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Optimising the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies
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  • Laura Walls,
  • Koray Malci,
  • Behnaz Nowrouzi,
  • Rachel Li,
  • Leopold d'Espaux,
  • Jeff Wong,
  • Jonathan Dennis,
  • Andrea Semiao,
  • Stephen Wallace,
  • José Martinez,
  • Jay Keasling,
  • Leonardo Rios-Solis
Laura Walls
The University of Edinburgh Institute for Bioengineering
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Koray Malci
The University of Edinburgh Institute for Bioengineering
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Behnaz Nowrouzi
The University of Edinburgh Institute for Bioengineering
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Rachel Li
Joint BioEnergy Institute
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Leopold d'Espaux
Joint BioEnergy Institute
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Jeff Wong
Joint BioEnergy Institute
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Jonathan Dennis
Centre for Synthetic and Systems Biology (SynthSys)
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Andrea Semiao
The University of Edinburgh School of Engineering Institute for Infrastructure and Environment
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Stephen Wallace
Centre for Synthetic and Systems Biology (SynthSys)
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José Martinez
Technical University of Denmark
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Jay Keasling
Joint BioEnergy Institute
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Leonardo Rios-Solis
The University of Edinburgh Institute for Bioengineering
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Peer review status:UNDER REVIEW

09 Jun 2020Submitted to Biotechnology and Bioengineering
09 Jun 2020Assigned to Editor
09 Jun 2020Submission Checks Completed
14 Jun 2020Reviewer(s) Assigned
11 Jul 2020Editorial Decision: Revise Major
11 Jul 2020Review(s) Completed, Editorial Evaluation Pending

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.