Metabolic fluxes in the central carbon metabolism of Dinoroseobacter shibae and Phaeobacter gallaeciensis, two members of the marine Roseobacter clade.

BACKGROUND: In the present work the central carbon metabolism of Dinoroseobacter shibae and Phaeobacter gallaeciensis was studied at the level of metabolic fluxes. These two strains belong to the marine Roseobacter clade, a dominant bacterial group in various marine habitats, and represent surface-associated, biofilm-forming growth (P. gallaeciensis) and symbiotic growth with eukaryotic algae (D. shibae). Based on information from recently sequenced genomes, a rich repertoire of pathways has been identified in the carbon core metabolism of these organisms, but little is known about the actual contribution of the various reactions in vivo. RESULTS: Using 13C labelling techniques in specifically designed experiments, it could be shown that glucose-grown cells of D. shibae catabolise the carbon source exclusively via the Entner-Doudoroff pathway, whereas alternative routes of glycolysis and the pentose phosphate pathway are obviously utilised for anabolic purposes only. Enzyme assays confirmed this flux pattern and link the lack of glycolytic flux to the absence of phosphofructokinase activity. The previously suggested formation of phosphoenolpyruvate from pyruvate during mixotrophic CO2 assimilation was found to be inactive under the conditions studied. Moreover, it could be shown that pyruvate carboxylase is involved in CO2 assimilation and that the cyclic respiratory mode of the TCA cycle is utilised. Interestingly, the use of intracellular pathways was highly similar for P. gallaeciensis. CONCLUSION: The present study reveals the first insight into pathway utilisation within the Roseobacter group. Fluxes through major intracellular pathways of the central carbon metabolism, which are closely linked to the various important traits found for the Roseobacter clade, could be determined. The close similarity of fluxes between the two physiologically rather different species might provide the first indication of more general key properties among members of the Roseobacter clade which may explain their enormous success in the marine realm.

Identification of cell culture conditions to control N-glycosylation site-occupancy of recombinant glycoproteins expressed in CHO cells.

The effect of different cell culture conditions on N-glycosylation site-occupancy has been elucidated for two different recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells, recombinant human tissue plasminogen activator (t-PA) and a recombinant enzyme (glycoprotein 2-GP2). Both molecules contain a N-glycosylation site that is variably occupied. Different environmental factors that affect the site-occupancy (the degree of occupied sites) of these molecules were identified. Supplementing the culture medium with additional manganese or iron increased the fraction of fully occupied t-PA (type I t-PA) by approximately 2.5-4%. Decreasing the cultivation temperature from 37 to 33 degrees C or 31 degrees C gradually increased site-occupancy of t-PA up to 4%. The addition of a specific productivity enhancer, butyrate, further increased site-occupancy by an additional 1% under each cultivation temperature tested. In addition, the thyroid hormones triiodothyronine and thyroxine increased site-occupancy of t-PA compared to control conditions by about 2%. In contrast, the addition of relevant nucleoside precursor molecules involved in N-glycan biosynthesis (e.g., uridine, guanosine, mannose) either had no effect or slightly reduced site-occupancy. For the recombinant enzyme (GP2), it was discovered that culture pH and the timing of butyrate addition can be used to control N-glycan site-occupancy within a specific range. An increase in culture pH correlated with a decrease in site-occupancy. Similarly, delaying the timing for butyrate addition also decreased site-occupancy of this molecule. These results highlight the importance of understanding how cell culture conditions and media components can affect the product quality of recombinant glycoproteins expressed in mammalian cell cultures. Furthermore, the identification of relevant factors will enable one to control product quality attributes, specifically N-glycan site-occupancy, within a specific range when applied appropriately.

Effect of different carbon sources on central metabolic fluxes and the recombinant production of a hydrolase from Thermobifida fusca in Bacillus megaterium.

The recombinant Bacillus megaterium strain WH323 was employed for the inducible production and secretion of recombinant Thermobifida fusca hydrolase (TFH). Continuous cultivations were carried out in a chemostat using either glucose or pyruvate as sole carbon source. A remarkable increase of produced TFH was detected for the pyruvate-dependent cultivation compared to glucose-dependent growth. Estimation of intracellular carbon fluxes through the central metabolism for both growth conditions using (13)C-labelled substrates revealed noticeable changes of the fluxes through the tricarboxylic acid cycle, the pentose phosphate pathway and around the pyruvate node when protein production was induced. With pyruvate as sole carbon source the observed alterations of the fluxes yielded an increased production of ATP and NADPH both required for the anabolism. Additionally, the analysis of the corresponding secretome revealed significantly reduced amounts of extracellular proteases in the medium compared to glucose-grown cultivations. Thus, pyruvate-dependent chemostat cultivation was identified as a favourable condition for production and secretion of recombinant TFH using B. megaterium as production host.

Proteome analysis of a recombinant Bacillus megaterium strain during heterologous production of a glucosyltransferase.

A recombinant B. megaterium strain was used for the heterologous production of a glucosyltransferase (dextransucrase). To better understand the physiological and metabolic responses of the host cell to cultivation and induction conditions, proteomic analysis was carried out by combined use of two-dimensional gel electrophoresis and mass spectrometry (2-DE/MS) for protein separation and identification. 2-DE method was optimized for the separation of intracellular proteins. Since the genome of B. megaterium is not yet available, peptide sequencing using peptide fragment information obtained from nanoelectrospray ionization quadrupole-time-of-flight tandem mass spectrometry (ESI-QqTOF MS/MS) was applied for protein identification. 167 protein spots were identified as 149 individual proteins, including most enzymes involved in the central carbon metabolic pathways and many enzymes related to amino acid synthesis and protein synthesis. Based on the results a 2-DE reference map and a corresponding protein database were constructed for further proteomic approaches on B. megaterium. For the first time it became possible to perform comparative proteomic analysis on B. megaterium in a batch culture grown on glucose with xylose induction for dextrasucrase production. No significant differences were observed in the expression changes of enzymes of the glycolysis and TCA cycle, indicating that dextransucrase production, which amounted to only 2 % of the entire protein production, did not impose notable metabolic or energetic burdens on the central carbon metabolic pathway of the cells. However, a short-term up-regulation of aspartate aminotransferase, an enzyme closely related to dextransucrase production, in the induced culture demonstrated the feasibility to use 2-DE method for monitoring dextransucrase production. It was also observed that under the cultivation conditions used in this study B. megaterium tended to channel acetyl-CoA into pathways of polyhydroxybutyrate production. No expression increases were found with cytosolic chaperones such as GroEL and DnaK during dextransucrase production and secretion, whereas a strong up-regulation of the oligopeptide-binding protein OppA was observed in correlation with an increased secretion of dextransucrase into the culture medium.

Systems biology of recombinant protein production in Bacillus megaterium.

Over the last two decades the Gram-positive bacterium Bacillus megaterium was systematically developed to a useful alternative protein production host. Multiple vector systems for high yield intra- and extracellular protein production were constructed. Strong inducible promoters were combined with DNA sequences for optimised ribosome binding sites, various leader peptides for protein export and N- as well as C-terminal affinity tags for affinity chromatographic purification of the desired protein. High cell density cultivation and recombinant protein production were successfully tested. For further system biology based control and optimisation of the production process the genomes of two B. megaterium strains were completely elucidated, DNA arrays designed, proteome, fluxome and metabolome analyses performed and all data integrated using the bioinformatics platform MEGABAC. Now, solid theoretical and experimental bases for primary modeling attempts of the production process are available.

Comparative study on central metabolic fluxes of Bacillus megaterium strains in continuous culture using 13C labelled substrates.

Fluxes of central carbon metabolism [glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA cycle), biomass formation] were determined for several Bacillus megaterium strains (DSM319, WH320, WH323, MS941) in C- and N-limited chemostat cultures by (13)C labelling experiments. The labelling patterns of proteinogenic amino acids were analysed by GC/MS and therefrom flux ratios at important nodes within the metabolic network could be calculated. On the basis of a stoichiometric metabolic model flux distributions were estimated for the different B. megaterium strains used at various cultivation conditions. Generally all strains exhibited similar metabolic flux distributions, however, several significant changes were found in (1) the glucose flux entering the PPP via the oxidative branch, (2) the reversibilities within the PPP, (3) the relative fluxes of pyruvate and acetyl-CoA fed to the TCA cycle, (4) the fluxes around the pyruvate node involving a futile cycle.