Abstract
Engineered microbes can produce valuable chemicals, however production strains often require extensive optimization before they can be used at industrial scales. To quantify production, metabolic engineers typically employ mass spectrometry, which offers excellent chemical specificity. However, with this approach samples are derived from bulk cultures, obscuring potential cell-to-cell differences in production, and precluding selection methods that rely on measurements of a single cell’s phenotype, such as directed evolution. Here, we use stimulated Raman scattering (SRS) based chemical imaging to directly visualize free fatty acids in metabolically engineered Escherichia coli. We uncover substantial heterogeneity in fatty acid production among and within colonies. We then demonstrate longitudinal SRS imaging, allowing fatty acids to be linked with the cells that generated them. Further, using the hyperspectral images, we develop an approach for compositional analysis that determines the chain length and unsaturation of the fatty acids in living cells. Our results demonstrate that cell-to-cell heterogeneity in production of metabolic targets can be substantial and indicate that controlling this to eliminate low producers represents a critical area for future optimization. Further, because SRS imaging is rapid, chemically specific, and can be acquired longitudinally, it is a promising method for metabolic engineers, allowing for direct imaging of biosynthesis at the single-cell level.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵† Co-first authors
