Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway
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....
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crspringernat:10.1186/1475-2859-11-22 2023-05-15T14:09:39+02:00 Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway Krainer, Florian W Dietzsch, Christian Hajek, Tanja Herwig, Christoph Spadiut, Oliver Glieder, Anton 2012 http://dx.doi.org/10.1186/1475-2859-11-22 https://link.springer.com/content/pdf/10.1186/1475-2859-11-22.pdf en eng Springer Science and Business Media LLC Microbial Cell Factories volume 11, issue 1 ISSN 1475-2859 Applied Microbiology and Biotechnology Bioengineering Biotechnology journal-article 2012 crspringernat https://doi.org/10.1186/1475-2859-11-22 2022-01-04T15:32:21Z 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 utilization pathway significantly affected the specific growth rate, the methanol uptake and the specific productivity of recombinant P. pastoris Mut S strains. A recently developed methodology to determine strain specific parameters based on dynamic batch cultivations proved to be a valuable tool for fast strain characterization and thus early process development. Article in Journal/Newspaper Antarc* Antarctica Springer Nature (via Crossref) Microbial Cell Factories 11 1 |
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Applied Microbiology and Biotechnology Bioengineering Biotechnology |
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Applied Microbiology and Biotechnology Bioengineering Biotechnology Krainer, Florian W Dietzsch, Christian Hajek, Tanja Herwig, Christoph Spadiut, Oliver Glieder, Anton Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway |
topic_facet |
Applied Microbiology and Biotechnology Bioengineering Biotechnology |
description |
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 utilization pathway significantly affected the specific growth rate, the methanol uptake and the specific productivity of recombinant P. pastoris Mut S strains. A recently developed methodology to determine strain specific parameters based on dynamic batch cultivations proved to be a valuable tool for fast strain characterization and thus early process development. |
format |
Article in Journal/Newspaper |
author |
Krainer, Florian W Dietzsch, Christian Hajek, Tanja Herwig, Christoph Spadiut, Oliver Glieder, Anton |
author_facet |
Krainer, Florian W Dietzsch, Christian Hajek, Tanja Herwig, Christoph Spadiut, Oliver Glieder, Anton |
author_sort |
Krainer, Florian W |
title |
Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway |
title_short |
Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway |
title_full |
Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway |
title_fullStr |
Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway |
title_full_unstemmed |
Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway |
title_sort |
recombinant protein expression in pichia pastoris strains with an engineered methanol utilization pathway |
publisher |
Springer Science and Business Media LLC |
publishDate |
2012 |
url |
http://dx.doi.org/10.1186/1475-2859-11-22 https://link.springer.com/content/pdf/10.1186/1475-2859-11-22.pdf |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_source |
Microbial Cell Factories volume 11, issue 1 ISSN 1475-2859 |
op_doi |
https://doi.org/10.1186/1475-2859-11-22 |
container_title |
Microbial Cell Factories |
container_volume |
11 |
container_issue |
1 |
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1766281675564646400 |