Biotechnology and Bioengineering, Vol.118, No.1, 279-293, 2021
Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors inSaccharomyces cerevisiaeusing advanced bioprocessing strategies
Taxadien-5 alpha-hydroxylase and taxadien-5 alpha-olO-acetyltransferase catalyze the oxidation of taxadiene to taxadien-5 alpha-ol and subsequent acetylation to taxadien-5 alpha-yl-acetate in the biosynthesis of the blockbuster anticancer drug, paclitaxel (Taxol (R)). Despite decades of research, the promiscuous and multispecific CYP725A4 enzyme remains a major bottleneck in microbial biosynthetic pathway development. In this study, an interdisciplinary approach was applied for the construction and optimization of the early pathway inSaccharomyces cerevisiae, across a range of bioreactor scales. High-throughput microscale optimization enhanced total oxygenated taxane titer to 39.0 +/- 5.7 mg/L and total taxane product titers were comparable at micro and minibioreactor scale at 95.4 +/- 18.0 and 98.9 mg/L, respectively. The introduction of pH control successfully mitigated a reduction of oxygenated taxane production, enhancing the potential taxadien-5 alpha-ol isomer titer to 19.2 mg/L, comparable with the 23.8 +/- 3.7 mg/L achieved at microscale. A combination of bioprocess optimization and increased gas chromatography-mass spectrometry resolution at 1 L bioreactor scale facilitated taxadien-5 alpha-yl-acetate detection with a final titer of 3.7 mg/L. Total oxygenated taxane titers were improved 2.7-fold at this scale to 78 mg/L, the highest reported titer in yeast. Critical parameters affecting the productivity of the engineered strain were identified across a range of scales, providing a foundation for the development of robust integrated bioprocess control systems.