eGFP Gene Integration in HO: A Metabolomic Impact?

Abstract

Integrating fluorescent genes including eGFP in the yeast genome is common practice for various applications, including cell visualization and population monitoring. The transformation of a commercial S. cerevisiae strain by integrating a cassette including a gene encoding an EGFP protein in the HO gene was carried out using CRISPR-Cas9 technology. Although this type of integration is often used and described as neutral at the phenotypic level of the cell, we have highlighted that under alcoholic fermentation (in a Chardonnay must), it has an impact on the exometabolome. We observed 41 and 82 unique biomarkers for the S3 and S3GFP strains, respectively, as well as 28 biomarkers whose concentrations varied significantly between the wild-type and the modified strains. These biomarkers were mainly found to correspond to peptides. Despite similar phenotypic growth and fermentation parameters, high-resolution mass spectrometry allowed us to demonstrate, for the first time, that the peptidome is modified when integrating this cassette in the HO gene.

Keywords: CRISPR-Cas9 technology; HO gene; S. cerevisiae; eGFP; metabolomics.

Figure 1

Two-photon microscopy observation of (A) the modified strain S3GFP and (B) the wild-type strain S3. The images (A2,B2) were obtained with channel FF01-492/SP (400–492 nm) and (A3,B3) with channel FF03-525/50 (500–550 nm). The images (A1,B1) provided were obtained by merging these two detection channels.

Figure 2

Fermentation profile comparison in Chardonnay must. Cell viability and sugar consumption were monitored for the Saccharomyces cerevisiae wild-type strain S3 (in grey) and the recombinant strain S3GFP (in green). Solid curves represent viable cell populations and dotted curves represent sugar concentrations. For each strain, the experiments were performed in triplicate and the error bars represent the confidence interval.

Figure 3

(A) Heatmap based on the 151 significatively different annotated compounds for S3 and S3GFP in Chardonnay must. (B1) and (B2) represent the van Krevelen diagrams for the compounds that are significatively more present in S3 and S3GFP (in red on the heatmap), respectively. (C1) and (C2) represent retention time versus mass (Da) for the compounds significatively more present in S3 and S3GFP, respectively. The area of bubbles corresponds to the square root of detected area divided by 10.