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Engineering high hydrophobin expression yeast strains: toward switchable biosurfactants

Presenters Name: 
Lauren Harkins
Co Presenters Name: 
Primary Research Mentor: 
Bryan Berger
Secondary Research Mentor: 
Nathanael Sallada
Time: 
2:00 - 3:15
Time of Presentation: 
2019 - 2:00pm to 3:15pm
Session: 
4
Location: 
Newcomb Hall Ballroom
Presentation Type: 
Poster
Presentations Academic Category: 
Engineering
Grant Program Recipient: 
Double Hoo Research Grant
Abstract: 

Hydrophobins are amphiphilic, non-immunogenic proteins characterized by four conserved disulfide bonds and high surface activity, making them valuable in diverse applications. The disulfide bonds are important for surface activity of class II hydrophobins. Our goal is to identify which disulfides are critical in class II hydrophobin HFBI, and replace them with pH-dependent salt bridges to create a pH-responsive biosurfactant. However, due to low expression levels of HFBI variants in Pichia pastoris, we investigated the effect of increased HFBI gene copy number in combination with chaperone protein overexpression (Kar2p, Pdi1p, and Ero1p) to improve yield. To compare HFBI expression after varying copy number with and without chaperone overexpression, integrated intensities from a dot blot were analyzed to determine fold changes in expression. Increasing the copy number alone did not increase HFBI concentrations, but expression synergistically increased with increased copy number and chaperone gene overexpression. The greatest increase was seen in 3-copy-HFBI strain with ERO1 overexpression, yielding a 30 ± 4.0-fold increase compared to 1-copy-HFBI without chaperone overexpression. All combinations of one, two, or three disulfide bond deletions in HFBI were successfully made using site-directed mutagenesis and verified by Sanger sequencing. Going forward, high expression yeast strains for each of the disulfide deletion constructs will be created using the synergistic approach of increasing gene copy number and overexpressing chaperone proteins. Following purification, HFBI variants will be analyzed for changes in surface activity using a variety of biophysical techniques to identify critical disulfide bonds.