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Optimising the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies
  • +9
  • 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

Corresponding Author:[email protected]

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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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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.
09 Jun 2020Submitted to Biotechnology and Bioengineering
09 Jun 2020Submission Checks Completed
09 Jun 2020Assigned to Editor
14 Jun 2020Reviewer(s) Assigned
11 Jul 2020Review(s) Completed, Editorial Evaluation Pending
11 Jul 2020Editorial Decision: Revise Major
27 Aug 20201st Revision Received
27 Aug 2020Submission Checks Completed
27 Aug 2020Assigned to Editor
28 Aug 2020Reviewer(s) Assigned
12 Sep 2020Review(s) Completed, Editorial Evaluation Pending
12 Sep 2020Editorial Decision: Accept
16 Sep 2020Published in Biotechnology and Bioengineering. 10.1002/bit.27569