Optimising the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies

Laura E. Walls, Koray Malci, Behnaz Nowrouzi, Rachel Li, Leo d'Espaux, Jeff Wong, Jonathan A. Dennis, Andrea J C Semiao, Stephen Wallace, José L. Martinez, Jay D. Keasling, Leonardo Rios-Solis*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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. This article is protected by copyright. All rights reserved.
Original languageEnglish
JournalBiotechnology and Bioengineering
ISSN0006-3592
DOIs
Publication statusAccepted/In press - 2020

Keywords

  • S. cerevisiae
  • Taxadien-5α-hydroxylase
  • High throughput microbioreactor
  • Taxol
  • Taxadien-5-alpha-ol O-acetyltransferase

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