Alvaro Díaz-Barrera

Endoplasmic Reticulum-Associated rht-PA Processing in CHO Cells: Influence of Mild Hypothermia and Specific Growth Rates in Batch and Chemostat Cultures.

Background Chinese hamster ovary (CHO) cells are the main host for producing recombinant proteins with human therapeutic applications mainly because of their capability to perform proper folding and glycosylation processes. In addition, mild hypothermia is one of the main strategies for maximising the productivity of these systems. However, little information is available on the effect of culture temperature on the folding and degradation processes of recombinant proteins that takes place in the endoplasmic reticulum. Methods In order to evaluate the effect of the mild hypothermia on processing/endoplasmatic reticulum-associated degradation (ERAD) processes, batch cultures of CHO cells producing recombinant human tissue plasminogen activator (rht-PA) were carried out at two temperatures (37°C and 33°C) and treated with specific inhibitors of glycosylation and ERAD I (Ubiquitin/Proteasome system) or ERAD II (Autophagosoma/Lisosomal system) pathways. The effect of mild hypothermia was analysed separately from its indirect effect on specific cell growth rate. To do this, chemostat cultures were carried out at the same incubation conditions as the batch cultures, controlling cell growth at high (0.017 h-1) and low (0.012 h-1) dilution rates. For a better understanding of the investigated phenomenon, cell behaviour was also analysed using principal component analysis (PCA). Results and Conclusion Results suggest that rht-PA is susceptible to degradation by both ERAD pathways studied, revealing that processing and/or ERAD processes are sensitive to temperature cultivation in batch culture. Moreover, by isolating the effect of culture temperature from the effect of cell growth rate verifyed by using chemostat cultures, we have found that processing and/or ERAD processes are more sensitive to reduction in specific growth rate than low temperature, and that temperature reduction may have a positive effect on protein processing. Interestingly, PCA indicated that the integrated performance displayed by CHO cells is modulated predominantly by specific growth rate, indicating that the culture temperature has a lower weighted effect within the range of conditions evaluated in this work.

Evaluation of Gene Expression and Alginate Production in Response to Oxygen Transfer in Continuous Culture of Azotobacter vinelandii

Alginates are polysaccharides used as food additives and encapsulation agents in biotechnology, and their functional properties depend on its molecular weight. In this study, different steady-states in continuous cultures of A. vinelandii were established to determine the effect of the dilution rate (D) and the agitation rate on alginate production and expression of genes involved in alginate polymerization and depolymerization. Both, the agitation and dilution rates, determined the partitioning of the carbon utilization from sucrose into alginate and CO2 under oxygen-limiting conditions. A low D (0.07 h−1) and 500 rpm resulted in the highest carbon utilization into alginate (25%). Quantitative real-time polymerase chain reaction was used to determine the transcription level of six genes involved in alginate polymerization and depolymerization. In chemostat cultures at 0.07 h−1, the gene expression was affected by changes in the agitation rate. By increasing the agitation rate from 400 to 600 rpm, the algE7 gene expression decreased tenfold, whereas alyA1, algL and alyA2 gene expression increased between 1.5 and 2.8 times under similar conditions evaluated. Chemostat at 0.07 h−1 showed a highest alginate molecular weight (580 kDa) at 500 rpm whereas similar molecular weights (480 kDa) were obtained at 400 and 600 rpm. The highest molecular weight was not explained by changes in the expression of alg8 and alg44 (genes involved in alginate polymerization). Nonetheless, a different expression pattern observed for lyases could explain the highest alginate molecular weight obtained. Overall, the results suggest that the control of alginate molecular weight in A. vinelandii cells growing in continuous mode is determined by a balance between the gene expression of intracellular and extracellular lyases in response to oxygen availability. These findings better our understanding of the biosynthesis of bacterial alginate and help us progress toward obtain tailor-made alginates.

Alginate Biosynthesis in Azotobacter vinelandii: Overview of Molecular Mechanisms in Connection with the Oxygen Availability

The Gram-negative bacterium Azotobacter vinelandii can synthetize the biopolymer alginate that has material properties appropriate for plenty of applications in industry as well as in medicine. In order to settle the foundation for improving alginate production without compromising its quality, a better understanding of the polymer biosynthesis and the mechanism of regulation during fermentation processes is necessary. This knowledge is crucial for the development of novel production strategies. Here, we highlight the key aspects of alginate biosynthesis that can lead to producing an alginate with specific material properties with particular focus on the role of oxygen availability linked with the molecular mechanisms involved in the alginate production.

Bacterial alginate production: an overview of its biosynthesis and potential industrial production

Alginate is a linear polysaccharide that can be used for different applications in the food and pharmaceutical industries. These polysaccharides have a chemical structure composed of subunits of (1–4)-β-d-mannuronic acid (M) and its C-5 epimer α-l-guluronic acid (G). The monomer composition and molecular weight of alginates are known to have effects on their properties. Currently, these polysaccharides are commercially extracted from seaweed but can also be produced by Azotobacter vinelandii and Pseudomonas spp. as an extracellular polymer. One strategy to produce alginates with different molecular weights and with reproducible physicochemical characteristics is through the manipulation of the culture conditions during fermentation. This mini-review provides a comparative analysis of the metabolic pathways and molecular mechanisms involved in alginate polymerization from A. vinelandii and Pseudomonas spp. Different fermentation strategies used to produce alginates at a bioreactor laboratory scale are described.

Molecular mass of Poly-3-hydroxybutyrate (P3HB) produced by Azotobacter vinelandii is influenced by the polymer content in the inoculum

Poly-3-hydroxybutyrate (P3HB) is a biopolymer produced by Azotobacter vinelandii. The physicochemical properties and applications of P3HB are strongly influenced by its weight-average molecular mass (Mw), and in A. vinelandii, it could be influenced by the culture conditions. The aim of this study was to evaluate the effect of the P3HB content of the inoculum on the Mw of the polymer produced by A. vinelandii OP in bioreactor cultures. A. vinelandii cells containing 20, 50 and 70% of P3HB were used as inoculum. The P3HB content in the inoculum affected the volumetric P3HB productivity (qP3HB) and the Mw of P3HB. Those cultures inoculated with cells containing 20% of P3HB, achieved the highest qP3HB (0.17±0.018gP3HBL-1h-1); whereas a P3HB content of 70% was reflected as a low qP3HB (0.021±0.002gP3HBL-1h-1). On the other hand, using an inoculum with 70% of polymer content, the Mw of the biopolymer remained stable at values close to 3200kDa; whereas, when an inoculum with 20% of P3HB was used, the Mw decreased drastically during early stages of cultivation. These results show that manipulating the P3HB content of the inoculum is possible to produce biopolymers with a suitable Mw.