Pathway analysis of Pichia pastoris to elucidate methanol metabolism and its regulation for production of recombinant proteins

Abstract

This research rationally analyzes metabolic pathways of Pichia pastoris to study the metabolic flux responses of this yeast under methanol metabolism. A metabolic model of P. pastoris was constructed and analyzed by elementary mode analysis (EMA). EMA was used to comprehensively identify the cell's metabolic flux profiles and its underlying regulation mechanisms for the production of recombinant proteins from methanol. Change in phenotypes and flux profiles during methanol adaptation with varying feed mixture of glycerol and methanol was examined. EMA identified increasing and decreasing fluxes during the glycerol-methanol metabolic shift, which well agreed with experimental observations supporting the validity of the metabolic network model. Analysis of all the identified pathways also led to the determination of the metabolic capacities as well as the optimum metabolic pathways for recombinant protein synthesis during methanol induction. The network sensitivity analysis revealed that the production of proteins can be improved by manipulating the flux ratios at the pyruvate branch point. In addition, EMA suggested that protein synthesis is optimum under hypoxic culture conditions. The metabolic modeling and analysis presented in this study could potentially form a valuable knowledge base for future research on rational design and optimization of P. pastoris by determining target genes, pathways, and culture conditions for enhanced recombinant protein synthesis. The metabolic pathway analysis is also of considerable value for production of therapeutic proteins by P. pastoris in biopharmaceutical applications.

Transcriptome analysis of Corynebacterium glutamicum in the process of recombinant protein expression in bioreactors

“…As the most important energy source for metabolic pathways, ATP plays a vital role in cell growth and the production of the target products by affecting peptide folding, stress response, transportation, metabolic flux, and signal transduction [33–35]. A prior study had shown that approximately four ATP equivalents were required to form each polypeptide bond of GFP, and the amount of ATP needed from the intermediate metabolism to produce one unit of GFP was 333 [35].…”

“…A prior study had shown that approximately four ATP equivalents were required to form each polypeptide bond of GFP, and the amount of ATP needed from the intermediate metabolism to produce one unit of GFP was 333 [35]. Thus, the requirement of ATP is larger in C .…”

Metabolic Network Analysis of Microbial Methane Utilization for Biomass Formation and Upgrading to Bio-Fuels

“…Metabolic networks of the different organisms (Komagataella phaffii formerly Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Cupriavidus necator formerly Ralstonia eutropha and Clostridium autoethanogenum) were modified from published stoichiometric network analyses (Melzer et al, 2009;Lopar et al, 2013Lopar et al, , 2014Ternon et al, 2014;Unrean, 2014;Kracke et al, 2016;Koch et al, 2017; to fulfill the requirements of this elementary flux mode analysis. Specifically, the networks were integrated with methane assimilation pathways, compiled as follows:…”

Cofactor engineering improved CALB production in Pichia pastoris through heterologous expression of NADH oxidase and adenylate kinase

“…Several studies about metabolic engineering in P . pastoris portrayed the regulatory aspects of intracellular metabolic reactions and development in recombinant protein production [ 3 , 4 ]. In these, it was reported that the metabolic stress response against overexpression of recombinant protein redirects the intracellular carbon metabolism towards precursor formation to satisfy the increased energy demand.…”