“…The higher oxygenated taxanes production compared to self-sufficient strains also denotes that potentially the uncoupling promoted the formation of the oxygenated taxanes. Hence, it is possible that besides the molecular oxygen, the formation of oxygenated taxanes by CYP725A4 was also mediated by other reaction intermediates which acted as the terminal electron and oxygen acceptor(s) [ 47 ]. On this matter, the 1.73-fold improved taxadiene titre in BN6 and 3.9-fold in BN5 compared to LRS5 might be due to activation of oxidative stress response [ 31 ] by uncoupling, and as a measure to combat the GGPP stress [ 48 ].…”
Section: Resultsmentioning
confidence: 99%
“…Therefore, at least at 5 mM level, the 1.4-fold decrease in total oxygenated taxanes production (16.7 ± 4.2 mg/L) could be a result of potentially poor enzymatic performance and lower substrate conversion [ 49 ]. The presence of uncoupling events might support the activity of Taxus CYP725A4 and POR interaction in yeast as in Kells et al [ 71 ] study, where the resulting reactive metabolites potentially act as the oxygen and electron donors to the P450 enzyme in addition to the molecular oxygen [ 47 ]. Fig.…”
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 higher oxygenated taxanes production compared to self-sufficient strains also denotes that potentially the uncoupling promoted the formation of the oxygenated taxanes. Hence, it is possible that besides the molecular oxygen, the formation of oxygenated taxanes by CYP725A4 was also mediated by other reaction intermediates which acted as the terminal electron and oxygen acceptor(s) [ 47 ]. On this matter, the 1.73-fold improved taxadiene titre in BN6 and 3.9-fold in BN5 compared to LRS5 might be due to activation of oxidative stress response [ 31 ] by uncoupling, and as a measure to combat the GGPP stress [ 48 ].…”
Section: Resultsmentioning
confidence: 99%
“…Therefore, at least at 5 mM level, the 1.4-fold decrease in total oxygenated taxanes production (16.7 ± 4.2 mg/L) could be a result of potentially poor enzymatic performance and lower substrate conversion [ 49 ]. The presence of uncoupling events might support the activity of Taxus CYP725A4 and POR interaction in yeast as in Kells et al [ 71 ] study, where the resulting reactive metabolites potentially act as the oxygen and electron donors to the P450 enzyme in addition to the molecular oxygen [ 47 ]. Fig.…”
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.
“…Therefore, at least at 5 mM level, the 1.4-fold decrease in total oxygenated taxanes production (16.7 ± 4.2 mg/L) could be a result of potentially poor enzymatic performance and lower substrate conversion (47). The presence of uncoupling events might support the activity of Taxus CYP725A4 and POR interaction in yeast as in Kells et al (69) study, where the resulting reactive metabolites potentially act as the oxygen and electron donors to the P450 enzyme in addition to the molecular oxygen (45).…”
Section: De Nitive Screening Design Interpretationmentioning
confidence: 86%
“…The higher oxygenated taxanes production compared to self-su cient strains also denotes that potentially the uncoupling promoted the formation of the oxygenated taxanes. Hence, it is possible that besides the molecular oxygen, the formation of oxygenated taxanes by CYP725A4 was also mediated by other reaction intermediates which acted as the terminal electron and oxygen acceptor(s) (45). On this matter, the 1.73-fold improved taxadiene titre in BN6 and 3.9-fold in BN5 compared to LRS5 might be due to activation of oxidative stress response (31) by uncoupling, and as a measure to combat the GGPP stress (46).…”
Section: Tandem Expression Of Taxus Cyp725a4 and Por And The Effect O...mentioning
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.
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.
“…He et al discussed recent progress in the identification and engineering of novel GTs for synthesis of plant natural products [ 4 ]. Cytochrome P450 enzymes (CYPs) catalyze a series of C–H and C C oxygenation reactions for biosynthesis of desired chemicals or pharmaceutical intermediates, a review article by Yan et al provided a comprehensive overview of CYP function for the C–H and C C oxygenation reactions and also various strategies for achieving higher selectivity and enzymatic activity [ 5 ]. Vitreoscilla hemoglobin (VHB) has been widely used to enhance cellular oxygen transfer and metabolite synthesis in fermentation.…”
Section: Enzyme Characterization and Engineeringmentioning
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