[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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 abstract][Hide abstract] ABSTRACT: To develop a functional phosphate-regulated promoter in Pichia pastoris, a phosphate-responsive gene, PHO89, which encodes a putative sodium (Na(+))-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter (P(PHO89)) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. P(PHO89) was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1alpha and the glyceraldehyde-3-phosphate dehydrogenase promoter, P(PHO89) exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple P(PHO89)-based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of P(PHO89) for controlled production of recombinant proteins in P. pastoris.
[Show abstract][Hide abstract] ABSTRACT: The gene encoding translation elongation factor 1-alpha from the yeast Pichia pastoris was cloned. The gene revealed an open reading frame of 1,380 bp with the potential to encode a polypeptide of 459 amino acids with a calculated mass of 50.1 kDa. The potential of the promoter (P (TEF1)) in P. pastoris was investigated with comparison to the glyceraldehyde-3-phosphate dehydrogenase promoter (P (GAP)) by using a bacterial lipase gene as a reporter gene. P (TEF1) demonstrated a tighter growth-associated expression mode, improved functioning in the presence of high glucose concentrations, and promoter activities that yielded recombinant protein at levels similar to or in one case greater than P (GAP). The sequence of the gene was deposited in GenBank under accession no. EF014948.