Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast
Artikel i vetenskaplig tidskrift, 2015

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 a-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.

droplet microfluidics

protein secretion

systems biology

yeast cell factories

random mutagenesis

Författare

Mingtao Huang

Chalmers, Biologi och bioteknik, Systembiologi

Y. Bai

Kungliga Tekniska Högskolan (KTH)

East China University of Science and Technology

S. L. Sjostrom

Kungliga Tekniska Högskolan (KTH)

B. M. Hallstrom

Kungliga Tekniska Högskolan (KTH)

Zihe Liu

Chalmers, Biologi och bioteknik, Systembiologi

Dina Petranovic Nielsen

Chalmers, Biologi och bioteknik, Systembiologi

M. Uhlen

Kungliga Tekniska Högskolan (KTH)

Danmarks Tekniske Universitet (DTU)

H. N. Joensson

Kungliga Tekniska Högskolan (KTH)

H. A. Svahn

Kungliga Tekniska Högskolan (KTH)

Jens B Nielsen

Chalmers, Biologi och bioteknik, Systembiologi

Proceedings of the National Academy of Sciences of the United States of America

0027-8424 (ISSN) 1091-6490 (eISSN)

Vol. 112 34 E4689-E4696

Industrial Systems Biology of Yeast and A. oryzae (INSYSBIO)

Europeiska kommissionen (FP7), 2010-01-01 -- 2014-12-31.

Styrkeområden

Livsvetenskaper och teknik

Ämneskategorier

Bioinformatik och systembiologi

DOI

10.1073/pnas.1506460112

PubMed

26261321

Mer information

Senast uppdaterat

2018-02-28