Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast.
There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.
Department of Biology and Biological Engineering, Chalmers University of Technology, SE41296 Gothenburg, Sweden; Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE41296 Gothenburg, Sweden;
Mingtao
M
Huang
Novo Nordisk Foundation Center for Biosustainability, KTH Royal Institute of Technology, SE10691 Stockholm, Sweden; Science for Life Laboratory, KTH Royal Institute of Technology, SE17165 Solna, Sweden; Division of Proteomics and Nanobiotechnology, KTH Royal Institute of Technology, SE10691 Stockholm, Sweden;
Yunpeng
Y
Bai
Novo Nordisk Foundation Center for Biosustainability, KTH Royal Institute of Technology, SE10691 Stockholm, Sweden; Science for Life Laboratory, KTH Royal Institute of Technology, SE17165 Solna, Sweden; Division of Proteomics and Nanobiotechnology, KTH Royal Institute of Technology, SE10691 Stockholm, Sweden;
Staffan L
SL
Sjostrom
Novo Nordisk Foundation Center for Biosustainability, KTH Royal Institute of Technology, SE10691 Stockholm, Sweden; Science for Life Laboratory, KTH Royal Institute of Technology, SE17165 Solna, Sweden; Division of Proteomics and Nanobiotechnology, KTH Royal Institute of Technology, SE10691 Stockholm, Sweden;
Björn M
BM
Hallström
Department of Biology and Biological Engineering, Chalmers University of Technology, SE41296 Gothenburg, Sweden; Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE41296 Gothenburg, Sweden;
Zihe
Z
Liu
Department of Biology and Biological Engineering, Chalmers University of Technology, SE41296 Gothenburg, Sweden; Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE41296 Gothenburg, Sweden;
Dina
D
Petranovic
Novo Nordisk Foundation Center for Biosustainability, KTH Royal Institute of Technology, SE10691 Stockholm, Sweden; Science for Life Laboratory, KTH Royal Institute of Technology, SE17165 Solna, Sweden; Division of Proteomics and Nanobiotechnology, KTH Royal Institute of Technology, SE10691 Stockholm, Sweden; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK2970 Hørsholm, Denmark.
Mathias
M
Uhlén
Novo Nordisk Foundation Center for Biosustainability, KTH Royal Institute of Technology, SE10691 Stockholm, Sweden; Science for Life Laboratory, KTH Royal Institute of Technology, SE17165 Solna, Sweden; Division of Proteomics and Nanobiotechnology, KTH Royal Institute of Technology, SE10691 Stockholm, Sweden;
Haakan N
HN
Joensson
Novo Nordisk Foundation Center for Biosustainability, KTH Royal Institute of Technology, SE10691 Stockholm, Sweden; Science for Life Laboratory, KTH Royal Institute of Technology, SE17165 Solna, Sweden; Division of Proteomics and Nanobiotechnology, KTH Royal Institute of Technology, SE10691 Stockholm, Sweden;
Helene
H
Andersson-Svahn
Department of Biology and Biological Engineering, Chalmers University of Technology, SE41296 Gothenburg, Sweden; Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE41296 Gothenburg, Sweden; Science for Life Laboratory, KTH Royal Institute of Technology, SE17165 Solna, Sweden; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK2970 Hørsholm, Denmark nielsenj@chalmers.se.
Jens
J
Nielsen
D016681:Genome, Fungal; D044085:Microfluidics; D009154:Mutation; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*
D029701:Saccharomyces cerevisiae Proteins
droplet microfluidics; protein secretion; random mutagenesis; systems biology; yeast cell factories