title: Genome-scale metabolic reconstruction and in silico analysis of methylotrophic yeast Pichia pastoris for strain improvement.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 28 Medical Drive, #05-01, 117456, Singapore. cheld@nus.edu.sg.
authors.forename: Bevan Ks
authors.initials: BK
authors.lastname: Chung
authors.forename: Suresh
authors.initials: S
authors.lastname: Selvarasu
authors.forename: Camattari
authors.initials: C
authors.lastname: Andrea
authors.forename: Jimyoung
authors.initials: J
authors.lastname: Ryu
authors.forename: Hyeokweon
authors.initials: H
authors.lastname: Lee
authors.forename: Jungoh
authors.initials: J
authors.lastname: Ahn
authors.forename: Hongweon
authors.initials: H
authors.lastname: Lee
authors.forename: Dong-Yup
authors.initials: DY
authors.lastname: Lee
mesh_terms: D002072:Butylene Glycols / Q000378:metabolism; D016681:Genome, Fungal; D053858:Metabolic Networks and Pathways; D000432:Methanol / Q000378:metabolism; D010641:Phenotype; D010843:Pichia / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002072:Butylene Glycols; D011994:Recombinant Proteins; C026978:2,3-butylene glycol; D000432:Methanol