Enhancing secreted production from yeast species with high throughput microdroplet screening methods

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dc.contributor.advisorAlper, Hal S.
dc.contributor.committeeMemberContreras, Lydia
dc.contributor.committeeMemberGeorgiou, George
dc.contributor.committeeMemberOtero, José Manuel
dc.creatorWagner, James Michael
dc.date.accessioned2023-05-09T00:20:10Z
dc.date.available2023-05-09T00:20:10Z
dc.date.created2019-05
dc.date.issued2023-05-01
dc.date.submittedMay 2019
dc.date.updated2023-05-09T00:23:33Z
dc.description.abstractMicrofluidics-based sorting of cells after encapsulation in water-in-oil or water/oil/water droplet emulsions has great potential to significantly accelerate the designbuild-test cycle for production strain engineering. This is especially true for non-conventional hosts and products that are secreted, as screening can be performed with minimal prior biological knowledge (relative to rational engineering) and entrapment of secreted product with the responsible producer cell can enable high throughput screening directly for extracellular product concentration. Microfluidic technology for droplet sorting has advanced and become more available in recent years, but gaps remain, including the limited number of droplet-compatible methods for transducing extracellular product titers into sortable fluorescence signals, and a lack of direct, matched comparisons to more established techniques such as fluorescence activated cell sorting (FACS). This research used yeast-based systems (Saccharomyces cerevisiae and Yarrowia lipolytica) as test beds for focusing on and addressing three areas that are related to these limitations. In the first focus area, we develop RNA aptamers in droplets (RAPID) screening, a method that expands the generality of high throughput microfluidic droplet screening. Using RNA aptamers, we transduce extracellular titer into fluorescence, enabling the screening of millions of yeast variants and ultimately enhancing secretion of a recombinant protein (streptavidin) from yeast. For the second focus area, we undertook a comparative analysis of traditional single cell fluorescence activated cell sorting (single cell FACS) and microdropletenabled FACS (droplet FACS) using water/oil/water (w/o/w) emulsions. To do so, we engineered and evolved the non-conventional yeast Y. lipolytica for high extracellular production of riboflavin (vitamin B2), an innately fluorescent product. In a parity-matched comparison ofsingle cell FACS and droplet FACS, both techniques led to significant increases in total riboflavin. However, single cell FACS favored intracellular accumulation, while droplet FACS favored extracellular product accumulation and yielded higher total riboflavin production. In the third focus area, a droplet-based bacterial-fungal consortium/co-culture approach was applied, wherein Escherichia coli biosensors were used to screen for improved extracellular polyketide production from Y. lipolytica. This droplet sorting strategy was used to screen genome-wide piggyBac transposon libraries, leading to the identification of several alleles for enhancing triacetic acid lactone (TAL) production.
dc.description.departmentChemical Engineering
dc.format.mimetypeapplication/pdf
dc.identifier.urihttps://hdl.handle.net/2152/118712
dc.identifier.urihttp://dx.doi.org/10.26153/tsw/45591
dc.language.isoen
dc.subjectYeast
dc.subjectBiosensor
dc.subjectDirected evolution
dc.subjectMicrofluidic droplet
dc.subjectRNA aptamer
dc.subjectRiboflavin
dc.subjectPolyketide
dc.titleEnhancing secreted production from yeast species with high throughput microdroplet screening methods
dc.typeThesis
dc.type.materialtext
thesis.degree.departmentChemical Engineering
thesis.degree.disciplineChemical Engineering
thesis.degree.grantorThe University of Texas at Austin
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy

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