(26c) Biomolecular Phase Separation Suppresses Gene Expression in Bacteria

After spontaneous protein phase separation (PS) in a living cell was observed for the first time, subsequent studies have revealed that the occurrence of biomolecular PS is ubiquitous in a living system. Past studies also focused on biophysical mechanisms behind the biomolecular PS, which significantly enhances our understandings of the PS process. At the same time, it has been suggested that PS can be used as a new engineering tool to control biochemical processes in a living cell. Specifically, once biomolecules undergo PS, they will self-assemble to form membraneless organelles (MLO), where their local concentrations become much higher than those outside the MLOs. Due to this unique feature, it has been speculated that PS and resulted MLOs play an important role in regulating biochemical processes such as gene regulations. This is because many proteins involved in gene regulations are found to undergo PS both in vitro and in vivo. By co-enriching these proteins in MLOs, PS may enhance the overall gene expression level. However, previous experimental studies with both natural and engineered systems yielded rather contradictory results on how PS regulates the gene expression.

Motivated by this, this study aims to unravel the relationship between PS and the gene expression quantitatively. With E. coli as a model organism, we constructed a variety of synthetic genetic circuits to probe the relationship. Specifically, each genetic circuit contains a transcription factor (TF) fused with a fluorescent protein and an intrinsically disordered protein (IDP). By fusing TF to IDP, TF will undergo PS. Also, there is another fluorescent protein placed under a promoter inducible by the corresponding TF. Consequently, by measuring the expression levels of two fluorescent proteins, one could quantitatively map the functional landscape of PS in terms of the gene expression. By testing various TF activators with different activation mechanisms and different IDPs reported in literature, we found that these transcriptional activators become transcriptional repressors once they undergo PS in E. coli. Such results demonstrate that PS does not guarantee the enhancement in gene expression as commonly expected. Based on this study, the biphasic nature of a transcriptional activator coupled with PS (i.e., from activator to repressor) will be used as a novel synthetic biology tool to engineer living cells in the future.