Behnaz Nowrouzi1,2, Rachel A Li3,4, Laura E Walls1,2, Leo d'Espaux3,4, Koray Malcı1,2, Lungang Liang1,2, Nestor Jonguitud-Borrego1,2, Albert I Lerma-Escalera5, Jose R Morones-Ramirez5, Jay D Keasling3,4,6,7,8, Leonardo Rios-Solis9,10. 1. Institute for Bioengineering, School of Engineering, The University of Edinburgh, Edinburgh, EH9 3BF, United Kingdom. 2. Centre for Synthetic and Systems Biology (SynthSys), The University of Edinburgh, Edinburgh, EH9 3BD, United Kingdom. 3. DOE Joint BioEnergy Institute, Emeryville, CA, 94608, USA. 4. Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA. 5. Centro de Investigación en Biotecnología y Nanotecnología, Facultad de Ciencias Químicas, Universidad Autónoma de Nuevo León, Apodaca, Mexico. 6. Departments of Chemical & Biomolecular Engineering and of Bioengineering, University of California, Berkeley, Berkeley, CA, 94720, USA. 7. Center for Biosustainability, Danish Technical University, Lyngby, Denmark. 8. Center for Synthetic Biochemistry, Institute for Synthetic Biology, Shenzhen Institutes for Advanced Technologies, Shenzhen, China. 9. Institute for Bioengineering, School of Engineering, The University of Edinburgh, Edinburgh, EH9 3BF, United Kingdom. Leo.Rios@ed.ac.uk. 10. Centre for Synthetic and Systems Biology (SynthSys), The University of Edinburgh, Edinburgh, EH9 3BD, United Kingdom. Leo.Rios@ed.ac.uk.
Abstract
BACKGROUND: Cost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to relieve this bottleneck. RESULTS: Multi-copy chromosomal integration of TASY harbouring a selection of fusion solubility tags improved taxadiene titres 22-fold, up to 57 ± 3 mg/L at 30 °C at microscale, compared to expressing a single episomal copy of TASY. The scalability of the process was highlighted through achieving similar titres during scale up to 25 mL and 250 mL in shake flask and bioreactor cultivations, respectively at 20 and 30 °C. Maximum taxadiene titres of 129 ± 15 mg/L and 127 mg/L were achieved through shake flask and bioreactor cultivations, respectively, of the optimal strain at a reduced temperature of 20 °C. CONCLUSIONS: The results of this study highlight the benefit of employing a combination of molecular biology and bioprocess tools during synthetic pathway development, with which TASY activity was successfully improved by 6.5-fold compared to the highest literature titre in S. cerevisiae cell factories.
BACKGROUND: Cost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to relieve this bottleneck. RESULTS: Multi-copy chromosomal integration of TASY harbouring a selection of fusion solubility tags improved taxadiene titres 22-fold, up to 57 ± 3 mg/L at 30 °C at microscale, compared to expressing a single episomal copy of TASY. The scalability of the process was highlighted through achieving similar titres during scale up to 25 mL and 250 mL in shake flask and bioreactor cultivations, respectively at 20 and 30 °C. Maximumtaxadiene titres of 129 ± 15 mg/L and 127 mg/L were achieved through shake flask and bioreactor cultivations, respectively, of the optimal strain at a reduced temperature of 20 °C. CONCLUSIONS: The results of this study highlight the benefit of employing a combination of molecular biology and bioprocess tools during synthetic pathway development, with which TASY activity was successfully improved by 6.5-fold compared to the highest literature titre in S. cerevisiae cell factories.
Authors: James Kirby; Minobu Nishimoto; J Genevieve Park; Sydnor T Withers; Farnaz Nowroozi; Dominik Behrendt; Elizabeth J Garcia Rutledge; Jeffrey L Fortman; Holly E Johnson; James V Anderson; Jay D Keasling Journal: Phytochemistry Date: 2010-06-30 Impact factor: 4.072
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