The synergetic effect from the combination of different adsorption resins in batch and semi-continuous cultivations ofS. cerevisiaecell 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 a 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 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.

Improved Production of Taxol® Precursors inS. cerevisiaeusing Combinatorialin silicoDesign and Metabolic Engineering

“…cerevisiae has been used as a platform to produce high-value biopharmaceuticals, ranging from recombinant therapeutic proteins to plant-derived natural products [10,11]. By integrating heterologous plant-derived genes, early-step precursors of Taxol® (paclitaxel), a leading anticancer drug with a market size over billions of USD [12], have been produced by yeast cell factories [13][14][15][16][17]. Geranylgeranyl diphosphate (GGPP), a product of the yeast mevalonate pathway, is the substrate of the cyclisation step, the first step in the Taxol® biosynthesis pathway catalyzed by the taxadiene synthase [18].…”