Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast
2015 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 34, E4689-E4696 p.Article in journal, Letter (Other academic) Published
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 highthroughput 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 wholegenome 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.
Place, publisher, year, edition, pages
NATL ACAD SCIENCES , 2015. Vol. 112, no 34, E4689-E4696 p.
protein secretion;yeast cell factories, droplet microfluidics, random mutagenesis, systems biology
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:kth:diva-159295DOI: 10.1073/pnas.1506460112ISI: 000360005600010PubMedID: 26261321ScopusID: 2-s2.0-84940521020OAI: oai:DiVA.org:kth-159295DiVA: diva2:784171
QC 201604292015-01-282015-01-282016-06-22Bibliographically approved