Optimising the biosynthesis of oxygenated and acetylated Taxol
precursors in Saccharomyces cerevisiae using advanced bioprocessing
strategies
Abstract
Taxadien-5α-hydroxylase and taxadien-5α-ol O-acetyltransferase catalyse
the oxidation of taxadiene to taxadien-5α-ol and subsequent acetylation
to taxadien-5α-yl-acetate in the biosynthesis of the blockbuster
anti-cancer drug, paclitaxel (Taxol). 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
optimisation of the early pathway in Saccharomyces cerevisiae, across a
range of bioreactor scales. High-throughput microscale optimisation
enhanced total oxygenated taxane titre to 39.0±5.7 mg/L and total taxane
product titres were comparable at micro and mini-bioreactor 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-5α-ol isomer titre to 19.2 mg/L,
comparable to the 23.8±3.7 mg/L achieved at microscale. A combination of
bioprocess optimisation and increased GC-MS resolution at 1L bioreactor
scale facilitated taxadien-5α-yl-acetate detection with a final titre of
3.7 mg/L. Total oxygenated taxane titres were improved 2.7-fold at this
scale to 78 mg/L, the highest reported titre 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.