Longitudinal single-cell chemical imaging of engineered strains reveals heterogeneity in fatty acid production

Understanding metabolic heterogeneity is critical for optimizing microbial production of valuable chemicals, but requires tools that can quantify metabolites at the single-cell level over time. Here, we develop longitudinal hyperspectral stimulated Raman scattering (SRS) chemical imaging to directly visualize free fatty acids in engineered Escherichia coli over many cell cycles. We also develop compositional analysis to determine the chain length and unsaturation of the fatty acids in living cells. Our method reveals substantial heterogeneity in fatty acid production among and within colonies that emerges over the course of many generations. Interestingly, the strains display distinct types of production heterogeneity in an enzyme-dependent manner. By pairing time-lapse and SRS imaging, we examine the relationship between growth and production at the single-cell level. Single-cell quantification does not show a significant growth-production tradeoff in a strain that exhibits high production heterogeneity. Our results demonstrate that cell-to-cell production heterogeneity is pervasive and provide a means to link single-cell and population-level production.