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Developing a Yeast Platform Strain for an Enhanced Taxadiene Biosynthesis by CRISPR/Cas9
Joseph C Utomo1, Fabio C Chaves2, Philippe Bauchart3
1Department of Biological Science, University of Calgary, Calgary, AB T2N1N4, Canada.
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.
Insights
We engineered yeast to produce taxadiene, a precursor to the anticancer drug paclitaxel. This yeast platform facilitates future research into microbial paclitaxel biosynthesis and production.
Area of Science:
- Biotechnology
- Synthetic Biology
- Metabolic Engineering
Background:
- Paclitaxel is a vital anticancer diterpenoid, but its biosynthesis is not fully understood.
- Challenges in plant transformation and precursor scarcity hinder paclitaxel production.
- Yeast Saccharomyces cerevisiae offers a promising platform for elucidating and producing paclitaxel precursors.
Purpose of the Study:
- To develop a yeast platform for taxadiene production.
- To identify optimal geranylgeranyl pyrophosphate (GGPP) synthases (GGPPS) for enhanced taxadiene yield.
- To optimize the terpenoid pathway flux and integrate genes using CRISPR/Cas9 for a stable production strain.
Main Methods:
- Screening of various GGPPS to identify the most effective enzyme for taxadiene production.
- Metabolic engineering strategies to increase flux towards the terpenoid pathway.
- CRISPR/Cas9 gene integration to create a marker-free yeast strain.
Main Results:
- Achieved a taxadiene titer of 2.02 ± 0.40 mg/L using a plasmid-based system.
- Obtained a taxadiene titer of 0.41 ± 0.06 mg/L with integrated genes in the yeast genome.
- Successfully developed a robust yeast platform for microbial paclitaxel biosynthesis research.
Conclusions:
- The engineered yeast strain serves as a valuable platform for future studies on paclitaxel biosynthesis.
- This work advances biotechnological approaches for producing complex natural products like paclitaxel.
- The developed platform enables efficient screening of gene candidates for microbial paclitaxel production.
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