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The Insect Cell-Baculovirus Expression Vector System (IC-BEVS) is broadly used for the production of recombinant proteins and vaccine manufacture, yet the host physiological aspects that contribute to productivity are to be disclosed. This work provides the first quantitative analysis of the metabolic fluxes of High Five cells. This analysis was conducted in comparison with Sf9 cells, another major host for biologicals production via BEVS. Moreover, herein is presented, for the first time, quantitative data of the relative contribution of sugars and amino acids catabolism to the activity of the TCA cycle in Sf9 and High Five cells. High Five cells metabolic activity was markedly influenced by the amino acids concentration in culture medium, which determine the rates of amino acid catabolism, carbon overflow and by-product formation. This characteristic of High Five cells was reflected in the activities of anaplerotic metabolism and the TCA cycle, which may not work as a true cycle as a function of medium composition. This was not the case for Sf9 cells, in which the glucose carbon incorporation in the TCA cycle was significantly higher and lactate production minor. Following infection, the decrease in by-product accumulation rates was accompanied by an increase in net ATP synthesis in Sf9 and High Five cells, although through distinct mechanisms cell-line dependent. The impact of baculovirus infection on cellular metabolic status highlights the capacity of this virus to re-direct the cellular fluxome toward ATP production to support replication and progeny generation. These results pave the way to deepen our knowledge on the relationship between a host cell and the virus, contributing to disclosing the metabolic determinants that contribute to productivity. Biotechnol. Bioeng. 2017;114: 674-684. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Baculovirus infection boosts the host biosynthetic activity towards the production of viral components and the recombinant protein of interest, hyper-productive phenotypes being the result of a successful adaptation of the cellular network to that scenario. Spodoptera frugiperda derived Sf9 and Trichoplusia ni derived High Five cell lines have a major track record for the production of recombinant proteins, with High Five cells presenting higher productivities. A metabolic profiling of the two insect cell lines was pursued to underpin specific cellular traits behind productive phenotypes. Multivariate analysis identified cell-line dependent metabolic signatures linked to productivity. Pathway analysis highlighted cellular pathways of paramount importance in supporting infection and protein production. Moreover, better producer phenotypes proved to be correlated with the capacity of cells to shift their metabolism in favor of energy-generating pathways to fuel biosynthesis, a scenario observed in the High Five cell line. Metabolomic profiling allowed us to identify metabolic pathways involved in infection and recombinant protein production, which can be selected as targets for further improvement of the system., (© 2013 Wiley Periodicals, Inc.)

The cell density effect (i.e., the drop in the specific productivity in the baculovirus-insect cells expression system when cells are infected at high cell densities) has been extensively described in the literature. In this article, a model for the central metabolism of serum-free suspension cultures of Spodoptera frugiperda Sf9 cells is proposed and used to investigate the metabolic basis for this phenomenon. The main metabolic pathways (glycolysis, pentose phosphate pathway, tricarboxylic acids cycle, glutaminolysis, and amino acids metabolism), cellular growth and energetics were considered. The analysis of the stoichiometric model allowed further understanding of the interplay of the consumption of carbon and nitrogen sources in insect cells. Moreover, metabolic flux analysis revealed that Sf9 cells undergo a progressive inhibition of central metabolism when grown to high cell densities, for which the incorporation of amino acids carbon backbones into the TCA cycle (mainly glutamine) and the down-regulation of glycolysis are partially responsible. Following infection by baculovirus and cellular division arrest, central energy metabolism depended on the infection strategy chosen (cell concentration at the moment of infection and multiplicity of infection), inhibition being observed at high cell densities. Interestingly, the energetic status of the culture correlated with the decrease in cellular production of baculovirus, meaning that there is room for process optimization through the application of metabolic engineering techniques., ((c) 2009 Wiley Periodicals, Inc.)

The aim was to understand how interaction of the central carbon and the secondary carnitine metabolisms is affected under salt stress and its effect on the production of L-carnitine by Escherichia coli. The biotransformation of crotonobetaine into L-carnitine by resting cells of E. coli O44 K74 was improved by salt stress, a yield of nearly twofold that for the control being obtained with 0.5 M NaCl. Crotonobetaine and the L-carnitine formed acted as an osmoprotectant during cell growth and biotransformation in the presence of NaCl. The enzyme activities involved in the biotransformation process (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA/acetate (pyruvate dehydrogenase, acetyl-CoA synthetase [ACS] and ATP/acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid cycle (isocitrate dehydrogenase [ICDH]) and glyoxylate shunt (isocitrate lyase [ICL]) were followed in batch with resting cells both in the presence and absence of NaCl and in perturbation experiments performed on growing cells in a high density cell recycle membrane reactor. Further, the levels of carnitine, crotonobetaine, gamma-butyrobetaine and ATP and the NADH/NAD(+) ratio were measured in order to know how the metabolic state was modified and coenzyme pools redistributed as a result of NaCl's effect on the energy content of the cell. The results provided the first experimental evidence of the important role played by salt stress during resting and growing cell biotransformation (0.5 M NaCl increased the L-carnitine production in nearly 85%), and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the main metabolic pathways and carbon flow operating during cell biotransformation was that controlled by the ICDH/ICL ratio, which decreased from 8.0 to 2.5, and the phosphotransferase/ACS ratio, which increased from 2.1 to 5.2, after a NaCl pulse fivefold the steady-state level. Resting E. coli cells were seen to be made up of heterogeneous populations consisting of several types of subpopulation (intact, depolarized, and permeabilized cells) differing in viability and metabolic activity as biotransformation run-time and the NaCl concentration increased. The results are discussed in relation with the general stress response of E. coli, which alters the NADH/NAD(+) ratio, ATP content, and central carbon enzyme activities.