Exploring optimal Taxol® CYP725A4 activity in Saccharomyces cerevisiae

Abstract

Background CYP725A4 catalyses the conversion of the first Taxol® precursor, taxadiene, to taxadiene-5α-ol (T5α-ol) and a range of other mono- and di-hydroxylated side products (oxygenated taxanes). Initially known to undergo a radical rebound mechanism, the recent studies have revealed that an intermediate epoxide mediates the formation of the main characterised products of the enzyme, being T5α-ol, 5(12)-oxa-3(11)-cyclotaxane (OCT) and its isomer, 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) as well as taxadienediols. Besides the high side product: main product ratio and the low main product titre, CYP725A4 is also known for its slow enzymatic activity, massively hindering further progress in heterologous production of Taxol® precursors. Therefore, this study aimed to systematically explore the key parameters for improving the regioselectivity and activity of eukaryotic CYP725A4 enzyme in a whole-cell eukaryotic biocatalyst, Saccharomyces cerevisiae. Results Investigating the impact of CYP725A4 and reductase gene dosages along with construction of self-sufficient proteins with strong prokaryotic reductases showed that a potential uncoupling event accelerates the formation of oxygenated taxane products of this enzyme, particularly the side products OCT and iso-OCT. Due to the harmful effect of uncoupling products and the reactive metabolites on the enzyme, the impact of flavins and irons, existing as prosthetic groups in CYP725A4 and reductase, were examined in both their precursor and ready forms, and to investigate the changes in product distribution. We observed that the flavin adenine dinucleotide improved the diterpenoids titres and biomass accumulation. Hemin was found to decrease the titre of iso-OCT and T5α-ol, without impacting the side product OCT, suggesting the latter being the major product of CYP725A4. The interaction between this iron and the iron precursor, δ-Aminolevulinic acid, seemed to improve the production of these diterpenoids, further denoting that iso-OCT and T5α-ol were the later products. While no direct correlation between cellular-level oxidative stress and oxygenated taxanes was observed, investigating the impact of salt and antioxidant on CYP725A4 further showed the significant drop in OCT titre, highlighting the possibility of enzymatic-level uncoupling event and reactivity as the major mechanism behind the enzyme activity. To characterise the product spectrum and production capacity of CYP725A4 in the absence of cell growth, resting cell assays with optimal neutral pH revealed an array of novel diterpenoids along with higher quantities of characterised diterpenoids and independence of the oxygenated product spectra from the acidity effect. Besides reporting on the full product ranges of CYP725A4 in yeast for the first time, the highest total taxanes of around 361.4 ± 52.4 mg/L including 38.1 ± 8.4 mg/L of T5α-ol was produced herein at a small, 10-mL scale by resting cell assay, where the formation of some novel diterpenoids relied on the prior existence of other diterpenes/diterpenoids as shown by statistical analyses. Conclusions This study shows how rational strain engineering combined with an efficient design of experiment approach systematically uncovered the promoting effect of uncoupling for optimising the formation of the early oxygenated taxane precursors of Taxol®. The provided strategies can effectively accelerate the design of more efficient Taxol®-producing yeast strains.

The synergetic effect from the combination of different adsorption resins in batch and semi‐continuous cultivations of S. Cerevisiae cell factories to produce acetylated Taxanes precursors of the anticancer drug Taxol

“…However, beyond total taxane recovery, it can be seen that the total oxygenated ratio [oxygenated taxanes (mg/L)/nonoxygenated taxanes (mg/L)] in every single treatment was lower (around 0.9) compared with the results given in treatment 9 which was 1.2 (Figure S10 from the Supporting Information Material and Figure 2b). This result is in agreement with previous studies focusing on the P450 for the synthesis of Taxadiene-5α-ol, highlighting that careful tuning is required to optimize the oxygenated ratio of taxanes Nowrouzi et al, 2022).…”

The synergetic effect from the combination of different adsorption resins in batch and semi-continuous cultivations ofS. cerevisiaecell factories to produce acetylated Taxanes precursors of the anticancer drug Taxol

“…However, beyond total taxane recovery, it can be seen that the oxygenated ratio in every single treatment was lower compared with the results given in treatment 9 (Figure S10 from the supplementary material). This result is in agreement with previous studies focusing on the P450 for the synthesis of Taxadiene-5α-ol, highlighting that careful tuning is required to optimize the oxygenated ratio of taxanes (Nowrouzi et al, 2022; Nowrouzi & Rios-Solis, 2021).…”

“…Besides this, the limitations of the dodecane overlay method (such as the evaporation of dodecane during cultivation) could make it harder for GC-MS detection. In previous studies using similar yeast strains for the recovery of taxanes and a dodecane overlay method, only 8 (Walls et al, 2021) and 7 (Nowrouzi et al, 2022) taxane compounds were detected in comparison to the 13 taxane compounds found in treatment 9. This highlights the significant benefits of this method for research focusing on the screening and discovery of new natural products.…”

Reconstitution of Early Paclitaxel Biosynthetic Network

“…The drug stabilizes microtubules and in turn blocks cell cycle progression. 12 It was first discovered from the Pacific yew tree ( Taxus brevifolia ) through an anti-cancer screening program conducted by National Cancer Institute (NCI) in 1962. 3 Paclitaxel belongs to a class of diterpenoids from Taxus called taxanes, of which more than 500 structures have been reported.…”

“…However, the observed catalytic promiscuity of T5αH on heterologous expression has been a bottleneck in paclitaxel biosynthetic pathway reconstitution for nearly two decades. Expression of T5αH in different heterologous systems, such as Escherichia coli [9][10][11] , Saccharomyces cerevisiae 12,13 and Nicotiana benthamiana 14 ( Supplementary Table 1 ), results in multiple oxidized taxadiene products. While up to 12 products have been reported, 10 only three have been structurally characterized, namely, taxadien-5α-ol ( 2 ), rearranged products 5 (12)-oxa-3 (11)-cyclotaxane (OCT, 3 ) 15 and 5 (11)-oxa-3 (11)-cyclotaxane (iso-OCT, 4 ) 16 .…”