Orthogonal Assays Clarify the Oxidative Biochemistry of Taxol P450

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Orthogonal assays clarify the oxidative biochemistry of Taxol P450 CYP725A4 Bradley Walters Biggs, John Edward Rouck, Amogh Kambalyal, William Arnold, Chin-Giaw Lim, Marjan de Mey, Mark O'Neil-Johnson, Courtney M. Starks, Aditi Das, and Parayil Kumaran Ajikumar ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00968 • Publication Date (Web): 01 Mar 2016 Downloaded from http://pubs.acs.org on March 6, 2016

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ACS Chemical Biology

Orthogonal assays clarify the oxidative biochemistry of Taxol P450 CYP725A4 Bradley Walters Biggs1,2,†, John Edward Rouck3,†, Amogh Kambalyal3, William Arnold3, Chin Giaw Lim1, Marjan De Mey1,4, Mark O’Neil-Johnson5, Courtney M. Starks5, *Aditi Das3, and *Parayil Kumaran Ajikumar1 1

Manus Biosynthesis, 1030 Massachusetts Avenue, Suite 300, Cambridge, MA 02138

2

Department of Chemical and Biological Engineering (Masters in Biotechnology Program), Northwestern University, Evanston, IL 60208 3

Department of Comparative Biosciences, Department of Biochemistry, Department of Bioengineering, Beckman Institute for Advanced Science and Engineering, University of Illinois Urbana-Champaign, Urbana, Illinois 61801

4

Centre for Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, B-9000, Belgium

5

Sequoia Sciences, 1912 Innerbelt Business Center Dr., Saint Louis, MO 63114

1 ACS Paragon Plus Environment


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Abstract Natural product metabolic engineering potentially offers sustainable and affordable access to numerous valuable molecules. However, challenges in characterizing and assembling complex biosynthetic pathways have prevented more rapid progress in this field. The anticancer agent Taxol represents an excellent case study. Assembly of a biosynthetic pathway for Taxol has long been stalled at its first functionalization, putatively an oxygenation performed by the cytochrome P450 CYP725A4, due to confounding characterizations. Here, through combined in vivo (Escherichia coli), in vitro (lipid nanodisc), and metabolite stability assays, we verify the presence and likely cause of this enzyme’s inherent promiscuity. Thereby, we remove the possibility that promiscuity simply existed as an artifact of previous metabolic engineering approaches. Further, spontaneous rearrangement and the stabilizing effect of a hydrophobic overlay suggest a potential role for non-enzymatic chemistry in Taxol’s biosynthesis. Taken together, this work confirms taxadiene-5α-ol as a primary enzymatic product of CYP725A4 and provides direction for future Taxol metabolic and protein engineering efforts.


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Plant specialized metabolisms are rich with chemically diverse and structurally complex molecules that hold value as pharmaceuticals and otherwise.1,2 Unfortunately, low accumulation in slow growing hosts often prevents traditional extraction methods from providing sufficient access to these compounds.3 Even more, the structural complexity of many plant natural products (PNPs) renders their chemical synthesis, if achievable, economically infeasible.4 As an alternative approach, heterologous biological production in quick growing and fermentable microbes such as Escherichia coli or Saccharomyces cerevisiae has been proposed.5,6 Though promising, this approach fundamentally requires the identification of a biochemical pathway for a molecule of interest, which can present a tremendous challenge.7 Such has been the case for the anticancer agent Taxol. Even after decades of work,8 characterization of Taxol’s biosynthetic pathway is incomplete, and efforts towards its heterologous biological synthesis remain in their initial stages (for a history of Taxol’s landmarks, please reference Figure S1). A major cause for this delay has been the confounding catalytic characterizations of the cytochrome P450 (P450) CYP725A4, Taxol’s putative first tailoring enzyme. Nearly twenty years ago Croteau and coworkers described CYP725A4 to perform the C-5 hydroxylation and concomitant allylic rearrangement of taxadiene as the first of several functionalizations of Taxol’s diterpene scaffold (Figure 1a).9,10 This ordering was determined according to the relative abundance of these structural features in subsequent, more decorated taxoids, and was confirmed by assimilation of labelled taxadiene-5α-ol (T-5α-ol) into downstream products upon feeding to Taxus cell cultures.9,10 Subsequent work in separate laboratory, however, claimed that CYP725A4 converts taxadiene instead to oxa-cyclotaxane (OCT) (Figure 1c).11 Later, upon our own studies, this enzyme was found to produce both T-5α-ol and OCT when incorporated into a taxadiene-overproducing E. coli strain.12 Even more, optimizations of this P450-containing strain have revealed additional oxygenated taxane species (Figure 1b, m/z 288). This apparent promiscuity was unexpected in light of these previous studies, uncharacteristic of discussions of P450 use in terpene biocatalysis,13 and undesirable with respect to productively channeling carbon flux to downstream Taxol metabolites. In addition, it was unclear if the observed promiscuity was inherent to this enzyme or an artifact of a particular assay background. Accordingly, further examination of CYP725A4’s biochemistry was warranted before continuing to engineer Taxol’s pathway. Characterizing plant P450s is challenging, however, and presents several technical hurdles. First, P450s are often intransigent to heterologous expression,14 requiring significant N-terminal modification.15 In addition, they require an electron donating cytochrome P450 reductase partner (CPR)16 and lipid bilayer17 for functional activity. This has precluded the development of a universal, robust, and facile platform for P450 screening. Instead, historical characterizations have predominantly been limited to native and heterologous in vivo assays and crude in vitro microsomal preparations, often with the inclusion of a heterologous reductase partner. Such assays allow complex and potentially nonnative biochemical interaction, which may alter enzyme performance in a context-dependent manner.18 We hypothesized such phenomena could have influenced the findings of previous characterizations of CYP725A4. Therefore, we sought to employ a purified in vitro assay to circumvent such issues and to probe this enzyme’s inherent catalytic properties. To this end, a handful of platforms have been developed,19,20 with lipid nanodiscs showing particular promise. Nanodisc technology has been optimized and used to analyze several mammalian P450s and one plant P450.21,22 Accordingly, herein we have paired the self-assembling, amphipathic scaffold protein-belted nanodisc assay with our previously developed E. coli metabolic engineering platform12 to examine CYP725A4.

RESULTS AND DISCUSSION



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In vivo characterization of CYP725A4. We began with fermentations of the previously described CYP725A4-containing E. coli strain,12 carried out in the presence of a dodecane overlay to capture hydrophobic terpene products. GC-MS analysis of the overlay shows both singly (m/z 288) and potentially doubly oxygenated (m/z 304) taxanes, with four monooxygenated species representing the majority of the total fermentation product (Figure 1b, Figures S2-7). Comparing with previous reports,9,11 two products possess mass spectra (MS) matching those for the validated structures of T-5α-ol and OCT (Figure 1, Figures S2-4). Two major products, however, remain unidentified, and so successive shake flask fermentations were carried out in order to generate material for further characterization. Purification efforts successfully isolated three of the four major oxygenated species, indicated by matched GC-MS identity before and after isolation from dodecane (Figures S2-5). Once isolated, T-5αol’s structure was reconfirmed with 2D NMR (Table S1). In addition, a previously uncharacterized oxygenated taxane, hereafter referred to as iso-oxa-cyclotaxane or “iso-OCT,” was successfully isolated and its structure was also determined by 2D NMR (Figure 1c, Table S1). OCT’s NMR has been previously reported,11 however in our hands this compound was insufficiently stable to obtain NMR, as described below. Combining NMR and GC-MS characterization, three of the four major oxygenated products were identified (Figure 1). A novel oxygenated taxane identified from E. coli fermentations with hydrophobic overlays. Interestingly, after multiple purification attempts, the final major oxygenated taxane species, hereafter referred to as UOTX (unstable, structurally unsolved oxygenated taxane), could not be isolated, as no fractions possessed a compound with matched retention time (Figure 2a). Instead, compounds with previously unobserved retention times emerge (Figure 2a, Figures S8-11), potentially indicating that UOTX degrades or isomerizes during purification. To evaluate if UOTX requires a hydrophobic overlay as a protecting environment, additional fermentations were completed without an overlay and with a vegetable oil used in substitute for dodecane. UOTX is only observed in the presence of an overlay (Figure 2b, Figures S12-13), indicating that an overlay is required to stably sequester and detect this metabolite from aqueous media. This result concerts with a recent work, wherein solvent extraction was also found to alter a natural product metabolite.23 Although its instability prevents structural solution, UOTX’s MS has been determined (Figure 2c). This MS bears similarity to that of T-5α-ol (Figure S3), which may indicate that UOTX represents a similar single oxygenation of a nearby carbon, such as C-20. The requirement of a hydrophobic overlay to detect UOTX suggests a possible role for a lipophilic environment in planta to sequester and shield this metabolite long enough for it to be catalyzed by a downstream enzyme. Regardless of a definitive role, this finding may interest plant biochemists who rarely employ hydrophobic overlays for enzymatic characterization. As seen here, their use in biochemical characterization may allow for additional unstable intermediate metabolite species to be observed. In vitro characterization of CYP725A4. We next studied the product distribution of CYP725A4 emanating from purified enzyme reconstituted in lipid nanodiscs. For these assays, additional considerations were taken into account. First, both linked and unlinked P450 constructions were used in order to eliminate protein fusion as the cause of promiscuity.24 Linked constructions constituted CYP725A4 fused to its native reductase partner.12 Unlinked assays were completed using both a CPR from Stevia rebaudiana, chosen from among three plant CPRs due to its favorable solubility, and a CPR from rat liver.25 Second, extraneous biochemical interactions were minimized by purifying each enzyme before its incorporation into the assay by adding a His-tag to chimeric and individual P450 proteins and taking advantage of CPR’s ability to be purified using ADP-agarose. This circumvented the need for crude microsomal preparations. Thus, the final nanodisc assays included only the nanodisc materials, P450, CPR, NADPH, and taxadiene (Figure 3a), and was characterized by UV-VIS to determine active protein concentration (Figure S14)


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Notably, linked and unlinked proteins assay show a nearly identical array of products by GC-MS (Figure 3b, Figures S15-21, Table S2). In comparison with the in vivo data, T-5α-ol and OCT are observed as consistent products. Interestingly, the ratio of OCT to T-5α-ol shifts from approximately 1:1 to favor the production of OCT when moving from the linked assay to the two unlinked assays. This could potentially be a function of a more transient CPR-P450 interaction for the unlinked constructions in vitro. Several oxygenated taxanes (m/z 288) previously not observed in the in vivo screening also appear (Figure 3b ND1-7, Figure S21). The minute yields and scaling restrictions of these systems prohibit NMR characterization, but the data indicate that P450 functionality does not vary between the linked and unlinked protein constructions, even when exchanging plant for mammalian CPR (Figure 3b, Figure S20). Accordingly, these findings provide continued confidence for a “share your parts” principle applied to selecting plant CPR with favorable properties such as solubility.16 In addition, they indicate that the dramatic catalytic variation observed during CPR fusion in another P450 assay24 was in some way unique. Lastly, these data demonstrate a cross-platform promiscuity for CYP725A4, while simultaneously confirming T-5α-ol as a consistent major product for both in vivo and in vitro assay systems. Metabolite instability and spontaneous rearrangement of OCT to iso-OCT. Motivated by the metabolite instability observed during purification, we probed the stability of the other major taxane species. To this end, isolated taxanes were subjected to a range of conditions (Table S3). Initial stability assays revealed no measureable degradation for taxadiene, T-5α-ol, or iso-OCT (Figures S22-24). However, OCT did show significant change (Figures S25-26). Whereas purified samples of OCT analyzed immediately after removal from -20°C show predominantly one peak, over time and at a range of temperatures, increasing amounts of iso-OCT are observed (Figure 4, Table S4). As this spontaneous rearrangement is irreversible, iso-OCT is concluded to be thermodynamically favored. Iso-OCT’s structure shows substantial bond rearrangement compared to OCT, and a scheme for this isomerization is proposed (Figure 4a). This observation of rearrangement is noteworthy, as iso-OCT was previously assumed to be a direct enzymatic product. Moreover, OCT’s instability rationalizes the difficulty in obtaining clean NMR for this compound. Further stability assays revealed T-5α-ol to be stable under all but acidic conditions (Figure S27). Acidic conditions appear to degrade the compound, potentially removing the hydroxyl group, as a previously unobserved diterpene (m/z 272) peak appears (Figures S27-30). However, none of T-5α-ol’s acid degradation products overlap with the major fermentation products. Hypothesizing that some oxygenated taxane products could be derived from a peroxide attack of one of taxadiene’s carboncarbon double bonds, with peroxide species stemming from an incomplete P450 catalytic cycle,26 we exposed taxadiene to hydrogen peroxide. Strikingly, this assay shows oxygenated products, with OCT representing a majority species, confirmed by MS spectra match (Figure 5, Figure S31-32). To ensure that the peroxide activity was a result of CYP725A4’s presence and not a system artifact, we reconfirmed that the taxadiene-overproducing E. coli strain without a P450 produces no oxygenated taxanes (Figure S33). Meaning, E. coli’s natively present reactive oxygen species are not sufficient to react with taxadiene to produce OCT. Negative controls for the nanodisc experiments similarly confirm that, without a P450 present, taxadiene does not react to form OCT in these systems (Figure S16). In addition, the aforementioned degradation assays show that taxadiene does not spontaneously form OCT under multiple other conditions (Figure S22). Therefore, we believe that the substrate bound P450, upon generation of a peroxide species, positions this specific reaction optimally. In addition, we would be careful to highlight that our proposed mechanism differs from H2O2-dependent peroxygenase generation of the reactive compound I.27 Resolving CYP725A4’s oxidative biochemistry. Confounding characterizations of CYP725A4 have long stalled Taxol biosynthesis efforts and raised doubts about this enzyme’s appropriateness for taxadiene’s first functionalization. In addition, CYP725A4’s observed promiscuity is somewhat 5 ACS Paragon Plus Environment


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unexpected. Though P450s are known to accept structurally diverse substrates and catalyze a variety of chemistries,28,29 including oxidation cascades and the highly multispecific activity of hepatic P450s on PNP pharmaceuticals,30,31 terpene biosynthetic P450s have infrequently been characterized to perform multiple distinct oxygenations on a single substrate,13 with limited examples.32,33 Thus, it was reasonable to question if CYP725A4’s observed promiscuity was inherent to the enzyme or introduced as an artifact of previous characterization assays, including our metabolic engineering platform. By observing CYP725A4’s promiscuity in the purified nanodisc in vitro assay, we can confirm that this behavior is inherent to the enzyme. It is possible that CYP725A4’s promiscuity is observed only now because of the improved P450 flux of our assay systems, wherein buried, physiologically-irrelevant, side activities have been made manifest.18 Metabolic engineering approaches have recently begun to encounter similar phenomena,14,34,35 especially with increasing oxygenation capacity.14 Such a finding would accord with a growing understanding that promiscuity is necessary for evolvability and plays a major role in PNP specialized metabolism.36–38 Yet, this explanation does not account for the absence of the promiscuous products OCT and iso-OCT in nature. If, however, we include our discovery that a peroxide chemistry forms OCT, understanding that peroxide species can result from incomplete P450 cycles, we gain a clearer picture of this enzyme and its future use. Elimination of the peroxide activity would mean narrowing the fermentation product profile, potentially to only one major compound if UOTX is also peroxide derived. CYP725A4 in the future. As OCT has not been observed in nature, it is possible that native protein-protein complexes, such as a metabolon,39 safely channel taxadiene and T-5α-ol to downstream metabolites and prevent peroxide interaction or other promiscuous catalytic functions. Accordingly, engineering a scaffold-like environment may help to heterologously recreate this effect.40 The inclusion of cytochrome b5 in such a complex may also assist in preventing incomplete P450 catalytic cycles by aiding in a more rapid transfer of the second NADPH derived electron to the heme complex.41 It is worth noting that a recent study did find Taxus cuspidata cell cultures spiked with methyl jasmonate and taxadiene could produce OCT.42 However, as methyl jasmonate is known to induce significant production of reactive oxygen species,43 it is difficult to confidently assign the OCT in that study to inherent plant activity and not an artifact of spiking. Other alternatives for improving CYP725A4’s specificity include directed evolution approaches44– 46 or adapting a heterologous P450 with favorable properties to perform the desire chemistry.47 Ultimately, the goal is to assemble an efficient biosynthetic pathway and not to recreate the native one. Accordingly, the native pathway serves as a template and not a constraint. At the same time, the currently limited information on Taxol’s native biosynthesis leaves unsolved whether a single linear or multiple, matrix-like pathways are biosynthetically viable.36 Obtaining such information would provide useful design rules for the “chemical logic” of such a complex molecule’s biosynthesis. Ultimately, our present work takes one step forward by providing design considerations for Taxol’s gateway P450’s functionalization.

CONCLUSION Natural product metabolic engineering holds tremendous promise. As it advances, numerous valuable chemicals will be made affordably and sustainably accessible to the public. Critical for the success of any chemical synthesis approach, though, is efficient selective oxygenation of inert carbons,48–50 and for biosynthesis this will likely be a function of how the field utilizes nature’s favored oxygenase, the cytochrome P450.51 P450s potentially render biosynthesis a competitive advantage,52,53 but only so far as they productively channel carbon flux. As seen here, catalytic promiscuity can significantly undermine this goal, and other recent works may hint that P450 promiscuity is a broader 6 ACS Paragon Plus Environment

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phenomenon of growing concern, especially with increased oxygenation capacity in metabolic engineering systems.14,34,35 Therefore, it will be necessary to develop tools for rapid screening and resolution of P450 promiscuity. The use of lipid nanodiscs for such a purpose has been foreshadowed,17,20,52 and this study highlights the utility of this platform, representing the first use of nanodiscs for the novel plant P450 characterization. The expression and purification of enzyme, specifically P450s, still represents the bottleneck in this workflow. However, with pure enzyme in hand, this method takes only on the order of two to three weeks to execute and could easily be parallelized. Moreover, our discovery of CYP725A4’s cross-platform promiscuity, and specifically the role of peroxide species, ought to interest both plant P450 biochemists and metabolic engineers. It remains to be seen how prominent a role peroxide species may play in the greater superfamily, especially considering that epoxide chemistries can also function productively within a pathway.54 Regardless, recognition of this possibility should help lead to more rapid resolution of its effects. In addition, the impact of non-enzymatic chemistry including spontaneous rearrangement,55,56 hydrophobic overlay sequestration,34 and PNP protecting groups,57 will need to be increasingly considered by PNP metabolic engineers and biochemists alike.58 Valuing intermediate metabolite instability may also encourage metabolic engineers to simultaneous engineer multiple enzymatic steps, perhaps as a metabolon-like complex, to obtain a stable product, along with considering if every observed specie is a direct enzymatic product. Altogether, once the appropriate tools are established, we will be able to assemble the pathways necessary to realize sustainable and affordable access to not only Taxol but a wealth of other natural products.



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Associated Content Materials, methods, supporting GC-MS figures, and gene and protein information can be found in associated supporting information. This material is free of charge via the Internet at http://pubs.acs.org. Author Information Corresponding author: *[email protected]; [email protected] Notes †

Authors contributed equally. C.G.L, M.D.M., and P.K.A. have financial interests in Manus Biosynthesis, Inc. Acknowledgments. The authors would like to thank N. Dudareva and C. N. S. Santos for their careful readings and comments on this manuscript. Keywords: C-H activation; Nanodisc; Natural products; P450 enzymes; Taxol;


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Figure Legends: Figure 1. Taxol pathway and E. coli fermentation products. a) Taxol’s previously established pathway, wherein CYP725A4 performs the first decoration of the diterpene scaffold taxadiene with the selective oxygenation of the fifth carbon. b) GC product profile of total ion count, m/z 288, and m/z 304 from the E. coli fermentation completed with a dodecane overlay. Numbered compounds correspond to structurally solved molecules. 5 is iso-OCT (20.85 min). 4 is OCT (21.60 min). 2 is T-5α-ol (21.85 min). UOTX (21.15 min) stands for the unstable, structurally unsolved oxygenated taxane. MOTX1 (23.25 min) and MOTX2 (23.60 min) are the minor monooxygenated taxanes. DOTX (24.25 min) the potentially double oxygenated taxane. c) Confirmed structures for two of the major monooxygenated taxanes, in addition to taxadiene-5α-ol. Figure 2. Taxane purification, overlay protection, and UOTX. a) GC for purification fractions. Each fraction is filtered by m/z 288 ion, except Fraction 4 for which the total ion count is shown, as its major compound does not contain a 288 ion. As can be seen, no fraction contains a compound with matched retention time to UOTX. Full GC-MS data for the newly observed purification compounds PTX1.1-6 can be found in supplemental Figures S8-11. b) Comparison of fermentations carried out with dodecane, vegetable oil, and no overlay. As can be seen, fermentations without an overlay do not show UOTX. c) MS for UOTX, which shows characteristic terpene fragmentation. Figure 3. Lipid nanodisc assay. a) Schematic for the in vitro lipid nanodisc assay, which contains only essential proteins, cofactors, substrate, and lipids. The P450 is in grey, CPR is in red, the lipids are in cyan, and the membrane scaffold protein are blue. b) Nanodisc assay GC for monooxygenated taxanes (m/z 288), referenced to metabolic engineering platform. Overlapping products T-5α-ol and OCT are observed in both assays, along with several previously unobserved compounds (ND1-7). Full GCMS for ND1-7 can be found in supplemental Figure S21. Notably, unlinked and linked in vitro assays closely share product profiles, even for the mammalian rat CPR. c) Lipid nanodisc products of taxadiene metabolism by CYP725A4 that overlap with the metabolic engineering platform. Figure 4. OCT’s spontaneous rearrangement to iso-OCT. a) OCT’s Spontaneous structural rearrangement. b) Total ion count GC for OCT at different temperatures and durations of time. As can be seen, over time and at a range of temperature, OCT spontaneously isomerizes to iso-OCT. c) Histogram representation of GC for OCT’s spontaneous isomerization to iso-OCT at different times and temperatures. Figure 5. Peroxide activity on taxadiene. a) Schematic of describe peroxide activity wherein taxadiene’s double bond is attacked by H2O2 to form OCT. b) GC (m/z 272, 288) showing taxadiene with and without exposure to peroxide species. As can be seen, with exposure to peroxide, OCT is formed. c) MS confirming observed species in peroxide experiment matches previously characterized OCT.


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N/A. 254x190mm (96 x 96 DPI)

ACS Paragon Plus Environment


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Figure 1. Taxol pathway and E. coli fermentation products. a) Taxol’s previously established pathway, wherein CYP725A4 performs the first decoration of the diterpene scaffold taxadiene with the selective oxygenation of the fifth carbon. b) GC product profile of total ion count, m/z 288, and m/z 304 from the E. coli fermentation completed with a dodecane overlay. Numbered compounds correspond to structurally solved molecules. 5 is iso-OCT (20.85 min). 4 is OCT (21.60 min). 2 is T-5α-ol (21.85 min). UOTX (21.15 min) stands for the unstable, structurally unsolved oxygenated taxane. MOTX1 (23.25 min) and MOTX2 (23.60 min) are the minor monooxygenated taxanes. DOTX (24.25 min) the potentially double oxygenated taxane. c) Confirmed structures for two of the major monooxygenated taxanes, in addition to taxadiene-5αol. 254x190mm (96 x 96 DPI)


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Figure 2. Taxane purification, overlay protection, and UOTX. a) GC for purification fractions. Each fraction is filtered by m/z 288 ion, except Fraction 4 for which the total ion count is shown, as its major compound does not contain a 288 ion. As can be seen, no fraction contains a compound with matched retention time to UOTX. Full GC-MS data for the newly observed purification compounds PTX1.1-6 can be found in supplemental Figures S8-11. b) Comparison of fermentations carried out with dodecane, vegetable oil, and no overlay. As can be seen, fermentations without an overlay do not show UOTX. c) MS for UOTX, which shows characteristic terpene fragmentation. 254x190mm (96 x 96 DPI)



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Figure 3. Lipid nanodisc assay. a) Schematic for the in vitro lipid nanodisc assay, which contains only essential proteins, cofactors, substrate, and lipids. The P450 is in grey, CPR is in red, the lipids are in cyan, and the membrane scaffold protein are blue. b) Nanodisc assay GC for monooxygenated taxanes (m/z 288), referenced to metabolic engineering platform. Overlapping products T-5α-ol and OCT are observed in both assays, along with several previously unobserved compounds (ND1-7). Full GC-MS for ND1-7 can be found in supplemental Figure S21. Notably, unlinked and linked in vitro assays closely share product profiles, even for the mammalian rat CPR. c) Lipid nanodisc products of taxadiene metabolism by CYP725A4 that overlap with the metabolic engineering platform. 254x190mm (96 x 96 DPI)


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Figure 4. OCT’s spontaneous rearrangement to iso-OCT. a) OCT’s Spontaneous structural rearrangement. b) Total ion count GC for OCT at different temperatures and durations of time. As can be seen, over time and at a range of temperature, OCT spontaneously isomerizes to iso-OCT. c) Histogram representation of GC for OCT’s spontaneous isomerization to iso-OCT at different times and temperatures. 254x190mm (96 x 96 DPI)



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Figure 5. Peroxide activity on taxadiene. a) Schematic of describe peroxide activity wherein taxadiene’s double bond is attacked by H2O2 to form OCT. b) GC (m/z 272, 288) showing taxadiene with and without exposure to peroxide species. As can be seen, with exposure to peroxide, OCT is formed. c) MS confirming observed species in peroxide experiment matches previously characterized OCT. 254x190mm (96 x 96 DPI)


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Orthogonal Assays Clarify the Oxidative Biochemistry of Taxol P450

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