Promoter selection reduces phenotypic heterogeneity and improves bioproduction

Cell-to-cell heterogeneity leads to the formation of subpopulations within an isogenic population, resulting in the coexistence of high and low producer cells. However, the impact of this phenotypic heterogeneity on bioproduction has been overlooked. Using flow cytometry to analyse commonly used yeast promoters, we found that many display macro-heterogeneity, generating multiple discrete subpopulations. Additionally, by regressing single-cell protein expression on single-cell RNA sequencing data, we explored the use of predictive models as a guide for selecting promoters with more homogeneous expression at desired expression levels. As a proof of concept, we engineered an improved lycopene pathway by substituting homogeneous promoters for macro-heterogeneous ones and implemented single-cell Raman spectroscopy to demonstrate up to a threefold increase in single-cell and bulk lycopene production. Overall, our results show that promoter heterogeneity can have a large impact on microbial bioprocesses and that the selection of homogeneous promoters can achieve more efficient production in a metabolically engineered pathway.