Increased expression of the fluorescent reporter protein ymNeonGreen in Saccharomyces cerevisiae by reducing RNA secondary structure near the start codon

Authors: Hector, Ronald - Ron; Mertens, Jeffrey; Nichols, Nancy

Submitted to: Biotechnology Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/27/2021
Publication Date: 12/31/2021
Citation: Hector, R.E., Mertens, J.A., Nichols, N.N. 2021. Increased expression of the fluorescent reporter protein ymNeonGreen in Saccharomyces cerevisiae by reducing RNA secondary structure near the start codon. Biotechnology Reports. 33: Article e00697. https://doi.org/10.1016/j.btre.2021.e00697.
DOI: https://doi.org/10.1016/j.btre.2021.e00697

Interpretive Summary: Fluorescent proteins are essential as reporters for studying cellular proteins. They make it possible to see a protein’s location in the cell and investigate the dynamics of how much of, and when, a protein is made. A newer fluorescent protein, reported to have increased brightness and faster maturation time compared to other reporter proteins, is useful for analyzing proteins even when they are present in small amounts. While the current version of this fluorescent protein expresses well in many cell lines and microorganisms, it does not function as expected when used in Brewer’s yeast, Saccharomyces cerevisiae. Our research created a version of the protein that more than doubled its brightness in yeast. The results will allow scientists increased ability to visualize and study essential proteins that are produced at very low levels. The new fluorescent protein will also work better for studying proteins with rapid increases or decreases in expression. Improving the understanding of how cells control and regulate protein expression makes it easier to engineer yeast strains to produce fuels and chemicals. This research will benefit the large community of scientists using Brewer’s yeast as a model organism as well as producers of renewable products seeking to optimize yields.

Technical Abstract: Expression of a new fluorescent reporter protein called mNeonGreen, that is not based on the jellyfish green fluorescent protein (GFP) sequence, shows increased brightness and folding speed compared to enhanced GFP. However, in vivo brightness of mNeonGreen and its yeast-optimized variant ymNeonGreen in S. cerevisiae is lower than expected, limiting the use of this high quantum yield, fast-folding reporter in budding yeast. This study shows that secondary RNA structure near the start codon in the ymNeonGreen ORF inhibits expression in S. cerevisiae. Removing secondary structure, without altering the ymNeonGreen protein sequence, led to a 2 and 4-fold increase in fluorescence when expressed in S. cerevisiae and E. coli, respectively. In S. cerevisiae, increased fluorescence was seen with strong and weak promoters and led to higher transcript levels suggesting greater transcript stability and improved expression in the absence of stable secondary RNA structure near the start codon.