Y.

Morales1
1.
2.
3.

M. Tortajada2

J. Pic3

J. Veh1

F. Llaneras1

Institut dInformatica i Aplicacions, Universitat de Girona, Spain

BIPOLIS S.L., C/ Catedrtico Agustn Escardino Benlloch, 9, 46980, Paterna, Valencia, Spain.
Instituto Universitario de Automtica e Informtica Industrial, Universidad Politcnica de Valencia, Spain

This work describes an experimental validation of a small-size FBA

model of Pichia pastoris, a methylotrophic yeast widely recognized as a
suitable expression system in industrial application.
Flux Balance Analysis (FBA) is a methodology extensively used in the
last years to analyse and predict the behaviour of living cells.
Our main objective is testing how suitable is the hypothesis of
maximum growth to predict the behaviour of P. Pastoris in different
conditions.

Constraint Based Model

Standard constraint-based
model (Palsson, 2006;
Llaneras, 2008)

Nv 0
M OC

Dv 0

Experimental data set

81 experimental datasets from P. pastoris chemostats were collected in literature.
Each dataset is defined by a set of experimental measurements of extracellular fluxes. Biomass growth, substrate
uptakes (glucose, glycerol, methanol and mixtures), O2, CO2 and by products (EthOH, Citrate, Pyruvate)
The consistency of each dataset has been evaluated using a C-balance and a possibilistic consistency analysis
(Llaneras,2011; Tortajada,2012)

Validation 1: Prediction of growth and yields on single substrates

Predicted yields are in reasonably good agreement with the
experimental data and also with a genome-scale model
(Caspeta,2012).
The best carbon source was glucose, then glycerol and
methanol. This agrees with literature (Inan & Meagner, 2001).

Methanol
Yx/s YS/O2 YS/CO2

Glucose
Yx/s YS/O2 YS/CO2

Glycerol
Yx/s YS/O2 YS/CO2

Cmmol

mmol

mmol

Cmmol

mmol

mmol

Cmmol

Mmol

mmol

mmol-1

mmol-1

mmol-1

mmol-1

mmol-1

mmol-1

mmol-1

mmol-1

mmol-1

(this work)

0.66 0.83 0.34

3.97

1.97

2.03

2.26

1.21

0.74

FBA
(Caspeta)

0.49 1.43 0.49

3.91

1.53

1.96

2.23

0.95

0.68

Exp.
(average)

0.41 1.05 0.63

0.08 0.06 0.15

3.49 1.42 1.82 1.99 1.42 1.00

0.64 0.54 0.39 0.18 0.27 0.20

FBA

Validation 2: FBA predictions in real scenarios

Predictions of growth are remarkably accurate growing on glycerol and glucose.
The model also predicts the by-products production rate, The O2 uptake rate and the CO2 production rate.
In methanol discrepancies are larger. These could be explained:
The model may have errors or limitations in methanol pathways.
The model do not consider the resources devoted for recombinant protein production.
FBA hypothesis could be less suitable in these case, Methanol is a less frequent substrate.
A

A Glycerol
B Glucose
C Methanol

Model contains 47
metabolites and 48 metabolic
reactions (Tortajada, 2010).

3748 N matrix
17 reversible reaction

Complete FBA Model

Constraints at given context.
Z represents cells objective, assuming that
cells have evolved to be optimal as result of
evolutionary pressure (Orth, 2010;
Varma, 1994).

Max Z=dv
In this work the
objective function is
maximize growth

QUESTION

Validation 3: Behavior under oxygen limitation

FBA model predicts different aerobic/anaerobic behavior. Only In anaerobic byproducts are formed. This is in agreement with literature (Baumann,2008;
Charoenrat,2005)

Validation 4: Predicting substrate preferences and anaerobic behavior

FBA assumption by itself modeling the complex machinery is sufficient to predict substrate preference, and use
of fermentative or respiratory pathways. (Garcia, 2013)

Is the hypothesis of maximum

growth capable and enough for
predict the cells behaviour ?

CONCLUSION
Our predictions based on growth maximisation assumption are
reasonably accurate: even if our model is small and imposes a strong
assumption regarding how cells regulate their fluxes, model provides
good predictions regarding growth and substrate preference.
Predictions are remarkable accurate when P. pastoris grows in glycerol
and glucose; however, in methanol are presented larger deviations.
In summary , small FBA models can be useful in data scarcity conditions
with strains and organisms not particularly well-known, which are
frequent in industry

ACKNOWLEDGEMENTS
This work has been partially supported by the Spanish Government (cicyt: DPI2011-28112-C04-01) and Biopolis S.L. (R.C.055/12).
Yeimy Morales is grateful for the BR Grants of the University of Girona (BR2012/26).
The authors are grateful to the company Biopolis S.L. for his support to this research.

However our model fail in predicting

co-consumptions, which do not
occur in actual cultures.