ABSTRACT The microalgae Tetradesmus obliquus is able to maintain a high photosynthetic efficiency under nitrogen limitation and is considered a promising green microalgae for sustainable production of diverse compounds, including biofuels. Here, we report the first draft whole-genome shotgun sequencing of T. obliquus . The final assembly comprises 108,715,903 bp with over 1,368 scaffolds.

In this study, stearoyl-ACP desaturase (SAD), the enzyme that converts stearic acid into oleic acid, is silenced by artificial microRNA in the green microalga Chlamydomonas reinhardtii. Two different constructs, which target different positions on the mRNA of stearoyl-ACP desaturase, were tested. The mRNA levels for SAD were reduced after the silencing construct was induced. In one of the strains, the reduction in SAD mRNA resulted in a doubling of the stearic acid content in triacylglycerol molecules, which shows that stearic acid production in microalgae is possible.

In this study the influence of day length on circadian rhythms of N. oleoabundans was investigated. N. oleoabundans was grown in a photobioreactor continuously operated (turbidostat), under various day/night (D/N) cycles; 20D4N, 16D8N and 12D12N. The following variables showed to be regulated by the circadian clock: maximum growth rate, start of starch synthesis and DNA replication to 4 and 8 copies. Timing of these processes was not influenced by day length. The length of the photoperiod caused changes in biomass composition, especially due to variations in starch content. In longer days, more starch was accumulated. Starch was used for cell division probably also when cell division occurred in the light. Therefore, also the timing of cell division should be considered in the production of microalgae biomass. This timing mechanism can be explored to produce biomass with a desired concentration of protein, lipids, carbohydrates or pigments.

Microalgae are a promising future source for sustainable edible oils. To make microalgal oil a cost-effective alternative for common vegetable oils, increasing TAG productivity and TAG content are of high importance. Fulfilling these targets requires proper understanding of lipid metabolism in microalgae. Here, we provide an overview of our current knowledge on the biology of TAG accumulation as well as the latest developments and future directions for increasing oil production in microalgae, considering both metabolic engineering techniques and cultivation strategies.

ABSTRACT Influenza viruses unable to express NS1 protein (delNS1) replicate poorly and induce large amounts of interferon (IFN). They are therefore considered candidate viruses for live-attenuated influenza vaccines. Their attenuated replication is generally assumed to result from the inability to counter the antiviral host response, as delNS1 viruses replicate efficiently in Vero cells, which lack IFN expression. In this study, delNS1 virus was parallel passaged on IFN-competent MDCK cells, which resulted in two strains that were able to replicate to high virus titers in MDCK cells due to adaptive mutations especially in the M-gene segment but also in the NP and NS gene segments. Most notable were clustered U-to-C mutations in the M segment of both strains and clustered A-to-G mutations in the NS segment of one strain, which presumably resulted from host cell-mediated RNA editing. The M segment mutations in both strains changed the ratio of M1 to M2 expression, probably by affecting splicing efficiency. In one virus, 2 amino acid substitutions in M1 additionally enhanced virus replication, possibly through changes in the M1 distribution between the nucleus and the cytoplasm. Both adapted viruses induced levels of IFN equal to that of the original delNS1 virus. These results show that the increased replication of the adapted viruses is not primarily due to altered IFN induction but rather is related to changes in M1 expression or localization. The mutations identified in this paper may be used to enhance delNS1 virus replication for vaccine production.

Baby hamster kidney (BHK21) cells are used to produce vaccines against various viral veterinary diseases, including rabies and foot-and-mouth-disease. Although particular influenza virus strains replicate efficiently in BHK21 cells the general use of these cells for influenza vaccine production is prohibited by the poor replication of most strains, including model strain A/PR/8/34 [H1N1] (PR8). We now show that in contrast to PR8, the related strain A/WSN/33 [H1N1] (WSN) replicates efficiently in BHK21 cells. This difference is determined by the haemagglutinin (HA) protein since reciprocal reassortant viruses with swapped HAs behave similarly with respect to growth on BHK21 cells as the parental virus from which their HA gene is derived. The ability or inability of six other influenza virus strains to grow on BHK21 cells appears to be similarly dependent on the nature of the HA gene since reassortant PR8 viruses containing the HA of these strains grow to similar titres as the parental virus from which the HA gene was derived. However, the growth to low titres of a seventh influenza strain was not due to the nature of the HA gene since a reassortant PR8 virus containing this HA grew efficiently on BHK21 cells. Taken together, these results suggest that the HA gene often primarily determines influenza replication efficiency on BHK21 cells but that in some strains other genes are also involved. High virus titres could be obtained with reassortant PR8 strains that contained a chimeric HA consisting of the HA1 domain of PR8 and the HA2 domain of WSN. HA1 contains most antigenic sites and is therefore important for vaccine efficacy. This method of producing the HA1 domain as fusion to a heterologous HA2 domain could possibly also be used for the production of HA1 domains of other viruses to enable the use of BHK21 cells as a generic platform for veterinary influenza vaccine production.

Influenza A viruses lacking the gene encoding the non-structural NS1 protein (delNS1) have potential use as live attenuated vaccines. However, due to the lack of NS1, virus replication in cell culture is considerably reduced, prohibiting commercial vaccine production. We therefore established two stable MDCK cell lines that show inducible expression of the allele B NS1 protein. Upon induction, both cell lines expressed NS1 to about 1000-fold lower levels than influenza virus-infected cells. Nevertheless, expression of NS1 increased delNS1 virus titres to levels comparable to those obtained with an isogenic virus strain containing an intact NS1 gene. Recombinant NS1 expression increased the infectious virus titres 244 to 544-fold and inhibited virus induced apoptosis. However, NS1 expression resulted in only slightly, statistically not significant, reduced levels of interferon-β production. Thus, the low amount of recombinant NS1 is sufficient to restore delNS1 virus replication in MDCK cells, but it remains unclear whether this occurs in an interferon dependent manner. In contrast to previous findings, recombinant NS1 expression did not induce apoptosis, nor did it affect cell growth. These cell lines thus show potential to improve the yield of delNS1 virus for vaccine production.

Madin–Darby canine kidney (MDCK) cells are currently considered for influenza vaccine manufacturing. A drawback of these cells is their anchorage dependent growth, which greatly complicates process scale-up. In this paper a novel MDCK cell line (MDCK-SFS) is described that grows efficiently in suspension and retained high expression levels of both α-2,6 and α-2,3 sialic acid receptors, which bind preferably to human and avian influenza viruses, respectively. The production of avian influenza virus by BHK21, Vero and MDCK-SFS cell lines was compared. Although BHK21 cells consisted of two populations, one of which lacks the α-2,3 receptor, they supported the replication of two influenza strains to high titres. However, BHK21 cells are generally not applicable for influenza production since they supported the replication of six further strains poorly. MDCK-SFS cells yielded the highest infectious virus titres and virus genome equivalent concentration for five of the eight influenza strains analyzed and the highest hemagglutination activity for all eight virus strains. Taken together with their suitability for suspension growth this makes the MDCK-SFS cell line potentially useful for large scale influenza virus production.

A licensed pharmaceutical process is required to be executed within the validated ranges throughout the lifetime of product manufacturing. Changes to the process, especially for processes involving biological products, usually require the manufacturer to demonstrate that the safety and efficacy of the product remains unchanged by new or additional clinical testing. Recent changes in the regulations for pharmaceutical processing allow broader ranges of process settings to be submitted for regulatory approval, the so‐called process design space, which means that a manufacturer can optimize his process within the submitted ranges after the product has entered the market, which allows flexible processes. In this article, the applicability of this concept of the process design space is investigated for the cultivation process step for a vaccine against whooping cough disease. An experimental design (DoE) is applied to investigate the ranges of critical process parameters that still result in a product that meets specifications. The on‐line process data, including near infrared spectroscopy, are used to build a descriptive model of the processes used in the experimental design. Finally, the data of all processes are integrated in a multivariate batch monitoring model that represents the investigated process design space. This article demonstrates how the general principles of PAT and process design space can be applied for an undefined biological product such as a whole cell vaccine. The approach chosen for model development described here, allows on line monitoring and control of cultivation batches in order to assure in real time that a process is running within the process design space. Biotechnol. Bioeng. 2009; 104: 492–504 © 2009 Wiley Periodicals, Inc.

Metabolic flux analysis has become a standard tool for analyzing metabolism and optimizing bioprocesses. Metabolic flux analysis makes use of a metabolic reaction network in combination with extra-cellular measurements and mass balancing to calculate flux distributions in metabolism. It is a useful tool to analyze metabolism of cells and can be used to optimize the bioprocess in terms of medium design and metabolic engineering of the cells. In this chapter first the fundamental aspects of metabolic networks and the mathematical methods are described. Next a metabolic model for mammalian cells is discussed, Finally, applications of metabolic flux balancing are reviewed. Further extension of the metabolic network models, possibly towards genome-scale models, will further increase the value of these models

There is a need for novel protein sources. Insects are a possible interesting source of protein. They are nutritious in terms of protein (40-75 g/100g dry weight) and minerals. Insect protein is of high quality and has a high digestibility (77-98%) and concentration of essential amino acids (46-96% of the nutritional profile). Also insect cells may be a promising novel source of protein. Choice of cell line, growth conditions and use of the baculovirus expression system opens up possibilities to engineer the nutritional value of the biomass. The technological limits as well as consumer acceptance of insect cell based food remains to be investigated.

Glucose addition and subsequent run-out experiments were compared to simulations with a detailed glycolytic model of Lactococcus lactis. The model was constructed largely on bases of enzyme kinetic data taken from literature and not adjusted for the specific simulations shown here. Upon glucose depletion a rapid increase in PEP, inorganic phosphate and a gradual decrease in fructose 1,6-bisphosphate (FBP) were measured and predicted by simulation. The dynamic changes in these and other intermediate concentrations as measured in the experiments were well predicted by the kinetic model.

The scale-up of insect cell cultures and the production of baculovirus with these cultures is dependent on the inoculation density applied. The effect of applying a low inoculation on the specific growth rate and on the duration of the lag phase was tested. Three different cell lines, HzAm1, Ha2302, and Sf21 were tested in a total of five cell line/medium combinations. Growth in suspension culture was examined, and data obtained were fitted with the Gompertz equation. A significant decline in specific growth rate with decreasing inoculation density was observed in all cell line/medium combinations, except for HzAm1. No critical inoculation density, below which no growth would occur, was found. In suspension culture in shake flasks, an inoculation density of 5 x 10(4) cells/mL is achievable, without severely influencing the overall growth rate. A lower inoculation density in suspension culture results in less steps in the scale-up process and might be a tool in bypassing the viral passage effect.

The death rate of Vero cells grown on Cytodex-3 microcarrierswas studied as a function of the gas flow rate in a smallair-lift loop reactor. The death rate may be described byfirst-order death-rate kinetics. The first-order death-rateconstant as calculated from the decrease in viable cells, theincrease in dead cells and the increase in LDH activity islinear proportional to the gas flow rate, with a specifichypothetical killing volume in which all cells are killed ofabout 2.10-3m3 liquid per m3 of air bubbles.In addition, an experiment was conducted in the sameair-lift reactor with Vero cells grown inside porous Asahimicrocarriers. The specific hypothetical killing volumecalculated from this experiment has a value of 3.10-4m3 liquid per m3 of air bubbles, which shows thatthe porous microcarriers were at least in part able to protectthe cells against the detrimental hydrodynamic forcesgenerated by the bubbles.

In order to optimize the production of monoclonal antibodies several homogeneous culture systems are studied. Data are presented on cultivation of hybridoma cell line MN12 in a continuous culture system with cell retention. A computerized measurement and control system was used for monitoring and control of process parameters as pH, dissolved oxygen concentration (DO), stirrer speed and temperature. Various analytical methods were used to study parameters as cell growth, viability, metabolism, oxygen consumption, monoclonal antibody (Mab) production, integrity and carbohydrate moiety of the monoclonal antibodies. During cultivation a change from aerobic to a more anaerobic metabolism was observed which not only affected the levels of nutrients and metabolites but also the glycosylation pattern of the antibodies produced. The antibody production and the number of antibody producing cells remained unchanged.