Orthogonal Assays Clarify the Oxidative Biochemistry of Taxol P450CYP725A4Bradley 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*,†

†Manus Biosynthesis, 1030 Massachusetts Avenue, Suite 300, Cambridge, Massachusetts 02138, United States‡Department of Chemical and Biological Engineering (Masters in Biotechnology Program), Northwestern University, Evanston,Illinois 60208, United States§Department of Comparative Biosciences, Department of Biochemistry, Department of Bioengineering, Beckman Institute forAdvanced Science and Engineering, University of Illinois Urbana−Champaign, Urbana, Illinois 61801, United States∥Centre for Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, B-9000, Ghent, Belgium⊥Sequoia Sciences, 1912 Innerbelt Business Center Dr., Saint Louis, Missouri 63114, United States

*S Supporting Information

ABSTRACT: Natural product metabolic engineering potentiallyoffers sustainable and affordable access to numerous valuablemolecules. However, challenges in characterizing and assemblingcomplex biosynthetic pathways have prevented more rapid progressin this field. The anticancer agent Taxol represents an excellent casestudy. Assembly of a biosynthetic pathway for Taxol has long beenstalled at its first functionalization, putatively an oxygenationperformed by the cytochrome P450 CYP725A4, due to confoundingcharacterizations. Here, through combined in vivo (Escherichia coli), invitro (lipid nanodisc), and metabolite stability assays, we verify thepresence and likely cause of this enzyme’s inherent promiscuity.Thereby, we remove the possibility that promiscuity simply existed asan artifact of previous metabolic engineering approaches. Further,spontaneous rearrangement and the stabilizing effect of a hydro-phobic overlay suggest a potential role for nonenzymatic chemistry in Taxol’s biosynthesis. Taken together, this work confirmstaxadiene-5α-ol as a primary enzymatic product of CYP725A4 and provides direction for future Taxol metabolic and proteinengineering efforts.

Plant specialized metabolisms are rich with chemicallydiverse and structurally complex molecules that hold value

as pharmaceuticals and otherwise.1,2 Unfortunately, lowaccumulation in slow growing hosts often prevents traditionalextraction methods from providing sufficient access to thesecompounds.3 Even more, the structural complexity of manyplant natural products (PNPs) renders their chemical synthesis,if achievable, economically infeasible.4 As an alternativeapproach, heterologous biological production in quick growingand fermentable microbes such as Escherichia coli orSaccharomyces cerevisiae has been proposed.5,6 Thoughpromising, this approach fundamentally requires the identi-fication of a biochemical pathway for a molecule of interest,which can present a tremendous challenge.7 Such has been thecase for the anticancer agent Taxol. Even after decades ofwork,8 characterization of Taxol’s biosynthetic pathway isincomplete, and efforts toward its heterologous biologicalsynthesis remain in their initial stages (for a history of Taxol’slandmarks, please reference Figure S1). A major cause for this

delay has been the confounding catalytic characterizations ofthe cytochrome P450 (P450) CYP725A4, Taxol’s putative firsttailoring enzyme.Nearly 20 years ago Croteau and co-workers described

CYP725A4 to perform the C-5 hydroxylation and concomitantallylic rearrangement of taxadiene as the first of severalfunctionalizations of Taxol’s diterpene scaffold (Figure 1a).9,10

This ordering was determined according to the relativeabundance of these structural features in subsequent, moredecorated taxoids, and was confirmed by assimilation of labeledtaxadiene-5α-ol (T-5α-ol) into downstream products uponfeeding to Taxus cell cultures.9,10 Subsequent work in separatelaboratory, however, claimed that CYP725A4 convertstaxadiene instead to oxa-cyclotaxane (OCT) (Figure 1c).11

Later, upon our own studies, this enzyme was found to produce

Received: November 22, 2015Accepted: March 1, 2016Published: March 1, 2016

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both T-5α-ol and OCT when incorporated into a taxadiene-overproducing E. coli strain.12 Even more, optimizations of thisP450-containing strain have revealed additional oxygenatedtaxane species (Figure 1b, m/z 288). This apparent promiscuitywas unexpected in light of these previous studies, unchar-acteristic of discussions of P450 use in terpene biocatalysis,13

and undesirable with respect to productively channeling carbonflux to downstream Taxol metabolites. In addition, it wasunclear if the observed promiscuity was inherent to this enzymeor an artifact of a particular assay background. Accordingly,further examination of CYP725A4’s biochemistry was war-ranted before continuing to engineer Taxol’s pathway.Characterizing plant P450s is challenging, however, and

presents several technical hurdles. First, P450s are oftenintransigent to heterologous expression,14 requiring significantN-terminal modification.15 In addition, they require an electrondonating cytochrome P450 reductase partner (CPR)16 andlipid bilayer17 for functional activity. This has precluded thedevelopment of a universal, robust, and facile platform for P450screening. Instead, historical characterizations have predom-inantly been limited to native and heterologous in vivo assaysand crude in vitro microsomal preparations, often with theinclusion of a heterologous reductase partner. Such assays allowcomplex and potentially non-native biochemical interaction,which may alter enzyme performance in a context-dependentmanner.18 We hypothesized such phenomena could haveinfluenced the findings of previous characterizations ofCYP725A4. Therefore, we sought to employ a purified invitro assay to circumvent such issues and to probe this enzyme’sinherent catalytic properties. To this end, a handful of platformshave been developed,19,20 with lipid nanodiscs showingparticular promise. Nanodisc technology has been optimized

and used to analyze several mammalian P450s and one plantP450.21,22 Accordingly, herein we have paired the self-assembling, amphipathic scaffold protein-belted nanodiscassay with our previously developed E. coli metabolicengineering platform12 to examine CYP725A4.

■ RESULTS AND DISCUSSION

In Vivo Characterization of CYP725A4. We began withfermentations of the previously described CYP725A4-contain-ing E. coli strain,12 carried out in the presence of a dodecaneoverlay to capture hydrophobic terpene products. GC-MSanalysis of the overlay shows both singly (m/z 288) andpotentially doubly oxygenated (m/z 304) taxanes, with fourmonooxygenated species representing the majority of the totalfermentation product (Figure 1b, Figures S2−S7). Comparingwith previous reports,9,11 two products possess mass spectra(MS) matching those for the validated structures of T-5α-oland OCT (Figure 1, Figures S2−S4). Two major products,however, remain unidentified, and so successive shake flaskfermentations were carried out in order to generate material forfurther characterization. Purification efforts successfully isolatedthree of the four major oxygenated species, indicated bymatched GC-MS identity before and after isolation fromdodecane (Figures S2−S5). Once isolated, T-5α-ol’s structurewas reconfirmed with 2D NMR (Table S1). In addition, apreviously uncharacterized oxygenated taxane, hereafterreferred to as iso-oxa-cyclotaxane or “iso-OCT,” was success-fully isolated, and its structure was also determined by 2DNMR (Figure 1c, Table S1). OCT’s NMR has been previouslyreported;11 however, in our hands this compound wasinsufficiently stable to obtain NMR, as described below.Combining NMR and GC-MS characterization, three of thefour major oxygenated products were identified (Figure 1).

A Novel Oxygenated Taxane Identified from E. coliFermentations with Hydrophobic Overlays. Interestingly,after multiple purification attempts, the final major oxygenatedtaxane species, hereafter referred to as UOTX (unstable,structurally unsolved oxygenated taxane), could not be isolated,as no fractions possessed a compound with matched retentiontime (Figure 2a). Instead, compounds with previouslyunobserved retention times emerge (Figure 2a, Figures S8−S11), potentially indicating that UOTX degrades or isomerizesduring purification. To evaluate if UOTX requires a hydro-phobic overlay as a protecting environment, additionalfermentations were completed without an overlay and with avegetable oil used in substitute for dodecane. UOTX is onlyobserved in the presence of an overlay (Figure 2b, Figures S12,S13), indicating that an overlay is required to stably sequesterand detect this metabolite from aqueous media. This resultconcerts with a recent work, wherein solvent extraction was alsofound to alter a natural product metabolite.23 Although itsinstability prevents structural solution, UOTX’s MS has beendetermined (Figure 2c). This MS bears similarity to that of T-5α-ol (Figure S3), which may indicate that UOTX represents asimilar 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 tosequester and shield this metabolite long enough for it to becatalyzed by a downstream enzyme. Regardless of a definitiverole, this finding may interest plant biochemists who rarelyemploy hydrophobic overlays for enzymatic characterization. Asseen here, their use in biochemical characterization may allow

Figure 1. Taxol pathway and E. coli fermentation products. (a) Taxol’spreviously established pathway, wherein CYP725A4 performs the firstdecoration of the diterpene scaffold taxadiene with the selectiveoxygenation of the fifth carbon. (b) GC product profile of total ioncount, m/z 288, and m/z 304 from the E. coli fermentation completedwith a dodecane overlay. Numbered compounds correspond tostructurally solved molecules. Compound 5 is iso-OCT (20.85 min).Compound 4 is OCT (21.60 min). Compound 2 is T-5α-ol (21.85min). UOTX (21.15 min) stands for the unstable, structurallyunsolved oxygenated taxane. MOTX1 (23.25 min) and MOTX2(23.60 min) are the minor monooxygenated taxanes. DOTX (24.25min) is the potentially double oxygenated taxane. (c) Confirmedstructures for two of the major monooxygenated taxanes, in additionto taxadiene-5α-ol.

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for additional unstable intermediate metabolite species to beobserved.In Vitro Characterization of CYP725A4. We next studied

the product distribution of CYP725A4 emanating from purifiedenzyme reconstituted in lipid nanodiscs. For these assays,additional considerations were taken into account. First, bothlinked and unlinked P450 constructions were used in order toeliminate protein fusion as the cause of promiscuity.24 Linkedconstructions constituted CYP725A4 fused to its nativereductase partner.12 Unlinked assays were completed usingboth a CPR from Stevia rebaudiana, chosen from among threeplant CPRs due to its favorable solubility, and a CPR from ratliver.25 Second, extraneous biochemical interactions wereminimized by purifying each enzyme before its incorporationinto the assay by adding a His-tag to chimeric and individualP450 proteins and taking advantage of CPR’s ability to bepurified using ADP-agarose. This circumvented the need forcrude microsomal preparations. Thus, the final nanodisc assaysincluded only the nanodisc materials, P450, CPR, NADPH, andtaxadiene (Figure 3a), and was characterized by UV−vis todetermine active protein concentration (Figure S14)Notably, linked and unlinked protein assays show a nearly

identical array of products by GC-MS (Figure 3b, Figures S15−S21, Table S2). In comparison with the in vivo data, T-5α-oland OCT are observed as consistent products. Interestingly, theratio of OCT to T-5α-ol shifts from approximately 1:1 to favorthe production of OCT when moving from the linked assay tothe two unlinked assays. This could potentially be a function ofa more transient CPR-P450 interaction for the unlinkedconstructions 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 scalingrestrictions of these systems prohibit NMR characterization,but the data indicate that P450 functionality does not varybetween the linked and unlinked protein constructions, evenwhen exchanging plant for mammalian CPR (Figure 3b, FigureS20). Accordingly, these findings provide continued confidencefor a “share your parts” principle applied to selecting plant CPR

with favorable properties such as solubility.16 In addition, theyindicate that the dramatic catalytic variation observed duringCPR fusion in another P450 assay24 was in some way unique.Last, these data demonstrate a cross-platform promiscuity forCYP725A4, while simultaneously confirming T-5α-ol as aconsistent major product for both in vivo and in vitro assaysystems.

Metabolite Instability and Spontaneous Rearrange-ment of OCT to iso-OCT. Motivated by the metaboliteinstability observed during purification, we probed the stabilityof the other major taxane species. To this end, isolated taxaneswere subjected to a range of conditions (Table S3). Initialstability assays revealed no measurable degradation fortaxadiene, T-5α-ol, or iso-OCT (Figures S22−S24). However,OCT did show significant change (Figures S25, S26). Whereaspurified samples of OCT analyzed immediately after removalfrom −20 °C show predominantly one peak, over time and at arange of temperatures, increasing amounts of iso-OCT areobserved (Figure 4, Table S4). As this spontaneous rearrange-ment is irreversible, iso-OCT is concluded to be thermody-namically favored. Iso-OCT’s structure shows substantial bondrearrangement compared to OCT, and a scheme for thisisomerization is proposed (Figure 4a). This observation ofrearrangement is noteworthy, as iso-OCT was previouslyassumed to be a direct enzymatic product. Moreover, OCT’sinstability rationalizes the difficulty in obtaining clean NMR forthis compound.Further stability assays revealed T-5α-ol to be stable under all

but acidic conditions (Figure S27). Acidic conditions appear todegrade the compound, potentially removing the hydroxylgroup, as a previously unobserved diterpene (m/z 272) peakappears (Figures S27−S30). However, none of T-5α-ol’s aciddegradation products overlap with the major fermentationproducts. Hypothesizing that some oxygenated taxane productscould be derived from a peroxide attack of one of taxadiene’s

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

Figure 3. Lipid nanodisc assay. (a) Schematic for the in vitro lipidnanodisc assay, which contains only essential proteins, cofactors,substrate, and lipids. The P450 is in gray. CPR is in red. The lipids arein cyan, and the membrane scaffold proteins are blue. (b) Nanodiscassay GC for monooxygenated taxanes (m/z 288), referenced tometabolic engineering platform. Overlapping products T-5α-ol andOCT are observed in both assays, along with several previouslyunobserved compounds (ND1−7). Full GC-MS for ND1−7 can befound in Figure S21. Notably, unlinked and linked in vitro assaysclosely share product profiles, even for the mammalian rat CPR. (c)Lipid nanodisc products of taxadiene metabolism by CYP725A4 thatoverlap with the metabolic engineering platform.

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carbon−carbon double bonds, with peroxide species stemmingfrom an incomplete P450 catalytic cycle,26 we exposedtaxadiene to hydrogen peroxide. Strikingly, this assay showsoxygenated products, with OCT representing a majorityspecies, confirmed by MS spectra match (Figure 5, FiguresS31, S32).

To ensure that the peroxide activity was a result ofCYP725A4’s presence and not a system artifact, wereconfirmed that the taxadiene-overproducing E. coli strainwithout a P450 produces no oxygenated taxanes (Figure S33).This means that E. coli’s natively present reactive oxygen speciesare not sufficient to react with taxadiene to produce OCT.Negative controls for the nanodisc experiments similarlyconfirm that, without a P450 present, taxadiene does notreact to form OCT in these systems (Figure S16). In addition,the aforementioned degradation assays show that taxadienedoes not spontaneously form OCT under multiple otherconditions (Figure S22). Therefore, we believe that thesubstrate 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 differsfrom H2O2-dependent peroxygenase generation of the reactivecompound I.27

Resolving CYP725A4’s Oxidative Biochemistry. Con-founding characterizations of CYP725A4 have long stalledTaxol biosynthesis efforts and raised doubts about thisenzyme’s appropriateness for taxadiene’s first functionalization.In addition, CYP725A4’s observed promiscuity is somewhatunexpected. Though P450s are known to accept structurallydiverse substrates and catalyze a variety of chemistries,28,29

including oxidation cascades and the highly multispecificactivity of hepatic P450s on PNP pharmaceuticals,30,31 terpenebiosynthetic P450s have infrequently been characterized toperform multiple distinct oxygenations on a single substrate,13

with limited examples.32,33 Thus, it was reasonable to questionif CYP725A4’s observed promiscuity was inherent to theenzyme or introduced as an artifact of previous characterizationassays, including our metabolic engineering platform.By observing CYP725A4’s promiscuity in the purified

nanodisc in vitro assay, we can confirm that this behavior isinherent to the enzyme. It is possible that CYP725A4’spromiscuity is observed only now because of the improvedP450 flux of our assay systems, wherein buried, physiologicallyirrelevant, side activities have been made manifest.18 Metabolicengineering approaches have recently begun to encountersimilar phenomena,14,34,35 especially with increasing oxygen-ation capacity.14 Such a finding would accord with a growingunderstanding that promiscuity is necessary for evolvability andplays a major role in PNP specialized metabolism.36−38 Yet, thisexplanation does not account for the absence of thepromiscuous products OCT and iso-OCT in nature. If,however, we include our discovery that a peroxide chemistryforms OCT, understanding that peroxide species can resultfrom incomplete P450 cycles, we gain a clearer picture of thisenzyme and its future use. Elimination of the peroxide activitywould mean narrowing the fermentation product profile,potentially to only one major compound if UOTX is alsoperoxide derived.

CYP725A4 in the Future. As OCT has not been observedin nature, it is possible that native protein−protein complexes,such as a metabolon,39 safely channel taxadiene and T-5α-ol todownstream metabolites and prevent peroxide interaction orother promiscuous catalytic functions. Accordingly, engineeringa scaffold-like environment may help to heterologously recreatethis effect.40 The inclusion of cytochrome b5 in such a complexmay also assist in preventing incomplete P450 catalytic cyclesby aiding in a more rapid transfer of the second NADPHderived electron to the heme complex.41 It is worth noting thata recent study did find Taxus cuspidata cell cultures spiked withmethyl jasmonate and taxadiene could produce OCT.42

However, as methyl jasmonate is known to induce significantproduction of reactive oxygen species,43 it is difficult toconfidently assign the OCT in that study to inherent plantactivity and not an artifact of spiking.Other alternatives for improving CYP725A4’s specificity

include directed evolution approaches44−46 or adapting aheterologous P450 with favorable properties to perform thedesired chemistry.47 Ultimately, the goal is to assemble anefficient biosynthetic pathway and not to recreate the nativeone. Accordingly, the native pathway serves as a template andnot a constraint. At the same time, the currently limitedinformation on Taxol’s native biosynthesis leaves unsolvedwhether a single linear or multiple, matrix-like pathways are

Figure 4. OCT’s spontaneous rearrangement to iso-OCT. (a) OCT’sspontaneous structural rearrangement. (b) Total ion count GC forOCT at different temperatures and durations of time. As can be seen,over time and at a range of temperatures, OCT spontaneouslyisomerizes to iso-OCT. (c) Histogram representation of GC forOCT’s spontaneous isomerization to iso-OCT at different times andtemperatures.

Figure 5. Peroxide activity on taxadiene. (a) Schematic of describedperoxide activity wherein taxadiene’s double bond is attacked by H2O2to form OCT. (b) GC (m/z 272, 288) showing taxadiene with andwithout exposure to peroxide species. As can be seen, with exposure toperoxide, OCT is formed. (c) MS confirming observed species inperoxide experiment matches previously characterized OCT.

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biosynthetically viable.36 Obtaining such information wouldprovide useful design rules for the “chemical logic” of such acomplex molecule’s biosynthesis. Ultimately, our present worktakes one step forward by providing design considerations forTaxol’s gateway P450s functionalization.

■ CONCLUSION

Natural product metabolic engineering holds tremendouspromise. As it advances, numerous valuable chemicals will bemade 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 thefield utilizes nature’s favored oxygenase, the cytochromeP450.51 P450s potentially render biosynthesis a competitiveadvantage,52,53 but only so far as they productively channelcarbon flux. As seen here, catalytic promiscuity can significantlyundermine this goal, and other recent works may hint thatP450 promiscuity is a broader phenomenon of growingconcern, especially with increased oxygenation capacity inmetabolic engineering systems.14,34,35 Therefore, it will benecessary to develop tools for rapid screening and resolution ofP450 promiscuity. The use of lipid nanodiscs for such apurpose has been foreshadowed,17,20,52 and this study highlightsthe utility of this platform, representing the first use ofnanodiscs for the novel plant P450 characterization. Theexpression and purification of enzyme, specifically P450s, stillrepresents the bottleneck in this workflow. However, with pureenzyme in hand, this method takes only on the order of 2 to 3weeks to execute and could 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 metabolicengineers. It remains to be seen how prominent a role peroxidespecies may play in the greater superfamily, especiallyconsidering that epoxide chemistries can also functionproductively within a pathway.54 Regardless, recognition ofthis possibility should help lead to more rapid resolution of itseffects. In addition, the impact of nonenzymatic chemistryincluding spontaneous rearrangement,55,56 hydrophobic overlaysequestration,34 and PNP protecting groups57 will need to beincreasingly considered by PNP metabolic engineers andbiochemists alike.58 Valuing intermediate metabolite instabilitymay also encourage metabolic engineers to simultaneouslyengineer multiple enzymatic steps, perhaps as a metabolon-likecomplex, to obtain a stable product, along with considering ifevery observed species is a direct enzymatic product.Altogether, once the appropriate tools are established, we willbe able to assemble the pathways necessary to realizesustainable and affordable access to not only Taxol but awealth of other natural products.

■ ASSOCIATED CONTENT

*S Supporting Informationcan be found in associated Supporting Information. Thismaterial is free of charge via the Internet at The SupportingInformation is available free of charge on the ACS Publicationswebsite at DOI: 10.1021/acschembio.5b00968.

Materials, methods, supporting GC-MS figures, and geneand protein information (PDF)Sequoia NMR (ZIP)

■ AUTHOR INFORMATION

Corresponding Authors*E-mail: aditidas@illinois.edu.*E-mail: pkaji@manusbio.com.

Author Contributions#These authors contributed equally.

NotesThe authors declare the following competing financialinterest(s): C.G.L, M.D.M., and P.K.A. have financial interestsin Manus Biosynthesis, Inc.

■ ACKNOWLEDGMENTS

The authors would like to thank N. Dudareva and C. N. S.Santos for their careful readings and comments on thismanuscript.

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