Limits...
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.

Show MeSH

Related in: MedlinePlus

Chemostat culture simulation results. Data points for each chemostat experimental data point were generated by constraints-based flux analysis implemented with glucose uptake rate constrained at the respective experimentally determined values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2908565&req=5

Figure 4: Chemostat culture simulation results. Data points for each chemostat experimental data point were generated by constraints-based flux analysis implemented with glucose uptake rate constrained at the respective experimentally determined values.

Mentions: In the case of glucose minimal medium, we observed that the predicted cell growth, oxygen uptake and carbon dioxide evolution rates are highly consistent with the data obtained from our chemostat experiments (Figure 4). Discrepancies between experimental observations and model predictions are within 20% which can be considered acceptable. The constantly higher growth and gaseous exchange rate predictions can be due to possible errors in biomass composition data obtained from various sources (see Methods and Additional file 2). It should be noted that in order to mimic physiological states, we specified conditional parameters (see Methods). NGAM requirement (2.3 mmol ATP/gDCW-hr) was estimated from our chemostat data whereas we defined P/O ratio (1.48 mol ATP/mol O) based on previous studies [39,40].


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)

Chemostat culture simulation results. Data points for each chemostat experimental data point were generated by constraints-based flux analysis implemented with glucose uptake rate constrained at the respective experimentally determined values.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Chemostat culture simulation results. Data points for each chemostat experimental data point were generated by constraints-based flux analysis implemented with glucose uptake rate constrained at the respective experimentally determined values.
Mentions: In the case of glucose minimal medium, we observed that the predicted cell growth, oxygen uptake and carbon dioxide evolution rates are highly consistent with the data obtained from our chemostat experiments (Figure 4). Discrepancies between experimental observations and model predictions are within 20% which can be considered acceptable. The constantly higher growth and gaseous exchange rate predictions can be due to possible errors in biomass composition data obtained from various sources (see Methods and Additional file 2). It should be noted that in order to mimic physiological states, we specified conditional parameters (see Methods). NGAM requirement (2.3 mmol ATP/gDCW-hr) was estimated from our chemostat data whereas we defined P/O ratio (1.48 mol ATP/mol O) based on previous studies [39,40].

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