The synergetic effect from the combination of different adsorption resins in batch and semi‐continuous cultivations of S. Cerevisiae cell factories to produce acetylated Taxanes precursors of the anticancer drug Taxol

Abstract

In situ product recovery is an efficient way to intensify bioprocesses as it can perform adsorption of the desired natural products in the cultivation. However, it is common to use only one adsorbent (liquid or solid) to perform the product recovery. For this study, the use of an in situ product recovery method with three combined commercial resins (HP‐20, XAD7HP, and HP‐2MG) with different chemical properties was performed. A new yeast strain of Saccharomyces cerevisiae was engineered using CRISPR Cas9 (strain EJ2) to deliver heterologous expression of oxygenated acetylated taxanes that are precursors of the anticancer drug Taxol ® (paclitaxel). Microscale cultivations using a definitive screening design (DSD) were set to get the best resin combinations and concentrations to retrieve high taxane titers. Once the best resin treatment was selected by the DSD, semi‐continuous cultivation in high throughput microscale was performed to increase the total taxanes yield up to 783 ± 33 mg/L. The best T5α‐yl Acetate yield obtained was up to 95 ± 4 mg/L, the highest titer of this compound ever reported by a heterologous expression. It was also observed that by using a combination of the resins in the cultivation, 8 additional uncharacterized taxanes were found in the gas chromatograms compared to the dodecane overlay method. Lastly, the cell‐waste reactive oxygen species concentrations from the yeast were 1.5‐fold lower in the resin's treatment compared to the control with no adsorbent aid. The possible future implications of this method could be critical for bioprocess intensification, allowing the transition to a semi‐continuous flow bioprocess. Further, this new methodology broadens the use of different organisms for natural product synthesis/discovery benefiting from clear bioprocess intensification advantages.

Increased paclitaxel recovery from Taxus baccata vascular stem cells using novel in situ product recovery approaches

“…While in our previous work (Santoyo-Garcia et al 2023 ) we reported the effectiveness of the combination of resins HP-20, XAD7HP and HP-2MG in recovering taxanes from yeast cell factories, it is important to mention that our findings have not been directly applied to plant VSCs cells. The physical–chemical differences of these macro-porous commercial resins can be found in Santoyo-Garcia et al ( 2023 ). In addition, the main physical–chemical properties are described in Table 1 .…”

“…Next, we determined the impact of combinations of these resins on in situ cultivation of VSCs at different resin concentrations and configurations to test the impact on both cell growth and taxane production, as the combination of these three resins has shown favourable results for purification of taxanes from recombinant yeast expressing some of the known paclitaxel pathway enzymes (Santoyo-Garcia et al 2023 ). Treatments consisting of three combinations of resins were employed (Table 1 ).…”

“…Treatment A had a majority of the polar resin HP-2MG. This specific combination was selected as it has been shown to be effective in the recovery of taxanes from yeast cell factories (Santoyo-Garcia et al 2023 ). As demonstrated in previous studies, the total concentration of the resins in the culturable cells is a critical variable for cell growth (Yin et al 2005 ; Santoyo-Garcia et al 2022 ).…”

“…In the context of microbial cells, cultures of Aspergillus fumigatus and Alternaria tenuissima have been immobilised using different entrapment carriers (gelatin, agar and Arabic gum), increasing paclitaxel yields by 1.3- and 1.8-fold, respectively, compared to free cultures (El-Sayed et al 2019 ). In engineered yeast, the effectiveness of combining different macro-porous resins with different surface polarity increased the titre recovery of different taxanes reaching an eightfold increase using this ISPR approach (Santoyo-Garcia et al 2023 ).…”

“…1 . Our primary objective was to increase taxanes yields, not only in yeasts, as previously reported (Santoyo-Garcia et al 2023 ), but also in various other productive organisms, such as plants cells. This process aims to elucidate the resin's influence on taxanes recovery, based on its adsorption characteristics for the particular metabolites generated by the plant cell line.…”