Single-cell metabolic heterogeneity dynamics and optimization of valencene biosynthesis by hyperspectral stimulated Raman scattering microscopy

Understanding metabolic heterogeneity and dynamics is crucial for optimizing microbial production of valuable chemicals in synthetic biology. However, current quantification methods often lack single-cell resolution or require destructive sampling. Here, we report engineering valencene-producing Yarrowia lipolytica ( Y. lipolytica ) and integrate hyperspectral stimulated Raman scattering (SRS) microscopy, a label-free and non-invasive imaging technique, with a Lasso unmixing algorithm to quantify valencene biosynthesis at the single-cell level, revealing previously unresolved metabolic heterogeneity in production yields. To demonstrate the utility of this approach, we perform time-resolved tracking of valencene metabolism and a substrate co-feeding strategy, which reveals that cells preferentially utilize acetic acid rather than glucose for valencene biosynthesis. In addition, by optimizing the carbon/nitrogen (C/N) ratio in the nutrient and applying fed-batch cultivation, the valencene yield is further increased to 125 mg/L. This work establishes hyperspectral SRS microscopy as a powerful tool for in situ visualization of microbial metabolism, offering a label-free and non-destructive alternative to current destructive methods in synthetic biology.