Developing a Yeast Platform Strain for an Enhanced Taxadiene Biosynthesis by CRISPR/Cas9.

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, , as a platform to elucidate the paclitaxel biosynthesis. As taxadiene is a diterpenoid, its common precursor, geranylgeranyl pyrophosphate (), needs to be increased in yeast. In this study, we screened various synthases () to find the most suitable 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.