## 3 Results
### Effect of BHRF1 on Cell Metabolism: Flux Balance Analysis
Figure 3: Two L bioreactor batch cultures of the parental KB26.5 cell line (**A.1** and **A.2**) and the engineered KB26.5-BHRF1 (**B.1** and **B.2**). Napierian logarithm of viable cell density versus time (\(\bullet\)). Duplicate cultures are presented for each cell line. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Parameter (nmol/mg\(\cdot\)h\({}^{-1}\))** & **KB26.5** & **KB26.5-BHRF1** \\ \hline Histidine & \(-6.744\pm 3.693\) & \(-5.368\pm 3.796\) \\ Isoleucine & \(-24.989\pm 5.918\) & \(-18.098\pm 16.359\) \\ Lactate & \(1435.500\pm 348.745\) & \(654.556\pm 34.663\) \\ Leucine & \(-28.854\pm 6.149\) & \(-24.593\pm 17.043\) \\ Lysine & \(-22.759\pm 9.314\) & \(-21.579\pm 17.480\) \\ Methionine & \(-7.315\pm 0.948\) & \(-6.511\pm 4.841\) \\ NH4\({}^{+}\) & \(156.137\pm 11.064\) & \(85.090\pm 15.910\) \\ Phenylalanine & \(-9.685\pm 2.050\) & \(-10.009\pm 8.848\) \\ Proline & \(13.080\pm 4.084\) & \(1.993\pm 8.586\) \\ Oxygen & \(-701.500\pm 160.513\) & \(-674.500\pm 12.021\) \\ Serine & \(-15.150\pm 6.181\) & \(-13.913\pm 11.826\) \\ Threonine & \(-16.883\pm 3.216\) & \(-18.522\pm 18.500\) \\ Tryptophan & \(-2.585\pm 3.297\) & \(-1.078\pm 0.722\) \\ Tyrosine & \(-8.340\pm 1.832\) & \(-8.392\pm 7.974\) \\ Valine & \(-24.539\pm 4.938\) & \(-20.236\pm 18.013\) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont._  
Figure 4: Metabolic flux distribution of the parental KB26.5 hybridoma cell line. The arrows indicate the direction of the reaction; the arrow thickness is proportional to the flux rate. Different coloring was used for each pathway, and the model was structured in cytoplasmic and mitochondrial reactions (mitochondrion is limited by a discontinued line). The values in the boxes correspond to the flux rate in nmol metabolite/(mg DCW\(\cdot\)h). In this case, FBA shows that the rate of transport of reduction power from cytoplasm to mitochondria through the malate-aspartate shuttle seems to be a bit higher in KB26.5-BHRF1 than in KB26.5, with a 19% increase, i.e., a rate of 228 versus 192 nmol/(mg:h) in the aspartate-glutamate mitochondrial symptom transport. However, in both cases, the high glycolytic fluxes caused the cells to generate lactate, as the malate-aspartate shuttle seemed to be insufficient to couple with the cytoplasmic NADH regeneration. In addition, a slight increase in the pathways related with the formation of a biomass was observed in KB26.5-BHRF1 due to the higher growth rate; as Pentose Phosphate Pathway to generate nucleotides or citrate export from the mitochondria to generate lipids. Due to the high growth rate of KB26.5-BHRF1, an increase in the TCA fluxes was to be expected, producing more energy and, therefore, increased biomass. However, in comparing the TCA cycles for both cell lines, no significant increase was observed, which was in accordance with the similar observed oxygen consumption rate (Table 1). What is clear from the FBA is that there was a collapse between glycolysis and the TCA pathways in both cases, as the high glucose uptake rate collapses the transport of NADH to  
Figure 5: Metabolic flux distribution of the engineered hybridoma cell line KB26.5-BHRF1. The arrows indicate the direction of the reaction; the arrow thickness is proportional to the flux rate. Different coloring was used for each pathway, and the model was structured in cytoplasmic and mitochondrial reactions (mitochondrion is limited by a discontinued line). The values in the boxes correspond to the flux rate in nmol metabolite/mg DCW-h.  
mitochondria, and NADH have to be regenerated in cytoplasm using pyruvate by means of lactate dehydrogenase. The reduction of the glycolytic pathway in KB26.5-BHRF1 involved a reduction of ATP production in the cytoplasm, as well as a reduction of NADH formation. Consequently, the need to oxidize the NADH formed in the cytoplasm also decreased, and, together with the slightly higher rates related to Malate-Aspartate Shuttle, yielded to a reduction in the formation of lactate.