UNraveling, Identifying and COntrolling isogenic Heterogeneity

Isogenic heterogeneity is a universal phenomenon leading to the emergence of subpopulations in both prokaryotes and eukaryotes. It has numerous implications in areas including but not limited to bioproduction, bacterial persistence, ecology and cancer. In microbial bioproduction processes, heterogeneity is a major source of process instability, but has been historically overlooked due to the difficulty to measure transient, single-cell phenotypes in a high-throughput manner. With the recent progress in single-cell RNA sequencing (scRNAseq), subpopulations that emerge from an isogenic culture can now be quantified and studied in greater detail than ever before. It is now becoming clear that heterogeneity is important for microbial fitness in natural environment with the presence of subpopulations with an increased stress signature. In UNICOH, I will characterize and control the emergence of heterogeneity in the eukaryote model organism Saccharomyces cerevisiae. Characterization will rely on heterogeneity markers identified using recently published scRNAseq data for S. cerevisiae. These markers will first be used to probe the yeast knock-out (YKO) collection to identify genes that contribute to heterogeneity. Rapid prototyping of the YKO collection will be performed at the Imperial College London Biofoundry where high-throughput transformation and screening can be automated. Second, heterogeneous targets identified using the library or published scRNAseq data will be validated using optogenetics, where in silico feedback regulation will allow to maintain the protein of interest to desired levels while reducing its associated heterogeneity. The findings will be transferred to industrial strains to create more stable strains. Together, UNICOH will shed light on the emergence of isogenic heterogeneity and make a critical contribution towards strain stability improvement during bioprocess scale-up which will be vital for establishing a future bioeconomy.