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Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

Mingtao Huang (Institutionen för biologi och bioteknik, Systembiologi) ; Y. P. Bai ; S. L. Sjostrom ; B. M. Hallstrom ; Zihe Liu (Institutionen för biologi och bioteknik, Systembiologi) ; Dina Petranovic (Institutionen för biologi och bioteknik, Systembiologi) ; M. Uhlen ; H. N. Joensson ; H. Andersson-Svahn ; Jens B. Nielsen (Institutionen för biologi och bioteknik, Systembiologi)
Proceedings of the National Academy of Sciences of the United States of America (0027-8424). Vol. 112 (2015), 34, p. E4689-E4696.
[Artikel, refereegranskad vetenskaplig]

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.

Nyckelord: protein secretion, yeast cell factories, droplet microfluidics, random mutagenesis, systems biology



Denna post skapades 2015-09-15. Senast ändrad 2016-06-30.
CPL Pubid: 222485

 

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Institutioner (Chalmers)

Institutionen för biologi och bioteknik, Systembiologi

Ämnesområden

Bioinformatik och systembiologi

Chalmers infrastruktur

 


Projekt

Denna publikation är ett resultat av följande projekt:


Industrial Systems Biology of Yeast and A. oryzae (INSYSBIO) (EC/FP7/247013)