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

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.