In situ recovery of the anticancer drug "paclitaxel" and its precursors from engineered yeast and plant cells using different macro-porous resins

The production and recovery of valuable natural products (NPs) with economic and social interest from different natural sources is on the agenda of several research institutes, governments and industries. The recovery and purification of NPs typically account for 60-80% of the total operational costs at the industrial scale. One important natural product is paclitaxel, which is a taxane capable of treating different types of cancers (breast and ovarian mainly). In this work, a technique called in situ product recovery (ISPR) which is an effective bioprocess integration tool, was evaluated and optimized to perform the adsorption of the taxanes in the same cultivation step. In the first chapter, a single commercial solid adsorbent was used, which was the macro-porous adsorbent resin HP-20. After the in situ cultivation, another integration of the process was performed by making the cell-lysis and desorption of the taxanes into the extraction solvent (Acetone at 99 %). A maximum taxadiene titre of 76 ± 19 mg/L was obtained which meant a 1.4-fold improvement compared to the unsustainable dodecane overlay method. In the second chapter, the ISPR technique was refined to increase the acetylated and oxygenated taxanes by using three different macro-porous resins (HP-20, XAD7HP and HP-2MG). A Definitive Screening Design (DSD) was performed to know which combination of the resins could result in a better fit, taking as factors different resins' concentrations. It was found that the best resin combination was 0.5, 1 and 1.5 % w/v of HP-20, XAD7HP and HP-2MG respectively, reaching a maximum total taxane titre of 190 ± 20 mg/L. Further, it was discovered that the resins could remove cell waste such as the reactive oxidative species (ROS), finding 1.5-fold lower ROS concentration in the media cultivated with the resins compared to the control. Using the combination of the same resin, a semi-continuous cultivation was performed reaching 783 ± 33 mg/L total taxanes, making it the highest total taxanes concentration ever retrieved from a yeast cell factory. Finally, in the third chapter, by cultivating cambial meristematic plant cells (Taxus baccata) with the improved ISPR method, we obtained a paclitaxel yield of 234 ± 23 mg/Kg of biomass in batch cultivations and 276 ± 24 mg/Kg of biomass in semi-continuous cultivations, which were 13-fold and 16-fold higher than the paclitaxel titres respectively compared with control cultivations. The outputs of this thesis have many future implications, as the innovative approach developed here could be applied to a significant range of metabolites, enhancing the limit of detection for natural product discovery, while radically improving bioprocess intensification and increasing product titres. Moreover, this work could enable the transition towards continuous flow bioreactors with fixed adsorbents. Lastly, this could lead to an optimisation in the use and development of novel, selective and more sustainable adsorption materials and extraction solvents.