Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production.
The production of recombinant proteins is frequently enhanced at the levels of transcription, codon usage, protein folding and secretion. Overproduction of heterologous proteins, however, also directly affects the primary metabolism of the producing cells. By incorporation of the production of a heterologous protein into a genome scale metabolic model of the yeast Pichia pastoris, the effects of overproduction were simulated and gene targets for deletion or overexpression for enhanced productivity were predicted. Overexpression targets were localized in the pentose phosphate pathway and the TCA cycle, while knockout targets were found in several branch points of glycolysis. Five out of 9 tested targets led to an enhanced production of cytosolic human superoxide dismutase (hSOD). Expression of bacterial β-glucuronidase could be enhanced as well by most of the same genetic modifications. Beneficial mutations were mainly related to reduction of the NADP/H pool and the deletion of fermentative pathways. Overexpression of the hSOD gene itself had a strong impact on intracellular fluxes, most of which changed in the same direction as predicted by the model. In vivo fluxes changed in the same direction as predicted to improve hSOD production. Genome scale metabolic modeling is shown to predict overexpression and deletion mutants which enhance recombinant protein production with high accuracy.
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna, Austria.
Justyna
J
Nocon
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna, Austria; Austrian Centre of Industrial Biotechnology, Vienna, Austria.
Matthias G
MG
Steiger
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna, Austria.
Martin
M
Pfeffer
Bioinformatics Research Center, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea.
Seung Bum
SB
Sohn
Bioinformatics Research Center, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea.
Tae Yong
TY
Kim
School of Bioengineering, University of Applied Sciences FH Campus Wien, Vienna, Austria; Austrian Centre of Industrial Biotechnology, Vienna, Austria.
Michael
M
Maurer
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna, Austria; Austrian Centre of Industrial Biotechnology, Vienna, Austria.
Hannes
H
Rußmayer
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna, Austria; Austrian Centre of Industrial Biotechnology, Vienna, Austria.
Stefan
S
Pflügl
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna, Austria; Austrian Centre of Industrial Biotechnology, Vienna, Austria.
Magnus
M
Ask
Austrian Centre of Industrial Biotechnology, Vienna, Austria; Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
Christina
C
Haberhauer-Troyer
Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
Karin
K
Ortmayr
Austrian Centre of Industrial Biotechnology, Vienna, Austria; Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
Stephan
S
Hann
Austrian Centre of Industrial Biotechnology, Vienna, Austria; Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
Gunda
G
Koellensperger
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna, Austria; Austrian Centre of Industrial Biotechnology, Vienna, Austria.
Brigitte
B
Gasser
Bioinformatics Research Center, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea; Metabolic Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 plus program), BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea.
Sang Yup
SY
Lee
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna, Austria; Austrian Centre of Industrial Biotechnology, Vienna, Austria. Electronic address: diethard.mattanovich@boku.ac.at.
Diethard
D
Mattanovich
D002952:Citric Acid Cycle / Q000235:genetics; D015870:Gene Expression; D006019:Glycolysis / Q000235:genetics; D006801:Humans; D060847:Metabolic Engineering; D055442:Metabolome / Q000235:genetics*; D008954:Models, Biological; D009243:NAD / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013482:Superoxide Dismutase / Q000096:biosynthesis / Q000235:genetics; D000072105:Superoxide Dismutase-1
D011994:Recombinant Proteins; C000606290:SOD1 protein, human; D009243:NAD; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1
Flux analysis; GEM; Metabolic modeling; NADPH; Recombinant protein; Yeast