity for pyruvate (K(m)=8.3 mM). It appeared that valine and leucine inhibited the three first steps of the pathway (AHAS, AHAIR and DHAD). Moreover, the AHAS activity was inhibited by isoleucine. Considering the kinetic data collected during this work, AHAS would be a key enzyme for further strain improvement intending to increase the valine production by C. glutamicum.

Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release

The death of the hybridoma VO 208 in a continuous culture at pH 7 and 6.8 was investigated by measuring both the appearance of visible dead cells which do not exclude the trypan blue dye and the release of lactate dehydrogenase (LDH) in the culture medium. The intracellular LDH was found to be completely released either when live cells lysed or when they were transformed into visible dead cells. No significant lysis of blue dead cells could be observed at the two different pH. Using a LDH balance over the culture system, cell lysis was found negligible at pH 7, but accounted for 20% of the total cell death at pH 6.8. A methodology is proposed to evaluate the rate constants of hybridoma lysis and total death. For the investigated cell line in continuous culture, the calculated total cell death rate constant was found to increase from 0.002 h−1 to 0.01 h−1 when decreasing the pH from 7 to 6.8.

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. Under these conditions, a dose‐dependent effect of PF‐68 (0–0.1%) was shown not only to significantly enhance growth but also to reduce cell lysis. Interestingly, supplementing the culture medium with PF‐68 led to increased IFN‐γ production as a result of both higher cell densities and a higher specific production rate of IFN‐γ. If cells were grown with agitation, lack of PF‐68 in the culture medium decreased the fraction of the fully glycosylated IFN‐γ glycoform (2N) from 80% to 65–70% during the initial period. This effect appeared to be due to a lag phase in cell growth observed during this period. Finally, a global kinetic study of CHO cell metabolism indicated higher efficiency in the utilization of the two major carbon substrates when cultures were supplemented with PF‐68. Therefore, these results highlight the importance of understanding how media surfactant can affect cell growth as well as cell death and the product quality of a recombinant glycoprotein expressed in CHO cell cultures.

An improved temperature-triggered process for glutamate production with Corynebacterium glutamicum☆

An improved glutamate-producing fed-batch process, using a temperature-sensitive strain of Corynebacterium glutamicum, has been characterized. By a tight control of the culture temperature, it was possible to get industrially interesting performance as regards glutamate concentration, yield and productivity. A 24 h fermentation period enabled the production of 85 g/l of glutamate in the production phase induced after a temperature shift from 33°C to 39°C. The maximum specific production rate of glutamate was 0.63 g/g/h with a yield of 0.46 g of glutamate/gram of glucose. The two main co-products of the fermentation were lactate (11 g) and trehalose (12 g). Only trace amounts of other organic acids accumulated in the culture medium. This process offers an interesting alternative to currently employed fermentation strategies in which biotin limitation and/or surfactant addition is used to induce glutamate production. Simple control of fermentor cooling can be used to control the onset of the production phase, offering significant advantages from both an economic and a process robustness viewpoint.

Influence of intracellular nucleotide and nucleotide sugar contents on recombinant interferon-γ glycosylation during batch and fed-batch cultures of CHO cells

Both the macroheterogeneity of recombinant human IFN‐γ produced by CHO cells and intracellular levels of nucleotides and sugar nucleotides, have been characterized during batch and fed‐batch cultures carried out in different media. Whereas PF‐BDM medium was capable to maintain a high percentage of the doubly‐ glycosylated glycoforms all over the process, mono‐glycosylated and non‐glycosylated forms increased during the batch culture using SF‐RPMI medium. Intracellular level of UTP was higher in PF‐BDM all over the batch culture compared to the SF‐RPMI process. UDP‐Gal accumulated only during the culture performed in PF‐BDM medium, probably as a consequence of the reduced UDP‐Glc synthesis flux in SF‐RPMI medium. When the recombinant CHO cells were cultivated in fed‐batch mode, the UTP level remained at a relatively high value in serum‐containing RPMI and its titer increased during the fed‐phase indicating an excess of biosynthesis. Besides, an accumulation of UDP‐Gal occurred as well. Those results all together indicate that UTP and UDP‐Glc syntheses in CHO cells cultivated in SF‐RPMI medium in batch process, could be limiting during the glycosylation processes of the recombinant IFN‐γ. At last, the determination of the energetic status of the cells over the three studied processes suggested that a relationship between the adenylate energy charge and the glycosylation macroheterogeneity of the recombinant IFN‐γ may exist. Biotechnol. Bioeng. 2008;100: 721–733.

Promoting effect of rapeseed proteins and peptides on Sf9 insect cell growth.

The Baculovirus Expression Vector System has become widely used for the production of recombinant proteins for research and diagnostics. Serum-free culture media able to support high cell densities have been developed for the large scale culture of insect cells. While serum elimination aims at avoiding the risks associated with the introduction of an ill defined component of bovine origin, additives such as protein hydrolysates from animal sources are still used. An alternative could be the supplementation of culture media with protein hydrolysates derived from plants. In this study, we describe the replacement of lactalbumin hydrolysate with a laboratory produced hydrolysate of rapeseed proteins. Its effect on Sf9 cell growth kinetics, substrate consumption and by-product formation in low-serum or serum-free medium was evaluated. Cells were unable to grow in the presence of a rapeseed protein hydrolysate generated by PTN 3.0 Special® enzyme and containing only 24% of peptides under 1 kDa in size. On the other hand, serum-free medium supplementation with a rapeseed protein hydrolysate obtained with Orientase 90N® enzyme had a strong growth promoting effect, leading to a 60% increase in maximal cell density without affecting cell metabolism. This significant positive effect could be explained by the higher degree of hydrolysis of this digest, with 74% of peptides under 1 kDa in size.

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. This phenomenon appeared to be mainly due to the desialylation of mEPO as a result of a sialidase activity released upon cell lysis. These results demonstrate that though a higher EPO titer was obtained in serum supplemented conditions, sialylation of EPO was severely affected by the presence of serum in the culture medium.