Biology subjects

St. Onge, R. P.

Publications and source records attributed to St. Onge, R. P..

3 recordsLinked to original sources

bioRxiv2021

A CRISPRi screen of essential genes reveals that proteasome regulation dictates acetic acid tolerance in Saccharomyces cerevisiae

CRISPR interference (CRISPRi) is a powerful tool to study cellular physiology under different growth conditions and this technology provides a means for screening changed expression of essential genes. In this study, a Saccharomyces cerevisiae CRISPRi library was screened for growth in medium supplemented with acetic acid. Acetic acid is a growth inhibitor challenging the use of yeast for industrial conversion of lignocellulosic biomasses. Tolerance towards acetic acid that is released during biomass hydrolysis is crucial for cell factories to be used in biorefineries. The CRISPRi library screened consists of >9,000 strains, where >98% of all essential and respiratory growth-essential genes were targeted with multiple gRNAs. The screen was performed using the high-throughput, high-resolution Scan-o-matic platform, where each strain is analyzed separately. Our study identified that CRISPRi targeting of genes involved in vesicle formation or organelle transport processes led to severe growth inhibition during acetic acid stress, emphasizing the importance of these intracellular membrane structures in maintaining cell vitality. In contrast, strains in which genes encoding subunits of the 19S regulatory particle of the 26S proteasome were downregulated had increased tolerance to acetic acid, which we hypothesize is due to ATP-salvage through an increased abundance of the 20S core particle that performs ATP-independent protein degradation. This is the first study where a high-resolution CRISPRi library screening paves the way to understand and bioengineer the robustness of yeast against acetic acid stress. IMPORTANCEAcetic acid is inhibitory to the growth of the yeast Saccharomyces cerevisiae, causing ATP starvation and oxidative stress, which leads to sub-optimal production of fuels and chemicals from lignocellulosic biomass. In this study, where each strain of a CRISPRi library was characterized individually, many essential and respiratory growth essential genes that regulate tolerance to acetic acid were identified, providing new understanding on the stress response of yeast and new targets for the bioengineering of industrial yeast. Our findings on the fine-tuning of the expression of proteasomal genes leading to increased tolerance to acetic acid suggests that this could be a novel strategy for increasing stress tolerance, leading to improved strains for production of biobased chemicals.

synthetic biology↗

bioRxiv2018

Dissection of yeast pleiotropic drug resistance regulation reveals links between cell cycle regulation and control of drug pump expression

Eukaryotes utilize a highly-conserved set of drug efflux transporters to confer pleiotropic drug resistance (PDR). Despite decades of effort interrogating this process, multiple aspects of the PDR process, in particular PDR regulation, remain mysterious. In order to interrogate the regulation of this critical process, we have developed a small-molecule responsive biosensor that couples PDR transcriptional induction to growth rate in Saccharomyces cerevisiae. We applied this system to genome-wide screens for potential PDR regulators using the homozygous diploid deletion collection. These screens identified and characterized a series of genes with significant but previously uncharacterized roles in the modulation of the yeast PDR in addition to recapitulating previously-known factors involved in PDR regulation. Furthermore, we demonstrate that disruptions of the mitotic spindle checkpoint assembly lead to elevated PDR response in response to exposure to certain compounds. These results not only establish our biosensor system as a viable tool to investigate PDR in high-throughput, but also uncovers novel control mechanisms governing PDR response and a previously uncharacterized link between this process and cell cycle regulation.\n\nSignificancePleiotropic drug resistance (PDR) is a conserved mechanism by which cells utilize membrane bound pumps to transport chemicals out of the cell. Here, we develop a growth-based biosensor system in yeast that enables high-throughput identification of factors that transcriptionally regulate PDR. Among the novel PDR regulators identified here, we show that spindle assembly checkpoint (SAC) proteins, which are important for cell cycle regulation, inhibit hyperactivation of PDR upon drug treatment. This result provides insights into PDR regulation, as well as potential targets for therapeutic intervention, particularly in chemoresistant cancers where the cell cycle regulation is often disrupted.

genetics↗

bioRxiv2018

HEx: a heterologous expression platform for the discovery of fungal natural products

For decades, fungi have been a source of FDA-approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.\n\nSummaryHere we present the largest scale effort reported to date toward the complete refactoring and heterologous expression of fungal biosynthetic gene clusters utilizing HEx, a novel synthetic biology platform.

synthetic biology↗