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Genome-scale metabolic reconstruction and in silico analysis of methylotrophic yeast Pichia pastoris for strain improvement.

Chung BK, Selvarasu S, Andrea C, Ryu J, Lee H, Ahn J, Lee H, Lee DY - Microb. Cell Fact. (2010)

Bottom Line: Pichia pastoris has been recognized as an effective host for recombinant protein production.Subsequent in silico analysis further explored the effect of various carbon sources on cell growth, revealing sorbitol as a promising candidate for culturing recombinant P. pastoris strains producing heterologous proteins.This computational approach, combined with synthetic biology techniques, potentially forms a basis for rational analysis and design of P. pastoris metabolic network to enhance humanized glycoprotein production.

View Article: PubMed Central - HTML - PubMed

Affiliation: NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 28 Medical Drive, #05-01, 117456, Singapore. cheld@nus.edu.sg.

ABSTRACT

Background: Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.

Results: A fully compartmentalized metabolic model of P. pastoris (iPP668), composed of 1,361 reactions and 1,177 metabolites, was reconstructed based on its genome annotation and biochemical information. The constraints-based flux analysis was then used to predict achievable growth rate which is consistent with the cellular phenotype of P. pastoris observed during chemostat experiments. Subsequent in silico analysis further explored the effect of various carbon sources on cell growth, revealing sorbitol as a promising candidate for culturing recombinant P. pastoris strains producing heterologous proteins. Interestingly, methanol consumption yields a high regeneration rate of reducing equivalents which is substantial for the synthesis of valuable pharmaceutical precursors. Hence, as a case study, we examined the applicability of P. pastoris system to whole-cell biotransformation and also identified relevant metabolic engineering targets that have been experimentally verified.

Conclusion: The genome-scale metabolic model characterizes the cellular physiology of P. pastoris, thus allowing us to gain valuable insights into the metabolism of methylotrophic yeast and devise possible strategies for strain improvement through in silico simulations. This computational approach, combined with synthetic biology techniques, potentially forms a basis for rational analysis and design of P. pastoris metabolic network to enhance humanized glycoprotein production.

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Reconstruction of Pichia pastoris. A recently published genome of P. pastoris [35] and various online databases, including KEGG [71], MetaCyc [70], PubMed http://www.ncbi.nlm.nih.gov/pubmed/, BRENDA [68] and ExPASy ENZYME database [69], were used to reconstruct and manually curate the iPP668 metabolic model.
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Figure 1: Reconstruction of Pichia pastoris. A recently published genome of P. pastoris [35] and various online databases, including KEGG [71], MetaCyc [70], PubMed http://www.ncbi.nlm.nih.gov/pubmed/, BRENDA [68] and ExPASy ENZYME database [69], were used to reconstruct and manually curate the iPP668 metabolic model.

Mentions: Following the procedure illustrated in Figure 1, a genome-scale network of P. pastoris metabolism was reconstructed (see Methods). During the reconstruction process, significant efforts were required in the gap-filling process. Essential metabolic reactions are added sequentially to the draft model, through repeated cell growth simulations using constraints-based flux analysis, to ensure in silico viability. For example, a metabolic gap exists in the initial draft of the methonine biosynthetic pathway as the gene coding for the enzyme catalyzing transamination of 2-keto-4-methylbutyrate to form methionine [31] was not found and had to be added manually. Similarly, in order to allow mitochondrial localization of acyl-carrier protein for fatty acid synthesis, a mitochondrial transport reaction was introduced to the model, thus improving metabolic network connectivity.


Genome-scale metabolic reconstruction and in silico analysis of methylotrophic yeast Pichia pastoris for strain improvement.

Chung BK, Selvarasu S, Andrea C, Ryu J, Lee H, Ahn J, Lee H, Lee DY - Microb. Cell Fact. (2010)

Reconstruction of Pichia pastoris. A recently published genome of P. pastoris [35] and various online databases, including KEGG [71], MetaCyc [70], PubMed http://www.ncbi.nlm.nih.gov/pubmed/, BRENDA [68] and ExPASy ENZYME database [69], were used to reconstruct and manually curate the iPP668 metabolic model.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908565&req=5

Figure 1: Reconstruction of Pichia pastoris. A recently published genome of P. pastoris [35] and various online databases, including KEGG [71], MetaCyc [70], PubMed http://www.ncbi.nlm.nih.gov/pubmed/, BRENDA [68] and ExPASy ENZYME database [69], were used to reconstruct and manually curate the iPP668 metabolic model.
Mentions: Following the procedure illustrated in Figure 1, a genome-scale network of P. pastoris metabolism was reconstructed (see Methods). During the reconstruction process, significant efforts were required in the gap-filling process. Essential metabolic reactions are added sequentially to the draft model, through repeated cell growth simulations using constraints-based flux analysis, to ensure in silico viability. For example, a metabolic gap exists in the initial draft of the methonine biosynthetic pathway as the gene coding for the enzyme catalyzing transamination of 2-keto-4-methylbutyrate to form methionine [31] was not found and had to be added manually. Similarly, in order to allow mitochondrial localization of acyl-carrier protein for fatty acid synthesis, a mitochondrial transport reaction was introduced to the model, thus improving metabolic network connectivity.

Bottom Line: Pichia pastoris has been recognized as an effective host for recombinant protein production.Subsequent in silico analysis further explored the effect of various carbon sources on cell growth, revealing sorbitol as a promising candidate for culturing recombinant P. pastoris strains producing heterologous proteins.This computational approach, combined with synthetic biology techniques, potentially forms a basis for rational analysis and design of P. pastoris metabolic network to enhance humanized glycoprotein production.

View Article: PubMed Central - HTML - PubMed

Affiliation: NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 28 Medical Drive, #05-01, 117456, Singapore. cheld@nus.edu.sg.

ABSTRACT

Background: Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.

Results: A fully compartmentalized metabolic model of P. pastoris (iPP668), composed of 1,361 reactions and 1,177 metabolites, was reconstructed based on its genome annotation and biochemical information. The constraints-based flux analysis was then used to predict achievable growth rate which is consistent with the cellular phenotype of P. pastoris observed during chemostat experiments. Subsequent in silico analysis further explored the effect of various carbon sources on cell growth, revealing sorbitol as a promising candidate for culturing recombinant P. pastoris strains producing heterologous proteins. Interestingly, methanol consumption yields a high regeneration rate of reducing equivalents which is substantial for the synthesis of valuable pharmaceutical precursors. Hence, as a case study, we examined the applicability of P. pastoris system to whole-cell biotransformation and also identified relevant metabolic engineering targets that have been experimentally verified.

Conclusion: The genome-scale metabolic model characterizes the cellular physiology of P. pastoris, thus allowing us to gain valuable insights into the metabolism of methylotrophic yeast and devise possible strategies for strain improvement through in silico simulations. This computational approach, combined with synthetic biology techniques, potentially forms a basis for rational analysis and design of P. pastoris metabolic network to enhance humanized glycoprotein production.

Show MeSH
Related in: MedlinePlus