Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

• Published in: Microbial Cell Factories
• Main Authors: Krainer Florian W, Dietzsch Christian, Hajek Tanja, Herwig Christoph, Spadiut Oliver, Glieder Anton
• Format: Article in Journal/Newspaper
• Language: English
• Published: BMC 2012
• Subjects: Pichia pastoris; methanol utilization pathway; Mut +; Mut S; recombinant protein expression; dihydroxyacetone synthase; formaldehyde dehydrogenase; transketolase; horseradish peroxidase; Candida antarctica lipase B; Microbiology; QR1-502; Antarc*; Antarctica
• Online Access: https://doi.org/10.1186/1475-2859-11-22; https://doaj.org/article/c76b9ab2a0804d0787e438d98b33c7c1

Abstract Βackground The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains. Results A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with Mut S phenotype was found to be superior over a strain with Mut + phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in Mut S strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant Mut S strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production. Conclusions Co-overexpressing enzymes of the methanol ...