## 3 Results
### Effect of BHRF1 on Cell Metabolism: Flux Balance Analysis  
The metabolism of animal cells growing in high glucose media is characterized by high rates of glycolysis fluxes. The metabolism of glucose by aerobic microorganisms yields pyruvate, which is partially converted into acetyl-CoA, and then completely oxidized to CO\({}_{2}\) and H\({}_{2}\)O in the TCA cycle. However, in mammalian cell lines, pyruvate is primarily converted into lactate [24]. The generation and accumulation of large amounts of lactate, since glucose cannot be completely oxidized, is the major consequence of such high glycolytic fluxes, leading to unbalanced metabolism. This metabolic behavior has been observed in hybridoma cell lines, regardless of the level of dissolved oxygen in the culture. Lactate generation from pyruvate seems to be necessary to fulfill the NADH regeneration requirements in the cytoplasm [42] due to the limiting transport rates of NADH into the mitochondria, where it can also be regenerated, even though lactate formation is a much less energetically efficient process than its oxidation in the Krebs cycle [34]. Flux Balance Analysis (FBA) is a useful tool to obtain information about the redistribution of internal metabolic fluxes. It also helped us to generate some hypotheses about the effects of BHRF1 on metabolism. Figures 4 and 5 show the distribution of the metabolic fluxes using the genome-derived model for _Mus musculus_. Metabolic flux balances were conducted using the data obtained from the bioreactor cultures. FBA led to deregulated metabolism in both cell lines, characterized by high glycolytic fluxes and consequent lactate generation and secretion. However, glycolytic fluxes were reduced by 54% (bottom part of glycolysis as a reference) due to the reduction in glucose consumption (51%) in KB26.5-BHRF1. Lactate generation flux dropped more than 60%. Despite the lower fluxes in glycolysis presented by the KB26.5-BHRF1 strain compared to KB26.5, the rate of carbon influx from cytoplasm to mitochondria through pyruvate was 14% higher (320 versus 274 nmol/(mg-h)). In other words, when calculating the fluxes in mass units (mg metabolite/(mg DCW-h)), only 13% of the total glucose consumed had entered the mitochondria of KB26.5 cells, whereas this increased to 32% in the engineered  
Figure 2: 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**). Time profile of cell density (\(\blacktriangle\)), cell viability (\(\lozenge\)); glucose ( \(\circ\) ) and lactate ( \(\blacksquare\) ) and product (bars) concentration of batch cultures. Duplicate cultures are shown for each cell line. KB26.5-BHRF1. When performing similar calculations to estimate the lactate generation ratio regardless of glucose consumption, lactate formation represented about 87% and 69% of the consumed glucose in KB26.5 and KB26.5-BHRF1 respectively. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Parameter (nmol/mg: h\({}^{-1}\))** & **KB26.5** & **KB26.5-BHRF1** \\ \hline Alanine & \(132.493\pm 12.954\) & \(48.305\pm 4.158\) \\ Arginine & \(-14.726\pm 2.812\) & \(-15.517\pm 8.825\) \\ Asparagine & \(5.420\pm 1.302\) & \(3.058\pm 0.681\) \\ Aspartic acid & \(4.304\pm 6.017\) & \(0.487\pm 0.801\) \\ Biomass & \(0.036\pm 0.006\) & \(0.048\pm 0.007\) \\ Cysteine & \(-5.024\pm 2.111\) & \(-4.825\pm 4.079\) \\ Glucose & \(-933.447\pm 134.567\) & \(-542.206\pm 177.457\) \\ Glutamic acid & \(7.858\pm 0.265\) & \(0.841\pm 0.956\) \\ Glutamine & \(-258.541\pm 76.627\) & \(-179.660\pm 122.421\) \\ Glycine & \(-11.597\pm 6.102\) & \(-12.094\pm 6.784\) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Specific consumption rates of glucose, amino acids and oxygen, and production rates of lactate and ammonia for KB26.5 and KB26.5-BHRF1 cell lines. Cultures were run in 2 L bioreactors. The specific rates are part of the 38 external measured fluxes used as modeling constraints, and are expressed in nmol/(mg:h) units.