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Enhancing secreted production from yeast species with high throughput microdroplet screening methods
- Publication Year :
- 2023
- Publisher :
- The University of Texas at Austin, 2023.
-
Abstract
- Microfluidics-based sorting of cells after encapsulation in water-in-oil or water/oil/water droplet emulsions has great potential to significantly accelerate the design-build-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 microdroplet-enabled 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 of single 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.
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi...........7a6998d3048fc33435eb05ac6b787fcd
- Full Text :
- https://doi.org/10.26153/tsw/45986