Streamlined isolation of E. coli biofactory metabolites using centrifugal extraction and partition chromatographic techniques. New hydroxytyrosol derivatives.

Microbial production of bioactive molecules provides a promising alternative to traditional isolation or chemical synthesis approaches, enabling high-yield production of pure compounds without the need for heavy-metal catalysts, harsh conditions, or complex plant-based processes. Hydroxytyrosol (HT), a potent antioxidant with significant pharmacological properties, has attracted considerable interest due to its broad applications in pharmaceuticals, cosmetics, and food industries. While Escherichia coli systems have been effective for HT production, optimization of extraction yield and recovery process remain a challenge. Furthermore, isolation of HT and its metabolites continues to suffer from inefficiencies even when biosynthetic pathways are successful. Centrifugal techniques such as annular centrifugal extraction and partition chromatography methods e.g., Annular Centrifugal Extraction - ACE, Countercurrent chromatography - CCC, Centrifugal Partition Chromatography - CPC offer advantages over traditional methods, including the absence of solid stationary phases, reduced organic solvent use, speed, and scalability. However, these techniques have been underutilized in biotechnology workflows. Additionally, existing procedures often employ simplistic analytical methods, which lead to loss of valuable data for pathway monitoring, validation and metabolite discovery. This study presents a comprehensive workflow for the production, extraction, isolation, and identification of HT and derivatives from metabolically engineered E. coli biofactories. The workflow combines ACE for media extraction with CPC for high yield and purity HT isolation. It also emphasizes the analysis and structural elucidation of HT metabolites by means of HRMS/MS and 1 & 2D NMR, facilitating biosynthetic pathway monitoring and discovery of novel HT metabolites with potential enhanced biological activity. Using this approach, 23 compounds were isolated and identified contributing to the completion of the complex biosynthetic route of HT synthesis from l-tyrosine. This is first reported of direct isolation of pure HT metabolites from E. coli biofactories and to our knowledge the first application of liquid-liquid centrifugal techniques in the treatment of biotechnological materials.