Abstract
Paclitaxel is an important diterpenoid commonly used as an anticancer drug. Although the paclitaxel biosynthetic pathway has been mostly revealed, some steps remain to be elucidated. The difficulties in plant transformations and the scarcity of the precursor of paclitaxel, (+)-taxa-4(5), 11(12)-diene (taxadiene), have hindered the full comprehension of paclitaxel biochemistry and, therefore, its production by biotechnological approaches. One solution is to use the budding yeast, Saccharomyces cerevisiae, as a platform to elucidate the paclitaxel biosynthesis. As taxadiene is a diterpenoid, its common precursor, geranylgeranyl pyrophosphate (GGPP), needs to be increased in yeast. In this study, we screened various GGPP synthases (GGPPS) to find the most suitable GGPPS for taxadiene production in yeast. We also optimized the taxadiene production by increasing the flux toward the terpenoid pathway. Finally, to remove selection markers, we integrated the required genes using a CRISPR/Cas9 system in the yeast genome. Our result showed that a titer of 2.02 ± 0.40 mg/L (plasmid) and 0.41 ± 0.06 mg/L (integrated) can be achieved using these strategies. This platform strain can be used to readily test the gene candidates for microbial paclitaxel biosynthesis in the future.
Highlights
IntroductionDue to its selective and potent mechanism of action, paclitaxel-based drugs have been used in chemotherapy to treat various cancers, including breast, lung, ovarian, and sarcoma [2,5]
This result indicated that pIPP can be used to increase flux towards taxadiene production
Our results showed that overexpression of endogenous yeast GGPP synthases (GGPPS) (BTS1) led to the highest taxadiene production with 782 ± 14 μg/L, almost an 80-fold increase compared to the yeast without GGPPS overexpression (Figure 3)
Summary
Due to its selective and potent mechanism of action, paclitaxel-based drugs have been used in chemotherapy to treat various cancers, including breast, lung, ovarian, and sarcoma [2,5]. There are two approaches to achieve the supply of paclitaxel, chemical (semi-) synthesis [6,7] and plant cell cultivation, of which the latter is more popular due to the economic viability of cell culture [8]. Despite these commercial advances, the tools to improve paclitaxel bio-production are limited. These limitations hamper the cost-effective supply of paclitaxel through a biotechnological means [10]
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