New Insights Into Curcumin Behavior After Its Biosynthesis in Engineered Escherichia coli Through Spectroscopic and Spatial Analysis

Introduction Curcumin is a naturally occurring compound with anti‐inflammatory, cholesterol‐lowering, antidiabetic, and antioxidant properties. Curcumin can be biosynthesized in microbial hosts despite being found natively in Curcuma longa roots. Objectives It is unclear whether curcumin is transported out of microbial cells, binds to the inner or outer cell membrane, or accumulates inside. This study aims to gain a better understanding of curcumin's behavior after its formation in the host, which could lead to the development of new approaches to improve curcumin production. Material and Methods Advanced imaging techniques, including SEM, TEM, SHS, and TPF, were utilized to understand the behavior of curcumin within and outside of engineered Escherichia coli cells. Results Curcumin was biosynthesized from ferulic acid in engineered E. coli BL21(DE3) by coexpressing 4‐coumarate: CoA ligase and curcuminoid synthase. Second harmonic scattering (SHS) spectroscopy experiments utilized curcumin as a probe to investigate the surface binding of curcumin onto living E. coli cells with experimentally determined adsorption free energy, ΔG , values of −14.0 kcal/mol. By employing second harmonic and two‐photon fluorescence imaging methods, the spatial distribution of curcumin aggregates was determined. Electron microscopy images revealed the presence of curcumin aggregates within the cells, at the surface, and in the media. Conclusion These experiments demonstrate that curcumin biosynthesis from ferulic acid leads to significant product aggregation within the cells, which could ultimately halt production by inducing cell death. Understanding the localization, transport, and removal of curcumin is crucial in developing more efficient biosynthetic pathways to enhance its production in microbial systems.