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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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Related in: MedlinePlus

Comparison of metabolic model reconstructions. The P. pastoris metabolic model reconstruction iPP668 is being compared with S. cerevisiae [37] and E. coli [25] and it is found that there is no reaction that is shared by E. coli and P. pastoris. The number in each section of the Venn diagram indicates the number of reactions that are common or specific to the respective organism(s).
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Figure 2: Comparison of metabolic model reconstructions. The P. pastoris metabolic model reconstruction iPP668 is being compared with S. cerevisiae [37] and E. coli [25] and it is found that there is no reaction that is shared by E. coli and P. pastoris. The number in each section of the Venn diagram indicates the number of reactions that are common or specific to the respective organism(s).

Mentions: Unique and conserved features in P. pastoris metabolism were further elucidated by comparing iPP668 with two model organisms: S. cerevisiae (iMM904) [37] and E. coli (iAF1260) [25]. It should be noticed that we disregarded the sub-cellular compartmentalization of reactions for the comparative analysis of metabolic capability by eliminating inter-compartmental transport and metabolic reaction duplicates in different compartments. From this comparison, there are 292 reactions and 439 metabolites that are common to the three species (Figure 2). These reactions largely belong to the central carbon metabolism and amino acid biosynthetic pathways. The 415 reactions and 196 metabolites, shared only by P. pastoris and S. cerevisiae, are generally classified under the lipid and carbohydrate biosynthetic pathways while the 79 reactions and 46 metabolites unique to P. pastoris are mainly from the methanol utilization pathway (Figure 3) and certain parts of lipid metabolism which are yeast-specific. It is further observed that the lipid biosynthetic pathways of both yeasts are structurally identical, only differing in the composition of fatty acid chains.


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)

Comparison of metabolic model reconstructions. The P. pastoris metabolic model reconstruction iPP668 is being compared with S. cerevisiae [37] and E. coli [25] and it is found that there is no reaction that is shared by E. coli and P. pastoris. The number in each section of the Venn diagram indicates the number of reactions that are common or specific to the respective organism(s).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Comparison of metabolic model reconstructions. The P. pastoris metabolic model reconstruction iPP668 is being compared with S. cerevisiae [37] and E. coli [25] and it is found that there is no reaction that is shared by E. coli and P. pastoris. The number in each section of the Venn diagram indicates the number of reactions that are common or specific to the respective organism(s).
Mentions: Unique and conserved features in P. pastoris metabolism were further elucidated by comparing iPP668 with two model organisms: S. cerevisiae (iMM904) [37] and E. coli (iAF1260) [25]. It should be noticed that we disregarded the sub-cellular compartmentalization of reactions for the comparative analysis of metabolic capability by eliminating inter-compartmental transport and metabolic reaction duplicates in different compartments. From this comparison, there are 292 reactions and 439 metabolites that are common to the three species (Figure 2). These reactions largely belong to the central carbon metabolism and amino acid biosynthetic pathways. The 415 reactions and 196 metabolites, shared only by P. pastoris and S. cerevisiae, are generally classified under the lipid and carbohydrate biosynthetic pathways while the 79 reactions and 46 metabolites unique to P. pastoris are mainly from the methanol utilization pathway (Figure 3) and certain parts of lipid metabolism which are yeast-specific. It is further observed that the lipid biosynthetic pathways of both yeasts are structurally identical, only differing in the composition of fatty acid chains.

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