title: Improved production of a heterologous amylase in Saccharomyces cerevisiae by inverse metabolic engineering.
abstract: The increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeast Saccharomyces cerevisiae is widely used as a cell factory for recombinant protein production. However, many recombinant proteins are produced at only 1% (or less) of the theoretical capacity due to the complexity of the secretory pathway, which has not been fully exploited. In this study, we applied the concept of inverse metabolic engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be associated with higher amylase secretion were identified through whole-genome sequencing. Several single-point mutations, including an S196I point mutation in the VTA1 gene coding for a protein involved in vacuolar sorting, were evaluated by introducing these to the starting strain. By applying this modification alone, the amylase secretion could be improved by 35%. As a complement to the identification of genomic variants, transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis.
authors: Novo Nordisk Foundation Center for Biosustainability, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors: Zihe
authors: Z
authors: Liu
authors: Novo Nordisk Foundation Center for Biosustainability, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors: Lifang
authors: L
authors: Liu
authors: Novo Nordisk Foundation Center for Biosustainability, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors: Tobias
authors: T
authors: Österlund
authors: Novo Nordisk Foundation Center for Biosustainability, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors: Jin
authors: J
authors: Hou
authors: Novo Nordisk Foundation Center for Biosustainability, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors: Mingtao
authors: M
authors: Huang
authors: The School of Biotechnology, AlbaNova, University Center, Royal Institute of Technology, Stockholm, Sweden.
authors: Linn
authors: L
authors: Fagerberg
authors: Novo Nordisk Foundation Center for Biosustainability, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors: Dina
authors: D
authors: Petranovic
authors: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark The School of Biotechnology, AlbaNova, University Center, Royal Institute of Technology, Stockholm, Sweden.
authors: Mathias
authors: M
authors: Uhlén
authors: Novo Nordisk Foundation Center for Biosustainability, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark nielsenj@chalmers.se.
authors: Jens
authors: J
authors: Nielsen
mesh_terms: D000681:Amylases / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D012641:Selection, Genetic; D014466:Ultraviolet Rays
chemical_list: D011994:Recombinant Proteins; D000681:Amylases