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Metab Eng. 2018 May;47:346-356. doi: 10.1016/j.ymben.2018.04.015. Epub 2018 Apr 23.

A comparative analysis of single cell and droplet-based FACS for improving production phenotypes: Riboflavin overproduction in Yarrowia lipolytica.

Author information

1
McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 E Dean Keeton St. Stop C0400, Austin, TX 78712, USA.
2
Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, USA; California Institute for Quantitative Biosciences, University of California San Francisco, San Francisco, CA, USA.
3
Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, TX 78712, USA.
4
Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, USA; California Institute for Quantitative Biosciences, University of California San Francisco, San Francisco, CA, USA; Chan Zuckerberg Biohub, San Francisco, CA, USA.
5
McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 E Dean Keeton St. Stop C0400, Austin, TX 78712, USA; Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, TX 78712, USA. Electronic address: halper@che.utexas.edu.

Abstract

Evolutionary approaches to strain engineering inherently require the identification of suitable selection techniques for the product and phenotype of interest. In this work, we undertake a comparative analysis of two related but functionally distinct methods of high-throughput screening: 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 first engineer and evolve the non-conventional yeast Yarrowia lipolytica for high extracellular production of riboflavin (vitamin B2), an innately fluorescent product. Following mutagenesis and adaptive evolution, a direct parity-matched comparison of these two selection strategies was conducted. Both single cell FACS and droplet FACS led to significant increases in total riboflavin titer (32 and 54 fold relative to the parental PO1f strain, respectively). However, single cell FACS favored intracellular riboflavin accumulation (with only 70% of total riboflavin secreted) compared with droplet FACS that favored extracellular product accumulation (with 90% of total riboflavin secreted). We find that for the test case of riboflavin, the extent of secretion and total production were highly correlated. The resulting differences in production modes and levels clearly demonstrate the significant impact that selection approaches can exert on final evolutionary outcomes in strain engineering. Moreover, we note that these results provide a cautionary tale when intracellular read-outs of product concentration (including signals from biosensors) are used as surrogates for total production of potentially secreted products. In this regard, these results demonstrate that extracellular production is best assayed through an encapsulation technique when performing high throughput screening.

PMID:
29698778
DOI:
10.1016/j.ymben.2018.04.015
[Indexed for MEDLINE]

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