Flux Response of Glycolysis and Storage Metabolism during Rapid Feast/Famine Conditions in Penicillium Chrysogenum Using Dynamic 13C Labeling

The physiology, growth and product formation of a cellular system are the results of a complex interaction between the extracellular environment and the cellular metabolic and regulatory mechanisms [1]. Thus, the production capacity of an organism depends strongly on environmental conditions that could be a reason for unexpected scale-up behaviour. The scale-up of substrate limited cultivation processes results in a series of differences in environmental conditions that have their origin in transport limitations and non-ideal mixing. Especially relevant are oxygen limitation [2], increase in carbon dioxide concentrations and substrate starvation in certain areas of the large-scale reactor [1, 3, 4]. Cells circulating through the broth experience alternating environments with different substrate availability [5] and the cellular environment becomes highly dynamic (at the time scale of the mixing time), reaching from substrate excess to substrate starvation, henceforth called the feast/famine regime. These dynamic conditions can lead to reduced biomass yield and reduced product formation.

For Penicillium chrysogenum, intermittent feeding in 360 s cycles with 36 s feeding with fresh medium and 324 s of no feed were applied to mimic large-scale bioreactor conditions. This regime leads to a 50% decrease in Penicillin G production [5]. Concentration measurements of intracellular metabolites over the feast/famine cycles have shown drastic intracellular dynamics in several metabolite levels, including nucleotides [5]. The concentration of the penicillin G pathway precursors was lower than continuous cultures, but could not explain the 50% decrease in productivity. Also, enzyme activities were comparable in both conditions. De Jonge et al [5] therefore concluded that the dynamics and the kinetics of the reactions have a major impact on the penicillin production flux. Additionally, other pathways could consume more energy, reducing the amount of ATP available for penicillin G production. One hypothesis was an increased synthesis and degradation of storage compounds to buffer the extracellular dynamics in substrate supply. A cycle of synthesis and degradation results in net energy consumption (futile cycle).

Intracellular fluxes that form a cycle cannot be estimated from extracellular measurements and intracellular concentration measurements alone, but additional labeling measurements are required. Here we apply a recently developed metabolic dynamic approach using hybrid systems theory to identify dynamic flux profiles during the feast/famine cycle [6] from dynamic concentration and ¹³C labeling measurements. Results

The intermittent feeding led to fluctuations in the extracellular glucose concentration between 400 mM down to 6.5 mM at the end of the cycle. The intracellular metabolite concentrations responded strongly and showed up to 100 fold changes.

When comparing steady state and the feast/famine feeding regime, significant differences in the amount of storage metabolites were observed. The average trehalose concentration increased from 59.1 μmol/g_DCW to an average of 178 μmol/g_DCW under dynamic conditions. In contrast to the increase in trehalose, the mannitol concentration decreased from 374 μmol/g_DCW to 83 μmol/g_DCW when dynamic conditions were applied.

The changes in concentration during a single cycle (360s) were small compared to the measurement accuracy. Mass isotopomer measurements are relative measurements and the accuracy increases at high metabolite concentration as the signals become stronger. Trehalose reaches an enrichment of 2.5% (per carbon) within 360s and 4.5% after three cycles.

The trehalose enrichment increased from 1.1% to about 2.4% within 210 s. An increase was only seen after about 100 s, which is also in agreement with the observation that the precursor Tre6P enrichment rose after 40-70 s. In contrast to this late enrichment, the first intermediate of the trehalose/glycogen branch G1P had a labeling pattern very similar to G6P. It can be assumed, that the phosphoglucomutase (PGM) connecting G6P and G1P was operating close to equilibrium. The concentration of UDP-Glc increased after the addition of substrate, and also ¹³C enrichment was immediately measured. The enrichment of UDP-Glc and Tre6P reached a maximum at about 210 s and began to slowly decrease again. At this time point the extracellular substrate concentration already dropped to 15 μmol/L.

The average concentration of mannitol was lower (83 μmol/g_DCW). But, the pool reached a labeling enrichment of 7.0% after 360s, which corresponds to an approximated production flux equivalent to 6.3% of the added glucose. After 200 s a decrease in concentration was observed and the labeling enrichment remained constant, indicating that there was no further production of mannitol. The pathway intermediate M6P seems to be at fast equilibrium with F6P – concentration profile and labeling enrichment were very similar.

Based on the concentration and labeling measurements, a dynamic flux estimation was performed using piece-wise affine flux functions in time. For the upper glycolytic flux, it was observed that the rate of glucose uptake rapidly increased to 0.41 μmol/g_DCW/s at 18 s and reached a maximum of 0.48 μmol/g_DCW/s at 100 s and then decreased in steps to its starting value 0.02 μmol/g_DCW/s. The following reaction (PGI) increased rapidly and reached a value of 0.40 μmol/g/s at 18 s. For the next interval, the flux decreased, in contrast to the uptake rate – a trend also observed for the following glycolytic reaction steps.

A significant amount of the carbon was entering storage metabolism and to a smaller extent the non-oxidative Pentose-Phosphate pathway. Thus, the differences in the first steps of glycolysis clearly indicate that the connected fluxes, PPP and storage metabolism, have to be taken into account.

Looking at the average flux, about 18% of the carbon entering the cell was recycled in the trehalose node. Additionally, 11% of the incoming carbon was recycled via glycogen. Mannitol cycling reached 9.1% of the inflow. Thus, the synthesis of these pools added up to 38% of the carbon uptake. The average inflow into the oxidative pentose-phosphate pathway was estimated at only 1.7% of the uptake rate. The maximum flux through the oxidative pentose phosphate pathway was observed after 100 s (10 nmol/g_DCW/s), which is lower compared to steady-state conditions [7], suggesting that NADPH production might originate from another reaction.

References:

[1] Lapin, A., Klann, M., Reuss, M., Multi-scale spatio-temporal modeling: lifelines of microorganisms in bioreactors and tracking molecules in cells. Adv Biochem Eng Biotechnol 2010, 121, 23-43.

[2] Oosterhuis, N. M., Kossen, N. W., Dissolved oxygen concentration profiles in a production-scale bioreactor. Biotechnol Bioeng 1984, 26, 546-550.

[3] Lara, A. R., Galindo, E., Ramirez, O. T., Palomares, L. A., Living with heterogeneities in bioreactors: understanding the effects of environmental gradients on cells. Mol Biotechnol 2006, 34, 355-381.

[4] Larsson, G., Törnkvist, M., Wernersson, E. S., Trägårdh, C., et al., Substrate gradients in bioreactors: origin and consequences. Bioproc Biosyst Eng 1996, 14, 281-289.

[5] de Jonge, L. P., Buijs, N. A. A., ten Pierick, A., Deshmukh, A., et al., Scale-down of penicillin production in Penicillium chrysogenum. Biotechnol J 2011, 6, 944-958.

[6] Abate, A., Hillen, R. C., Aljoscha Wahl, S., Piecewise affine approximations of fluxes and enzyme kinetics from in vivo13C labeling experiments. Int J Robust Nonlin 2012, 22, 1120-1139.

[7] Zhao, Z., Ten Pierick, A., de Jonge, L., Heijnen, J. J., Wahl, S. A., Substrate cycles in Penicillium chrysogenum quantified by isotopic non-stationary flux analysis. Microb Cell Fact 2012, 11, 140.