PMID- 32603587 OWN - NLM STAT- MEDLINE DCOM- 20210527 LR - 20210527 IS - 2161-5063 (Electronic) IS - 2161-5063 (Linking) VI - 9 IP - 8 DP - 2020 Aug 21 TI - Yeast Intracellular Staining (yICS): Enabling High-Throughput, Quantitative Detection of Intracellular Proteins via Flow Cytometry for Pathway Engineering. PG - 2119-2131 LID - 10.1021/acssynbio.0c00199 [doi] AB - 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 beta 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 delta 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. FAU - Hill, Brett D AU - Hill BD AD - Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States. FAU - Prabhu, Ponnandy AU - Prabhu P AD - Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States. FAU - Rizvi, Syed M AU - Rizvi SM AD - Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States. FAU - Wen, Fei AU - Wen F AUID- ORCID: 0000-0001-7970-4796 AD - Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States. LA - eng PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, Non-P.H.S. DEP - 20200715 PL - United States TA - ACS Synth Biol JT - ACS synthetic biology JID - 101575075 RN - 0 (Actins) RN - 0 (Antibodies) RN - 0 (Saccharomyces cerevisiae Proteins) RN - EC 1.1.1.21 (Aldehyde Reductase) RN - EC 1.1.1.9 (D-Xylulose Reductase) RN - EC 1.2.1.9 (Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)) SB - IM MH - Actins/analysis/metabolism MH - Aldehyde Reductase/analysis/genetics/metabolism MH - Antibodies/immunology MH - Clustered Regularly Interspaced Short Palindromic Repeats/genetics MH - D-Xylulose Reductase/analysis/genetics/metabolism MH - *Flow Cytometry MH - Gene Editing MH - Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)/analysis/immunology/metabolism MH - Intracellular Space/metabolism MH - Metabolic Engineering MH - Saccharomyces cerevisiae/*metabolism MH - Saccharomyces cerevisiae Proteins/*analysis/immunology/metabolism MH - Staining and Labeling OTO - NOTNLM OT - CRISPR OT - flow cytometry OT - intracellular staining OT - pathway engineering OT - xylose OT - yeast EDAT- 2020/07/01 06:00 MHDA- 2021/05/28 06:00 CRDT- 2020/07/01 06:00 PHST- 2020/07/01 06:00 [pubmed] PHST- 2021/05/28 06:00 [medline] PHST- 2020/07/01 06:00 [entrez] AID - 10.1021/acssynbio.0c00199 [doi] PST - ppublish SO - ACS Synth Biol. 2020 Aug 21;9(8):2119-2131. doi: 10.1021/acssynbio.0c00199. Epub 2020 Jul 15.