Genome-scale metabolic model of methylotrophic yeast Pichia pastoris and its use for in silico analysis of heterologous protein production.

The methylotrophic yeast Pichia pastoris has gained much attention during the last decade as a platform for producing heterologous recombinant proteins of pharmaceutical importance, due to its ability to reproduce post-translational modification similar to higher eukaryotes. With the recent release of the full genome sequence for P. pastoris, in-depth study of its functions has become feasible. 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. The protein-producing model versions of PpaMBEL1254 were then analyzed to examine the impact on oxygen limitation on protein production.

[1]  Monica L. Mo,et al.  Global reconstruction of the human metabolic network based on genomic and bibliomic data , 2007, Proceedings of the National Academy of Sciences.

[2]  Brigitte Gasser,et al.  Versatile modeling and optimization of fed batch processes for the production of secreted heterologous proteins with Pichia pastoris , 2006, Microbial cell factories.

[3]  S. Lee,et al.  Metabolic engineering of Escherichia coli for the production of l-valine based on transcriptome analysis and in silico gene knockout simulation , 2007, Proceedings of the National Academy of Sciences.

[4]  S. Lee,et al.  Strategies for systems‐level metabolic engineering , 2008, Biotechnology journal.

[5]  T. Gerngross,et al.  Advances in the production of human therapeutic proteins in yeasts and filamentous fungi , 2004, Nature Biotechnology.

[6]  Diethard Mattanovich,et al.  Hypoxic fed-batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins. , 2008, Biotechnology and bioengineering.

[7]  L. Quek,et al.  AraGEM, a Genome-Scale Reconstruction of the Primary Metabolic Network in Arabidopsis1[W] , 2009, Plant Physiology.

[8]  S. Lee,et al.  Genome-scale metabolic network analysis and drug targeting of multi-drug resistant pathogen Acinetobacter baumannii AYE. , 2010, Molecular bioSystems.

[9]  B. Palsson,et al.  An expanded genome-scale model of Escherichia coli K-12 (iJR904 GSM/GPR) , 2003, Genome Biology.

[10]  Ian T. Paulsen,et al.  TransportDB: a comprehensive database resource for cytoplasmic membrane transport systems and outer membrane channels , 2006, Nucleic Acids Res..

[11]  Michael Sauer,et al.  Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus. , 2009, FEMS yeast research.

[12]  L. Harvey,et al.  Heterologous protein production using the Pichia pastoris expression system , 2005, Yeast.

[13]  T. Gerngross,et al.  Glycosylation engineering in yeast: the advent of fully humanized yeast. , 2007, Current opinion in biotechnology.

[14]  Brigitte Gasser,et al.  Yeast systems biotechnology for the production of heterologous proteins. , 2009, FEMS yeast research.

[15]  David P. Kreil,et al.  The response to unfolded protein is involved in osmotolerance of Pichia pastoris , 2010, BMC Genomics.

[16]  L. Nielsen,et al.  Modeling Hybridoma Cell Metabolism Using a Generic Genome‐Scale Metabolic Model of Mus musculus , 2008, Biotechnology progress.

[17]  J. Nielsen,et al.  Analysis of Aspergillus nidulans metabolism at the genome-scale , 2008, BMC Genomics.

[18]  J. Cregg,et al.  Recombinant protein expression in Pichia pastoris , 2000, Molecular biotechnology.

[19]  Teresa Mitchell,et al.  Production of monoclonal antibodies by glycoengineered Pichia pastoris. , 2009, Journal of biotechnology.

[20]  M. Kanehisa,et al.  Using the KEGG Database Resource , 2005, Current protocols in bioinformatics.

[21]  J. Nielsen,et al.  Mathematical modelling of metabolism. , 2000, Current opinion in biotechnology.

[22]  Yves Van de Peer,et al.  Genome sequence of the recombinant protein production host Pichia pastoris , 2009, Nature Biotechnology.

[23]  Michael Sauer,et al.  The effect of temperature on the proteome of recombinant Pichia pastoris. , 2009, Journal of proteome research.

[24]  Y. Jang,et al.  Metabolic engineering of Clostridium acetobutylicum M 5 for highly selective butanol production , 2009 .

[25]  Brigitte Gasser,et al.  Antibody production with yeasts and filamentous fungi: on the road to large scale? , 2007, Biotechnology Letters.

[26]  O. Demin,et al.  The Edinburgh human metabolic network reconstruction and its functional analysis , 2007, Molecular systems biology.

[27]  Bernhard O. Palsson,et al.  Connecting Extracellular Metabolomic Measurements to Intracellular Flux States in Yeast , 2022 .

[28]  J. Nielsen,et al.  Genome-scale analysis of Streptomyces coelicolor A3(2) metabolism. , 2005, Genome research.

[29]  Diethard Mattanovich,et al.  Macromolecular and elemental composition analysis and extracellular metabolite balances of Pichia pastoris growing at different oxygen levels , 2009, Microbial cell factories.

[30]  Markus J. Herrgård,et al.  Reconstruction and validation of Saccharomyces cerevisiae iND750, a fully compartmentalized genome-scale metabolic model. , 2004, Genome research.

[31]  Michael Sauer,et al.  Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris , 2009, Microbial cell factories.

[32]  M. Sauer,et al.  Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris , 2008, Microbial cell factories.

[33]  P. Rouzé,et al.  Open access to sequence: Browsing the Pichia pastoris genome , 2009, Microbial cell factories.

[34]  B. Palsson,et al.  Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110 , 1994, Applied and environmental microbiology.

[35]  B. Palsson,et al.  Large-scale evaluation of in silico gene deletions in Saccharomyces cerevisiae. , 2003, Omics : a journal of integrative biology.

[36]  Roland Contreras,et al.  Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology , 2008, Nature Protocols.

[37]  Jack Hoopes,et al.  Humanization of Yeast to Produce Complex Terminally Sialylated Glycoproteins , 2006, Science.

[38]  Shoshana J. Wodak,et al.  CYGD: the Comprehensive Yeast Genome Database , 2004, Nucleic Acids Res..