Design of microbial cell factories using integrated synthetic biology and systems biology for the production of Taxol®, an anticancer drug

The cytochrome P450 enzymes play indispensable roles in the biosynthesis of natural products. The industrially compatible development of these biocatalysts relies on understanding their functionalities in the context of their basic stoichiometric needs at both cellular and enzymatic levels. Following a holistic approach, this work aimed to improve the production of early precursors to the polycyclic diterpenoid anticancer drug, paclitaxel (Taxol®), in Saccharomyces cerevisiae. This included enhancing the activity of the early enzymatic steps, catalysed by taxadiene synthase and the cytochrome P450 CYP725A4. Herein, in the 1st chapter, Taxol® biosynthesis pathway and the major studies for the heterologous production of its early precursors were described. Then, the equal importance of the cytochrome P450 enzyme and redox homeostasis in terpenoid biosynthesis were highlighted. Ensuring about the substrate sufficiency is the first key step for optimisation of any enzymatic protocol. Therefore, in the 3rd chapter, a yeast strain expressing three copies of taxadiene synthase with protein solubility tags was developed, with which the taxadiene synthase products and their titres were characterised at different cultivation scales. Eventually, an overall 6.5-fold improvement in taxadiene titre was obtained compared to the previous works, and taxadiene concentration was found to be tightly coupled to biomass accumulation. Among the other obstacles for Taxol® heterologous biosynthesis is the higher selectivity of CYP725A4 towards the side diterpenoids formation in addition to its overall low product titre. As the sufficiency of taxadiene was established in the previous chapter, in the 4th chapter, the steps taken for optimising the activity of Taxus sp. CYP725A4 enzyme and its cognate reductase were described. The use of resting cell assays and different growth media showed that the yeast endogenous reactions do not affect the product distributions, and the diterpenoids production and biomass accumulation are not tightly coupled. Moreover, it was suggested that the side diterpenoids might play intermediary roles in the later steps of the Taxol® pathway. To increase the CYP725A4 and reductase expression, the effect of the cofactors of the prosthetic groups of these two enzymes, being irons and flavins, were evaluated through an augmented definitive screen design and regression modelling. The same flavins and irons were found significant for all main and side diterpenoids, where flavin adenine dinucleotide was found to promote the diterpenoids titres. To optimise the electron transfer, several fusion proteins were constructed with prokaryotic cytochrome P450 reductases with superior electron coupling capacity, which together with the biochemical assays, showed that the electron uncoupling likely promotes the diterpenoids formation which also results in the substrate accumulation. However, as the increased expression of the P450-reductase genes and diterpenoids production led to oxidative stress, any attempt on increasing the uncoupling rate, which produces stress at enzymatic level, could be diminishing to the cell. Therefore, in the 5th chapter, the procedure for evolving robust, oxidative stress-resistant taxadiene-producing yeast cell factories was described. To simulate the diterpenoids production, the oxidative stress was re-introduced into the continuous cultures of the non-evolved and the best evolved strain with three-fold higher taxadiene titre. The enrichment analyses of the differential gene expression and metabolic flux profiles revealed the strong contributions of the antioxidant machinery, respiration capacity and cross-protection mechanism in the evolved strain. This led to reduced overflow metabolism, besides improving the cofactor availability upon oxidative stress re-induction in the evolved cell culture. Such results indicate its greater fitness for increased diterpenoids production that also likely eliminates the need for the costly exogenous cofactors or further metabolic engineering upon constructing an optimal diterpenoid-producing strain. By combining the classic and modern strain engineering with biological modelling, this study initiated a new paradigm in Taxol® microbial biosynthesis. The findings can benefit the cytochrome P450-dominated terpenoid biosynthesis pathways including that of Taxol®