Abstract
BackgroundPichia 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.ResultsA 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.ConclusionThe 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.
Highlights
Pichia pastoris has been recognized as an effective host for recombinant protein production
We have shown through the analysis of carbon source utilization that glycerol and sorbitol are the best candidates for achieving high growth yields in P. pastoris cell culture (Figure 5)
In this study, we have reconstructed a genome-scale metabolic model of the methylotrophic yeast P. pastoris, which has been developed for recombinant protein production as well as whole-cell biotransformation
Summary
Pichia pastoris has been recognized as an effective host for recombinant protein production. Over 70% of the therapeutic proteins under preclinical and clinical development are glycosylated and there has been an increasing need for highly efficient glycoprotein expression systems. Mammalian systems such as Chinese hamster ovary (CHO) cells have been most widely used since they have been extensively characterized and are capable of humanlike glycosylation. A number of studies have been carried out for understanding and enhancing physiological properties of P. pastoris These studies have elucidated various favorable characteristics of the organism, which makes it more attractive for large-scale production of recombinant humanized glycoprotein [6,7,8,9,10]. The emerging paradigm of systems biotechnology can play an important role in identifying key targets for strain improvement [15]
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