| 1. |
- Malcı, Koray, et al.
(författare)
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Improved production of Taxol ® precursors in S. cerevisiae using combinatorial in silico design and metabolic engineering
- 2023
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Ingår i: Microbial Cell Factories. - 1475-2859. ; 22:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Integrated metabolic engineering approaches that combine system and synthetic biology tools enable the efficient design of microbial cell factories for synthesizing high-value products. In this study, we utilized in silico design algorithms on the yeast genome-scale model to predict genomic modifications that could enhance the production of early-step Taxol® in engineered Saccharomyces cerevisiae cells. Results: Using constraint-based reconstruction and analysis (COBRA) methods, we narrowed down the solution set of genomic modification candidates. We screened 17 genomic modifications, including nine gene deletions and eight gene overexpressions, through wet-lab studies to determine their impact on taxadiene production, the first metabolite in the Taxol® biosynthetic pathway. Under different cultivation conditions, most single genomic modifications resulted in increased taxadiene production. The strain named KM32, which contained four overexpressed genes (ILV2, TRR1, ADE13, and ECM31) involved in branched-chain amino acid biosynthesis, the thioredoxin system, de novo purine synthesis, and the pantothenate pathway, respectively, exhibited the best performance. KM32 achieved a 50% increase in taxadiene production, reaching 215 mg/L. Furthermore, KM32 produced the highest reported yields of taxa-4(20),11-dien-5α-ol (T5α-ol) at 43.65 mg/L and taxa-4(20),11-dien-5-α-yl acetate (T5αAc) at 26.2 mg/L among early-step Taxol® metabolites in S. cerevisiae. Conclusions: This study highlights the effectiveness of computational and integrated approaches in identifying promising genomic modifications that can enhance the performance of yeast cell factories. By employing in silico design algorithms and wet-lab screening, we successfully improved taxadiene production in engineered S. cerevisiae strains. The best-performing strain, KM32, achieved substantial increases in taxadiene as well as production of T5α-ol and T5αAc. These findings emphasize the importance of using systematic and integrated strategies to develop efficient yeast cell factories, providing potential implications for the industrial production of high-value isoprenoids like Taxol®.
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| 2. |
- Nowrouzi, Behnaz, et al.
(författare)
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Rewiring Saccharomyces cerevisiae metabolism for optimised Taxol® precursors production
- 2024
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Ingår i: Metabolic Engineering Communications. - 2214-0301. ; 18
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Tidskriftsartikel (refereegranskat)abstract
- Saccharomyces cerevisiae has been conveniently used to produce Taxol® anticancer drug early precursors. However, the harmful impact of oxidative stress by the first cytochrome P450-reductase enzymes (CYP725A4-POR) of Taxol® pathway has hampered sufficient progress in yeast. Here, we evolved an oxidative stress-resistant yeast strain with three-fold higher titre of their substrate, taxadiene. The performance of the evolved and parent strains were then evaluated in galactose-limited chemostats before and under the oxidative stress by an oxidising agent. The interaction of evolution and oxidative stress was comprehensively evaluated through transcriptomics and metabolite profiles integration in yeast enzyme-constrained genome scale model. Overall, the evolved strain showed improved respiration, reduced overflow metabolites production and oxidative stress re-induction tolerance. The cross-protection mechanism also potentially contributed to better heme, flavin and NADPH availability, essential for CYP725A4 and POR optimal activity in yeast. The results imply that the evolved strain is a robust cell factory for future efforts towards Taxol© production.
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