Capp, J.-P. | bioRxiv Science

bioRxiv2023

Long-term phenotypic and genomic instability of an industrial ethanol-producing and C5-utilizing Saccharomyces cerevisiae strain

The performance of a microbial fermentation for the production of a bio-based product requires long-term robustness and stability of the producer strain. Here, we explored the genetic and metabolic stability of an industrial ethanol-producer Saccharomyces cerevisiae strain that was engineered by chromosomal integration of several copies of transgenes allowing co-fermentation of D-xylose and L-arabinose with glucose. Through sequential batch cultures in controlled bioreactor, it was found that this strain exhibited significant fluctuation of xylose and arabinose consumption starting at 50th generation and onward. Also, clones that have partly or completely lost the ability to assimilate arabinose or xylose were isolated, and this alteration was due to reduced copy number of the transgenes encoding the enzymes of the C5 sugar assimilation. However, subpopulation enriched with low or high RAD52 expression, whose expression level was reported to be proportional to homologous recombination rate did not exhibit defect in C5-sugar assimilation, arguing that other mechanisms may be responsible for copy number variation of transgenes. Overall, this work underscored genetic and metabolic instabilities, which although modest under our conditions, could in the longer term and under more stringent industrial conditions lead to a reduction of production performance.

bioengineering↗

bioRxiv2019

Gene expression noise produces cell-to-cell heterogeneity in eukaryotic homologous recombination rate

Variation in gene expression among genetically identical individual cells (called gene expression noise) directly contributes to phenotypic diversity. Whether such variation can impact genome stability and lead to variation in genotype remains poorly explored. We addressed this question by investigating whether noise in the expression of genes affecting homologous recombination (HR) activity either directly (RAD52) or indirectly (RAD27) confers cell-to-cell heterogeneity in HR rate in Saccharomyces cerevisiae. Using cell sorting to isolate subpopulations with various expression levels, we show that spontaneous HR rate is highly heterogeneous from cell-to-cell in clonal populations depending on the cellular amount of proteins affecting HR activity. Phleomycin-induced HR is even more heterogeneous, showing that RAD27 expression noise strongly affects the rate of recombination from cell-to-cell. Strong variations in HR rate between subpopulations are not correlated to strong changes in cell cycle stage. Moreover, this heterogeneity occurs even when simultaneously sorting cells at equal expression level of another gene involved in DNA damage response (BMH1) that is upregulated by DNA damage, showing that the initiating DNA damage is not responsible for the observed heterogeneity in HR rate. Thus gene expression noise seems mainly responsible for this phenomenon. Finally, HR rate non-linearly scales with Rad27 levels showing that total amount of HR cannot be explained solely by the time- or population-averaged Rad27 expression. Altogether, our data reveal interplay between heterogeneity at the gene expression and genetic levels in the production of phenotypic diversity with evolutionary consequences from microbial to cancer cell populations.

molecular biology↗