Insights on the Control of Yeast Single-Cell Growth Variability by Members of the Trehalose Phosphate Synthase (TPS) Complex.

Single-cell variability of growth is a biological phenomenon that has attracted growing interest in recent years. Important progress has been made in the knowledge of the origin of cell-to-cell heterogeneity of growth, especially in microbial cells. To better understand the origins of such heterogeneity at the single-cell level, we developed a new methodological pipeline that coupled cytometry-based cell sorting with automatized microscopy and image analysis to score the growth rate of thousands of single cells. This allowed investigating the influence of the initial amount of proteins of interest on the subsequent growth of the microcolony. As a preliminary step to validate this experimental setup, we referred to previous findings in yeast where the expression level of Tsl1, a member of the Trehalose Phosphate Synthase (TPS) complex, negatively correlated with cell division rate. We unfortunately could not find any influence of the initial TSL1 expression level on the growth rate of the microcolonies. We also analyzed the effect of the natural variations of trehalose-6-phosphate synthase (TPS1) expression on cell-to-cell growth heterogeneity, but we did not find any correlation. However, due to the already known altered growth of the tps1Δ mutants, we tested this strain at the single-cell level on a permissive carbon source. This mutant showed an outstanding lack of reproducibility of growth rate distributions as compared to the wild-type strain, with variable proportions of non-growing cells between cultivations and more heterogeneous microcolonies in terms of individual growth rates. Interestingly, this variable behavior at the single-cell level was reminiscent to the high variability that is also stochastically suffered at the population level when cultivating this tps1Δ strain, even when using controlled bioreactors.

A GRX1 Promoter Variant Confers Constitutive Noisy Bimodal Expression That Increases Oxidative Stress Resistance in Yeast.

Higher noise in the expression of stress-related genes was previously shown to confer better resistance in selective conditions. Thus, evolving the promoter of such genes toward higher transcriptional noise appears to be an attractive strategy to engineer microbial strains with enhanced stress resistance. Here we generated hundreds of promoter variants of the GRX1 gene involved in oxidative stress resistance in Saccharomyces cerevisiae and created a yeast library by replacing the native GRX1 promoter by these variants at the native locus. An outlier clone with very strong increase in noise (6-times) at the same mean expression level as the native strain was identified whereas the other noisiest clones were only 3-times increased. This variant provides constitutive bimodal expression and consists in 3 repeated but differently mutated copies of the GRX1 promoter. In spite of the multi-factorial oxidative stress-response in yeast, replacement of the native promoter by this variant is sufficient alone to confer strongly enhanced resistance to H2O2 and cumene hydroperoxide. New replacement of this variant by the native promoter in the resistant strain suppresses the resistance. This work shows that increasing noise of target genes in a relevant strategy to engineer microbial strains toward better stress resistance. Multiple promoter replacement could synergize the effect observed here with the sole GRX1 promoter replacement. Finally this work suggests that combining several mutated copies of the target promoter could allow enhancing transcriptional-mediated noise at higher levels than mutating a single copy by providing constitutive bimodal and highly heterogeneous expression distribution.

Bimodality of gene expression from yeast promoter can be instigated by DNA context, inducing conditions and strain background.

Bimodality in gene expression is thought to provide a high phenotypic heterogeneity that can be favourable for adaptation or unfavourable notably in industrial processes that require stable and homogeneous properties. Whether this property is produced or suppressed in different conditions has been understudied. Here we identified tens of Saccharomyces cerevisiae genomic fragments conferring bimodal yEGFP expression on centromeric plasmid and studied some of these promoters in different DNA contexts, inducing conditions or strain backgrounds. First, we observed that the bimodal behaviour identified on plasmid is generally suppressed at the genomic level. Second, an inducible promoter such as the copper-regulated CUP1 promoter can produce bimodal expression in a time- and dose-dependent fashion. For a given copper sulphate concentration, a constant proportion of the subpopulation is induced and only the induction level of this subpopulation changed with induction duration, while for a same induction time, higher copper sulphate concentrations induced more cells at higher levels. Third, we showed that bimodality conferred by the CUP1 promoter in expression profile is strain background dependent, revealing epistasis in the generation of bimodality. The influence of these parameters on bimodality has to be taken into account when considering transgene expression for industrial microbial productions.