Visualization of a Limonene Synthesis Metabolon Inside Living Bacteria by Hyperspectral SRS Microscopy.

Monitoring biosynthesis activity at single-cell level is key to metabolic engineering but is still difficult to achieve in a label-free manner. Using hyperspectral stimulated Raman scattering imaging in the 670-900 cm region, localized limonene synthesis are visualized inside engineered Escherichia coli. The colocalization of limonene and GFP-fused limonene synthase is confirmed by co-registered stimulated Raman scattering and two-photon fluorescence images. The finding suggests a limonene synthesis metabolon with a polar distribution inside the cells. This finding expands the knowledge of de novo limonene biosynthesis in engineered bacteria and highlights the potential of SRS chemical imaging in metabolic engineering research.