Genome-scale metabolic model of methylotrophic yeast

Pichia pastoris and its use for in silico analysis of heterologous

protein production
Seung Bum Sohn, Alexandra B. Graf , Tae Yong Kim, Brigitte Gasser, Michael Maurer, Pau Ferrer,
Diethard Mattanovich and Sang Yup Lee

Abstract
Here we present the first reconstruction of the genome-scale
metabolic model of the eukaryote P. pastoris type strain DSMZ 70382,
PpaMBEL1254, consisting of 1254 metabolic reactions and 1147
metabolites compartmentalized into eight different regions to
represent organelles. Additionally, equations describing the production
of two heterologous proteins, human serum albumin and human
superoxide dismutase, were incorporated to examine the impact on
oxygen limitation on protein production.
 Metología
Model reconstruction
The initial reconstruction of the metabolic model was
performed using the set of biochemical reactions
annotated from the genome based on the gene to
protein to reaction (GPR) relationship. The biomass
reaction was assembled using biomass components,
such as carbohydrates, amino acids and fatty acids,
and their respective contributions to the formation of
biomass are indicated.
Strains
Two strains of P. pastoris were used in this work,
the type strain (DSMZ 70382, also known as
CBS704) and strain GS115.
Culture conditions
The cultivation was carried out in a 5.0-L
bioreactor. The fermentation temperature was
controlled at 25°C, and the pH was kept at 5.0
with addition of 25% ammonium hydroxide. The
stirrer speed between 250 and 1200 rpm and the
air flow between 2.0 and 5.0 L/min. Chemostat
cultures were performed by adjusting the flow rate
of chemostat medium [29] and the harvest rate to
maintain a dilution rate D of 0.1/h after the end of
the batch phase.
Sampling and analysis
Three 10-mL aliquots of culture broth were
centrifuged and the supernatant was saved for
HPLC analysis.
 Metología
Constraints-based flux analysis Aditional reactions
Constraints for glucose and oxygen uptake rates were set The production of heterologous proteins was
to values determined experimentally including the
constraint for the metabolic reaction representing the implemented by the formulation of additional
maintenance energy requirement. In carbon utilization reactions that describe biosynthesis of the
studies, glucose was replaced with the carbon substrate
of interest. proteins of interest.
The glucose uptake rate was set to 2.88 mmol/g DCW/h.
The upper limit for oxygen uptake rate wasset to 4.14
mmol/g DCW/h, which was also determined from the
fermentation results.

where a, b, and c are coefficients which represent

the ratios at which DNA, mRNA and protein are
found in the cell, respectively, and x is the amount
of cellular energy required to drive this process.
 Resultados y conclusiones
Comparison of PpaMBEL1254 Carbon source utilization

Simulation of heterologous protein production

Resultados y conclusiones

As a result, fermentation strategies can be designed to optimize protein production

and growth rate by limiting the oxygen supply.With this metabolic model as a basis,
further studies in understanding and engineering P. pastoris to increase protein
production can be explored, such as gene knockout and gene overexpression
simulations.