Enhanced production of taxadiene in Saccharomyces cerevisiae
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Microbial Cell Factories Open Access
RESEARCH
Enhanced production of taxadiene in Saccharomyces cerevisiae 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 and Leonardo Rios‑Solis1,2*
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 taxa‑ diene 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 6.5fold compared to the highest literature titre in S. cerevisiae cell factories. Keywords: Taxadiene synthase, Saccharomyces cerevisiae, Paclitaxel, Taxol™, Yeast metabolic engineering, Minibioreactor Background The highly complex diterpenoid drug Paclitaxel (Taxol™) first gained FDA approval in 1992 for the treatment of ovarian cancer and has since proven efficacious against a wide range of additional diseases [1]. Direct extraction from its natural source, the bark of Pacific yew (Taxus brevifolia), is both destructive and extremely low-yielding. As a result, paclitaxel is currently produced predominantly by semi-synthesis, involving the *Correspondence: [email protected] † Behnaz Nowrouzi, Rachel A. Li and Laura E. Walls contributed equally. 1 Institute for Bioengineering, School of Engineering, The University of Edinburgh, Edinburgh EH9 3BF, United Kingdom Full list of author information is available at the end of the article
chemical modification of late precursors extracted from plant cell culture. However, as such methods are high in cost and have limited scalability, the development of a more sustainable source is critical to meet growing global demands [2]. One potential solution involves the heterologous expression of the biosynthetic pathway in microbial cell factories. The first committed step in the paclitaxel biosynthetic pathway is the cyclisation of the diterpenoid intermediate, geranylgeranyl
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