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  • The synergetic effect from ...
    Santoyo‐Garcia, Jorge H.; Walls, Laura E.; Valdivia‐Cabrera, Marissa; Malcı, Koray; Jonguitud‐Borrego, Nestor; Halliday, Karen J.; Rios‐Solis, Leonardo

    Biotechnology and bioengineering, August 2023, Letnik: 120, Številka: 8
    Journal Article

    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. The production of T5α‐yl Acetate using engineered S. cerevisiae cells was significantly enhanced through the implementation of a novel in situ product recovery method utilizing three biocompatible and highly absorbent polymeric resins simultaneously. Optimization using Design of Experiments facilitated a semi‐continuous cultivation strategy, yielding a total taxanes concentration of 783 ± 33 mg/L, with T5α‐yl Acetate achieving levels up to 95 ± 4 mg/L. Furthermore, this method resulted in a 1.5‐fold reduction in cell‐waste reactive oxygen species