Yeast Intracellular Staining (yICS): Enabling High-Throughput, Quantitative Detection of Intracellular Proteins via Flow Cytometry for Pathway Engineering

ACS Synth Biol. 2020 Aug 21;9(8):2119-2131. doi: 10.1021/acssynbio.0c00199. Epub 2020 Jul 15.

Abstract

The complexities of pathway engineering necessitate screening libraries to discover phenotypes of interest. However, this approach is challenging when desirable phenotypes cannot be directly linked to growth advantages or fluorescence. In these cases, the ability to rapidly quantify intracellular proteins in the pathway of interest is critical to expedite the clonal selection process. While Saccharomyces cerevisiae remains a common host for pathway engineering, current approaches for intracellular protein detection in yeast either have low throughput, can interfere with protein function, or lack the ability to detect multiple proteins simultaneously. To fill this need, we developed yeast intracellular staining (yICS) that enables fluorescent antibodies to access intracellular compartments of yeast cells while maintaining their cellular integrity for analysis by flow cytometry. Using the housekeeping proteins β actin and glyceraldehyde 3-phophate dehydrogenase (GAPDH) as targets for yICS, we demonstrated for the first time successful antibody-based flow cytometric detection of yeast intracellular proteins with no modification. Further, yICS characterization of a recombinant d-xylose assimilation pathway showed 3-plexed, quantitative detection of the xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK) enzymes each fused with a small (6-10 amino acids) tag, revealing distinct enzyme expression profiles between plasmid-based and genome-integrated expression approaches. As a result of its high-throughput and quantitative capability, yICS enabled rapid screening of a library created from CRISPR-mediated XDH integration into the yeast δ site, identifying rare (1%) clones that led to an 8.4-fold increase in XDH activity. These results demonstrate the utility of yICS for greatly accelerating pathway engineering efforts, as well as any application where the high-throughput and quantitative detection of intracellular proteins is desired.

Keywords: CRISPR; flow cytometry; intracellular staining; pathway engineering; xylose; yeast.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Actins / analysis
  • Actins / metabolism
  • Aldehyde Reductase / analysis
  • Aldehyde Reductase / genetics
  • Aldehyde Reductase / metabolism
  • Antibodies / immunology
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • D-Xylulose Reductase / analysis
  • D-Xylulose Reductase / genetics
  • D-Xylulose Reductase / metabolism
  • Flow Cytometry*
  • Gene Editing
  • Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / analysis
  • Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / immunology
  • Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / metabolism
  • Intracellular Space / metabolism
  • Metabolic Engineering
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / analysis*
  • Saccharomyces cerevisiae Proteins / immunology
  • Saccharomyces cerevisiae Proteins / metabolism
  • Staining and Labeling

Substances

  • Actins
  • Antibodies
  • Saccharomyces cerevisiae Proteins
  • Aldehyde Reductase
  • D-Xylulose Reductase
  • Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)