UNraveling, Identifying and COntrolling isogenic Heterogeneity

同源异质性是导致原核生物和真核生物中出现亚群的普遍现象。它在包括但不限于生物生产、细菌持久性、生态学和癌症等领域有许多影响。在微生物生物生产过程中，异质性是过程不稳定的主要来源，但由于难以以高通量方式测量瞬时单细胞表型，历史上一直被忽视。随着单细胞RNA测序(ScRNAseq)的最新进展，从等基因培养中产生的亚群现在可以比以往任何时候都更详细地进行量化和研究。现在越来越清楚的是，异质性对于微生物在自然环境中的适合性是重要的，因为存在具有增加压力特征的亚群。在UNICOH中，我将描述和控制真核生物模式生物酿酒酵母中异质性的出现。鉴定将依赖于使用最近发表的酿酒酵母scRNAseq数据确定的异质性标记。这些标记将首先被用来探测酵母敲除(YKO)集合，以识别导致异质性的基因。YKO收藏品的快速原型将在帝国理工学院伦敦生物铸造厂进行，在那里可以实现高通量转化和筛选的自动化。其次，使用文库或已发表的scRNAseq数据确定的异质靶标将使用光遗传学进行验证，其中在电子反馈调节中，将允许将感兴趣的蛋白质保持在所需的水平，同时降低其相关的异质性。这些发现将被转移到工业菌株上，以创造更稳定的菌株。总之，UNICOH将阐明等基因异质性的出现，并在生物工艺扩大期间为提高菌株稳定性做出关键贡献，这将对建立未来的生物经济至关重要。

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.