BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Structural modification of herboxidiene by substrate-flexiblecytochrome P450 and glycosyltransferase

Amit Kumar Jha & Dipesh Dhakal & Pham Thi Thuy Van &

Anaya Raj Pokhrel & Tokutaro Yamaguchi &Hye Jin Jung & Yeo Joon Yoon & Jae Kyung Sohng

Received: 1 December 2014 /Revised: 21 January 2015 /Accepted: 22 January 2015# Springer-Verlag Berlin Heidelberg 2015

Abstract Herboxidiene is a natural product produced byStreptomyces chromofuscus exhibiting herbicidal activity aswell as antitumor properties. Using different substrate-flexible cytochrome P450s and glycosyltransferase, differentnovel derivatives of herboxidiene were generated with struc-tural modifications by hydroxylation or epoxidation or conju-gation with a glucose moiety. Moreover, two isomers ofherboxidiene containing extra tetrahydrofuran ortetrahydropyran moiety in addition to the existingtetrahydropyran moiety were characterized. The hydroxylatedproducts for both of these compounds were also isolated andcharacterized from S. chromofuscus PikC harboring pikC fromthe pikromycin gene cluster of Streptomyces venezuelae andS. chromofuscus EryF harboring eryF from the erythromycingene cluster of Saccharopolyspora erythraea. The com-pounds generated were characterized by high-resolutionquadrupole-time-of-flight electrospray ionization mass spec-trometry (HR-QTOF-ESI/MS) and 1H- and 13C-nuclear mag-netic resonance (NMR) analyses. The evaluation of antibacte-rial activity against three Gram-positive bacteria,Micrococcusluteus, Bacillus subtilis, and Staphylococcus aureus, indicatedthat modification resulted in a transition from anticancer toantibacterial potency.

Keywords Streptomyces . Glycosylation . Hydroxylation .

Epoxidation . Antibacterial

Introduction

Herboxidiene, characterized structurally by its cis-substitutedtetrahydropyran acetic acid moiety and side chain consistingof an E-E-conjugated diene (Edmunds et al. 1997), was iso-lated from Streptomyces chromofuscus A7847 (ATCC 49982)as a novel polyketide with effective herbicidal activity againstseveral annual weeds (Wideman et al. 1992; Isaac et al. 1992).There was revised interest in the biological activities of thiscompound, because of its anticholesterol (Koguchi et al.1997) and antitumor activities (Sakai et al. 2002; Hasegawaet al. 2011). Advanced studies at the molecular level haveshown that it exhibits antitumor activity by targeting thespliceosome, particularly by inhibiting constitutive splicingtargeting spliceosomal subunit SF3b (Gao et al. 2013;Effenberger et al. 2014; Hasegawa et al. 2011; Lagisettiet al. 2013). A flurry of reports revealed that genesencoding splicing factors, including the drug targetsplicing factor 3B subunit 1 (SF3B1), are among themost highly mutated in various malignancies. The drugstargeting the spliceosome generally alter gene expres-sion, including alternative splicing of genes that are im-portant in cancer progression (Bonnal et al. 2012).

Due to its high potency, several successful chemical syn-thetic approaches have been attempted for preparingherboxidiene (Blakemore et al. 1999; Edmunds et al. 2000;Ghosh and Li 2011; Murray and Forsyth 2008; Banwell et al.2000; Zhang and Panek 2007; Pellicena et al. 2011; Premrajet al. 2012; Yadav et al. 2014) and its totally synthetic biolog-ically active analog, 6-norherboxidiene (Lagisetti et al. 2013).

Electronic supplementary material The online version of this article(doi:10.1007/s00253-015-6431-6) contains supplementary material,which is available to authorized users.

A. K. Jha :D. Dhakal : P. T. T. Van :A. R. Pokhrel : T. Yamaguchi :H. J. Jung : J. K. Sohng (*)Institute of Biomolecule Reconstruction (iBR), Department ofBT-Convergent Pharmaceutical Engineering, Sun Moon University,70, Sunmoon-Ro221, Tangjeong-myeon, Asan, Chungnam 333-708,Republic of Koreae-mail: sohng@sunmoon.ac.kr

Y. J. YoonDepartment of Chemistry and Nano Science, Ewha WomansUniversity, Seoul 120-750, Republic of Korea

Appl Microbiol BiotechnolDOI 10.1007/s00253-015-6431-6

However, these multistep chemical processes may not be ef-ficient or flexible enough for the synthesis of newherboxidiene analogs. Thus, to open up new avenues for bio-chemical characterization and a surge in the possibilities ofgenerating new analogs of herboxidiene, the 53-kb biosynthe-sis gene cluster for herboxidiene was examined by genomesequencing and gene inactivation studies (Shao et al. 2012).Moreover, characterizations of some key steps in biosyntheticpathway were attempted and new analogs were generatedthrough gene inactivation (Yu et al. 2013, 2014). Similarly,enhanced production of herboxidiene itself by a metabolicengineering approach was also accomplished (Jha et al. 2014).

Developing a Bgood^ drug from a natural product is a greatchallenge, because there are stringent requirements for im-proving properties such as absorption, distribution, metabo-lism, excretion, and toxicity, while maintaining acceptablepotency, which can be time consuming and expensive(Menzella and Reeves 2007). Thus, some new emerging tech-niques such as Bcombinatorial biosynthesis,^ based on precisegenetic manipulation of the enzymes for natural products, mayhelp in developing products with better pharmacodynamicand pharmacokinetic properties. The basic concept of this ap-proach uses combining and recombining the metabolic path-ways of different organisms at a genetic level to create a de-sired genetic scaffold. These technologies mostly rely on en-zymes with broad substrate tolerances, such as glycosyltrans-ferases, to achieve combinatorial attachment of sugars to nat-ural product aglycons (Blanchard and Thorson 2006; Wohlertet al. 1998; Sanchez et al. 2005; Salas and Méndez 2009) orcombinatorial assembly of complex multi-modular enzymes,such as polyketide synthase (PKS) and NRPS (Menzella andReeves 2007; Wong and Khosla 2012). The cytochrome P450genes, integrated in biosynthetic pathways, are capable of al-iphatic and aromatic bond hydroxylation, double-bond epox-idation, heterocyclization, aryl and phenolic ring coupling,oxidative rearrangements of carbon skeletons, and C–C bondcleavage in different natural products, with high chemo-,regio-, and stereo-selectivity (Podust and Sherman 2012).

Glycosylation has also been an effective tool for the diver-sification of natural products (Simkhada et al. 2010) by en-hancing the solubility and stability, broadening the biologicalpotency and applications (Singh et al. 2012), and sometimeseven altering the biological properties of the compounds(Kren andMartinkova 2001). Thus, manipulations using thesegenes may provide a platform for introducing the desiredstructural variability and creating designer molecules withnew biological activities.

Here, we explored the generation of new herboxidiene an-alogs by applying combinatorial biosynthesis strategies toS. chromofuscus, where the host strain was crafted in termsof its in vivo catalytic machinery for modifying herboxidieneusing selected substrate-flexible cytochrome P450 from dif-ferent biosynthetic gene clusters prevalent in different

actinomycetes and a flexible glycosyltransferase. Here, thenative producer strain was engineered metabolically for hy-droxylation using a dedicated hydroxylase pikC from thepikromycin gene cluster of Streptomyces venezuelae ATCC15439 and eryF from the erythromycin gene cluster ofSaccharopolyspora erythraea NRRL 2338 or epoxidationusing epoxidase, epoF, from the epothilone gene cluster ofSorangium cellulosum strain So ce90 and glycosylation usingYjiC, a flexible glycosyltransferase from Bacilluslicheniformis DSM 13 (ATCC 14580).

From these approaches, we were able to generate differentderivatives of herboxidiene. Among them, some compounds(11, 4, 12, and 8) had remarkable antibacterial activity againstGram-positive bacteria, whereas other derivatives showedmild or no activity in comparison to the parent compound.The assessment of cytotoxicity induced by compounds indi-cated that there was a loss of cytotoxicity in all compounds,while the parent compound retained effective cytotoxicpotency.

Materials and methods

Microorganisms and vectors

All the plasmids and bacterial strains used in this study arelisted in Table S1. The pGEM-T Easy Vector (Promega,Madison, WI, USA) was used to clone the polymerase chainreaction (PCR) products. pGEM-3Zf + (Promega) was used asa subcloning vector. Similarly, pIBR25 (Sthapit et al. 2004)was used as the expression vector and pSET152 (Biermanet al. 1992) was used as the integration vector for the geneticengineering of S. chromofuscus. DNA manipulations werecarried out in Escherichia coli XL1 Blue MRF (Stratagene,La Jolla, CA, USA). E. coli JM110 was used for the propaga-tion of nonmethylated DNA. E. coli strains were cultivated at37 °C in LB medium supplemented with ampicillin (100 μg/mL) and apramycin (100 μg/mL) as required.

Culture conditions

S. chromofuscus A7847 (ATCC 49982) or its geneticallyengineered hosts were cultured in different media for differentpurposes. ISP medium 2 (yeast extract 0.4 %, malt extract1 %, and glucose 0.4 %) was used as a seed medium andR2YE as a regeneration medium for recombinants. The regen-eration medium was supplemented with apramycin orthiostrepton, as required. Previously optimized high produc-tion medium for S. chromofuscus, media No. 6A6 (Jha et al.2014), was used without further modification in all cases,while for the production of glycosylated derivatives, supple-mentation with 4 % glucose was used to increase the pool ofglucose by exogenous feeding.

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DNA manipulation and sequence analyses

For amplification of target DNA fragments, PCR premix(GenoTech, Seoul, Korea) and TaKaRa LA Taq (Takara,Shiga, Japan) were used according to the manufacturer’s in-structions. PCR was performed in a Thermal Cycler Dice(Takara). The amplification conditions for PCR were as fol-lows: an initial denaturation at 94 °C for 7 min, 30 cycles ofdenaturation at 94 °C for 1 min, annealing at 55–65 °C for1 min, extension at 72 °C for 1 min, and a final extension at72 °C for 7 min. The PCR products were purified and clonedinto the pGEM-T Easy Vector for DNA amplification andsequencing. DNA preparation, digestion, ligation, and otherDNA manipulations were performed using standard tech-niques for E. coli. The chemicals and enzymes used in thisstudy were purchased from Sigma (St. Louis, MO, USA). Insilico analyses and comparisons of nucleotide and protein se-quences were performed using BLAST, FASTA, andClustalW.

Construction of the recombinant vector

The pIBR25 and pSET152, under the strong ermE* promoter,were used for the construction of recombinants. The primersused for amplification of specific DNA fragments are listed inTable S2. The PCR products of yjiC (AE017333.1) and galU(NC_007779.1) were isolated from the genomic DNA ofB. licheniformis DSM 13 and E. coli K12, respectively.Similarly, pikC (AF079139.1), eryF (M54983.1), and epoF(AF210843.1) with orf6 (AF210843.1) (Park et al. 2008) wereisolated from the genomic DNA of S. venezuelae ATCC15439, S. erythraea NRRL 2338, and S. cellulosum Soce90, respectively. The PCR products were purified andcloned into the pGEM-T Easy Vector and sequenced prior tocloning in the expression vector and integrative vector to ver-ify that no mutation had been introduced during the PCRamplification. After the sequence was analyzed, pikC anderyF were cloned into the BamHI and XbaI sites ofpSET152 to form the recombinant plasmids pPikC152 andpEryF152, respectively. The epoF along with orf6was clonedinto PacI and HindIII sites of pSET152 to form the recombi-nant plasmid pEpoF152. Similarly, yjiC was cloned inBamHI/EcoRI and galU in EcoRI/HindIII sites, respectively,for expression of both genes from a single vector, pIBR25, toconstruct pGYIBR.

Transformation and generation of recombinant strains

The preparation of protoplasts, transformation, and selectionof S. chromofuscus transformants were carried out accordingto standard protocols (Kieser et al. 2000). All therecombinants pGYIBR, pEryF152, pPikC152, andpEpoF152 including empty vectors pIBR25 and pSET152

were propagated in E. coli JM110 to obtain demethylatedDNA for transformation in the Streptomyces strain. After de-methylation of all the recombinant vectors, they were intro-duced into S. chromofuscus by polyethylene glycol (PEG)-mediated protoplast transformation. For protoplast transfor-mation, the native producer strain S. chromofuscus was cul-tured in 50 mL of seed medium. After 60 h, the myceliumwasharvested by centrifugation (3200 rpm, 12 min, 4 °C) andwashed with 15 mL of sucrose solution (10.3 %) and furtherwashed with 15 mL of P buffer. Finally, 10 mL of lysozymesolution (2 mg/mL in P buffer) was added to the cell pelletsand the content was incubated for 50 min at 37 °C. Afterincubation, the mix was filtered and centrifuged (6000 rpm,12 min), then washed with P buffer twice, and mixed with1 mL of P buffer. Next, 100 μL of the resulting mix was addedto 20 μL of plasmid DNA and 200 μL of 40 % PEG 1000 andcentrifuged for 1 min. The supernatant was partially discardedand then mixed with 100 μL of P buffer. Finally, it was platedon R2YE plates. The plates were incubated at 28 °C for 24 hand then overlaid with 0.3 % agar solution containing 10 μg/mL thiostrepton or 60 μg/mL apramycin for selectingrecombinants containing the expression or integrative vector,respectively. After 1 week, thiostrepton- or apramycin-resistant colonies, as appropriate, were selected and cul-tured in liquid ISP medium 2. Transformation of eachstrain was confirmed by isolation of the plasmid, PCR,and restriction enzyme digestion. The transformantswere designated as S. chromofuscus pGYIBR,S. chromofuscus EryF, S. chromofuscus PikC,S. chromofuscus EpoF, S. chromofuscus IBR25, andS. chromofuscus SET152, respectively (Table S1).

Morphological analysis of S. chromofuscus and transformant

S. chromofuscus, S. chromofuscus IBR25, S. chromofuscuspGYIBR, S. chromofuscus SET152, S. chromofuscus EryF,S. chromofuscus PikC, and S. chromofuscus EpoF werestreaked on a R2YE agar plate and incubated at 28 °C for8 days under continuous observation. The growth patternand morphological attributes were analyzed by visual obser-vation by the naked eye.

Production, extraction, and purification of novel herboxidiene

To analyze the novel herboxidiene production, 5 % of the seedof S. chromofuscus pGYIBR, S. chromofuscus EryF,S. chromofuscus PikC, and S. chromofuscus EpoFwere grownin optimized production medium 6A6. At the end of 8th day,culture broth from each sample was centrifuged (3000 rpm,15 min) to remove the cell pellets. The supernatant was ex-tracted with a double volume of ethyl acetate, and the extractwas dried under reduced pressure using a rotary evaporatorand reconstituted in 1 mL of methanol. Finally, the samples

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were analyzed by HPLC using a reverse phase C18 column(4.6×250 mm, 50 μm; Kanto Reagents, Japan) connected to aUV detector (236 nm) with a solvent system consisting ofwater (0.025 % trifluoroacetic acid) and 100 % methanol witha flow rate of 1 mL/min for 71 min. The methanol concentra-tions during HPLC were as follows: 10 % (0–0.10 min), 30 %(0.1–7.0 min), 60 % (7–20 min), 65 % (20–35 min), 87 %(35–55 min), 90 % (55–65 min), and 10 % (65–70 min).

Preparative HPLC for purification of novel herboxidiene

The products were purified by prep-HPLC with a C18 column(YMC-Pack ODS-AQ) (150×20 mm ID, 10 μm) connectedto a UV detector (236 nm) over 81 min using binary condi-tions with water (0.025 % trifluoroacetic acid) and methanolof 10 % (0–0.10 min), 30 % (0.1–7.0 min), 60 % (7–15 min),65 % (15–35 min), 87 % (35–60 min), 90 % (60–75 min), and10 % (75–80 min).

Detection and mass analysis

Ultra-pressure liquid chromatography (UPLC)-photodiode ar-ray (PDA) analysis was performed using a reversed-phaseUPLC-PDA with a C18 column (ACQUITY UPLC BEH,C18, 1.7 μm) connected to a PDA (UPLC LG 500 nm) at anabsorbance of 236 nm. The binary mobile phases were com-posed of same solvents as described previously. Total flowwas maintained at 0.3 μL/min for 15 min. The flow of meth-anol was 0–100 % from 0 to 9 min and maintained to 100 %until 9–12 min followed by 100–0 % in 12–15 min and thenstopped at 15 min. For exact mass analysis, a high-resolutionquadrupole-time-of-flight electrospray ionization mass spec-trometry (HR-QTOF-ESI/MS) analysis was performed in pos-itive ion mode using an ACQUITY (Billerica, USA) columncoupled with a SYNAPT G2-S (Waters Corp.) column.

Nuclear magnetic resonance analysis

To characterize the novel structure of the herboxidienes, eachpurified product was dried under reduced pressure using arotary evaporator followed by freeze-drying. To remove thetrace of water, the samples were treated with deuterium oxiderepeatedly, followed by freeze-drying. Finally, the freeze-dried samples were resuspended in 500 μL dimethyl sulfox-ide-d6 (DMSO-d6) and assessed in a 700-MHz spectrometerto determine 1H-nuclear magnetic resonance (NMR), 13C-NMR, two-dimensional NMR correlation spectroscopy(COSY), nuclear Overhauser effect spectroscopy (NOESY),rotational frame NOE spectroscopy (ROESY), heteronuclearsingle quantum correlation (HSQC), and heteronuclearmultiple-bond correlation (HMBC).

Evaluation of anticancer activities of compounds

To evaluate the effects of the different novel analogs ofherboxidiene on the proliferation and viability of B16F10melanoma and HeLa cervical cancer cells, cells were grownin a Dulbecco’s modified Eagle’s medium (DMEM;Invitrogen, Grand Island, NY, USA) supplemented with10 % fetal bovine serum (FBS; Invitrogen). All cells weremaintained at 37 °C in a humidified 5 % CO2 incubator. Forcell growth assays, cells seeded at 2000 cells/well in 96-wellplates (SPL Life Sciences, Gyeonggi, Korea) were treatedwith each compound at various concentrations for 72 h. Cellgrowth was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay.

Evaluation of antibacterial activities of compounds

To evaluate the antibacterial activity, each novel analog ofherboxidiene purified from different transformants ofS. chromofuscus was assayed for antibacterial activity againstthree different pathogenic Gram-positive bacteria—Staphylococcus aureus subsp. aureus KCTC 1916, Bacillussubtilis KACC 17047, and Micrococcus luteus KACC13377—using a paper disc diffusion method on Mueller-Hinton agar (MHA) plates. The inocula containing107 CFU/mL were spread on MHA plates for the bioassay.The sterile filter paper discs (6 mm in diameter) containing5 μL of 100 mM compounds were placed on the surface of theinoculated agar plates. The plates were incubated at 37 °C forup to 2 days under continuous observation. Each compoundwas tested in triplicate, and the zone of inhibition was mea-sured in millimeters in diameter.

Result

Construction of recombinant vectors and recombinant strainsof S. chromofuscus

The glucose-1-phosphate uridylyltransferase (galU) fromE. coli K12 and the UDP-glucosyltransferase (yjiC) fromB. licheniformis DSM 13 were cloned together into pIBR25(high copy expression vector) under the control of a strongpromoter, ermE*, to construct the recombinant plasmidpGYIBR. The pikC, cytochrome P450 monooxygenase inthe pikromycin biosynthetic gene cluster from S. venezuelae,was cloned into pSET152 (integration vector) under the con-trol of a strong promoter, ermE*, to construct pPikC152.Similarly, eryF, a cytochrome P450, characterized as havinghydroxylation activity in the biosynthetic pathway of erythro-mycin to yield erythronolide B, was also cloned into pSET152under the control of the strong promoter, ermE*, to constructpEryF152. The epoF, a cytochrome P450 epoxidase along

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with orf6 from the epothilone gene cluster in S. cellulosum,was cloned into pSET152 under the control of the strong pro-moter ermE* to construct the pEpoF152 recombinant plasmid(Park et al. 2008). All the recombinant vectors were confirmedby restriction digestion analysis and transformed intoS. chromofuscus . The result ing recombinants ofS. chromofuscus were designated as S. chromofuscuspGYIBR, S. chromofuscus IBR25, S. chromofuscus PikC,S. chromofuscus EryF, S. chromofuscus EpoF, andS. chromofuscus SET152.

Effect of recombinant plasmids on growth of recombinantstrains of S. chromofuscus

Surprisingly, we observed that the morphologies ofS. chromofuscus PikC, S. chromofuscus EryF, andS. chromofuscus EpoF were markedly different fromS. chromofuscus wild type and S. chromofuscus SET152 onR2YE plates and were found to grow very slowly, whereasS. chromofuscus SET152 exhibited similar growth to that ofS. chromofuscus (Fig. S1A). Similarly, S. chromofuscuspGYIBR was significantly different and grew slowly on theplate, whereas S. chromofuscuswild type and S. chromofuscusIBR25 exhibited similar growth (Fig. S1B).

HR-QTOF-ESI/MS analysis of derivatives of herboxidiene

To analyze the novel herboxidienes, S. chromofuscus PikCand S. chromofuscus EryF were cultured in 3 L of productionmedia No. 6A6. The culture broths were harvested, centri-fuged, extracted, and injected into a HPLC system. TheHPLC, coupled with HR-QTOF-ESI/MS analysis, showeddifferent novel hydroxylated herboxidiene products represent-ed by herboxidiene A1 (11) (Edmunds et al. 1997), 5-hydroxy-herboxidiene A1 (4), 5-hydroxy-25-demethyl-herboxidiene A1 (3), 5,6-dihydroxy-herboxidiene A1 (6), 5,6-dihydroxy-25-demethyl-herboxidiene A1 (5), herboxidieneB1 (12), 5-hydroxy-herboxidiene B1 (8), 5-hydroxy-25-demethyl-herboxidiene B1 (7), 5,6-dihydroxy-herboxidieneB1 (10), and 5,6-dihydroxy-25-demethyl-herboxidiene B1(9). The observed exact masses (m/z+) were as follows: (11)461.287 [M + Na]+ (Fig. S2A), (4) 477.282 [M + Na]+

(Fig. S2B), (3) 463.267 [M + Na]+ (Fig. S2B), (6) 493.277[M + Na]+ (Fig. S2C), (5) 479.262 [M + Na]+ (Fig. S2C), (12)461.287 [M + Na]+ (Fig. S3A), (8) 477.282 [M + Na]+

(Fig. S3B), (7) 463.267 [M + Na]+ (Fig. S3B), (10) 493.277[M + Na]+ (Fig. S3C), and (9) 479.262 [M + Na]+ (Fig. S3C).Analysis of extract from S. chromofuscus PikC andS. chromofuscus EryF showed the same HPLC profile andmass spectrum. Finally, compounds 11 (~40 mg), 4(~35 mg), 12 (~42 mg), and 8 (~34 mg) were obtained afterpurification and characterized by NMR analysis. Compounds3, 6, 5, 7, 10, and 9 were not produced in sufficient amounts

for a detailed analysis by NMR. On the basis of these results, apathway was proposed for hydroxylation (Fig. 1).

Analysis of extracted compound from S. chromofuscusEpoF showed different novel herboxidiene products, repre-sented by herboxidiene (1), 25-demethyl-herboxidiene (2),herboxidiene C (14), and 25-demethyl-herboxidiene C (13).The observed exact masses (m/z+) were as follows: (1)461.287 [M + Na]+ (Fig. S4A), (2) 447.272 [M + Na]+

(Fig. S4A), (14) 477.282 [M + Na]+ (Fig. S4B), and (13)463.267 [M + Na]+ (Fig. S4B). Compounds 2, 13, and 14were not produced in sufficient quantities for analysis byNMR, whereas compound 1 was reported previously (Jhaet al. 2014). These results supported the proposed pathwayof epoxidation (Fig. 2).

Analysis of compounds extracted from S. chromofuscuspGYIBR showed different novel herboxidiene products, rep-resented by 18-O-β-D-glucopyranoside herboxidiene (16) and18-O-β-D-glucopyranoside-25-demethyl-herboxidiene (15).The observed exact masses (m/z+) were as follows: (16)623.340 [M + Na]+ (Fig. S5) and (15) 609.325 [M + Na]+

(Fig. S5). These results are evidence for the proposed pathwayof glycosylation (Fig. 3).

Structural elucidation of novel herboxidiene analogs by NMR

Purified herboxidiene A1 (11) and 5-hydroxy-herboxidieneA1 (4) were subjected to NMR analyses at 700 MHz inDMSO-d6. We determined the 1H-NMR of herboxidiene A1(11) (Fig. S6A) and 1H-NMR of 5-hydroxy-herboxidiene A1(4) (Fig. S7A), followed by 13C-NMR of herboxidiene A1(11) (Fig. S6B) and 13C-NMR of 4 (Fig. S7B). For furtherstructural determination, two-dimensional NMR analyses of11 and 4 were performed, which included COSY (Figs. S6Cand S7C), ROESY (Figs. S6D and S7D), HMQC-DEPT(Figs. S6E and S7E), HMBC (Figs. S6F and S7F), andHMBC/HMQC-DEPT (Figs. S6G and S7G) overlappingstudies. By these analyses, herboxidiene A1 (11) was identi-fied, which has been synthesized previously by chemicalmethods (Edmunds et al. 1997). Similarly, 5-hydroxy-herboxidiene A1 (4) (2-((2R,4R,5S,6S)-4-hydroxy-6-((2E,4E,6 S , 8R ) - 8 - h y d r o x y - 8 - ( ( 4R , 5R ) - 4 -m e t h o x y - 5 -methyltetrahydrofuran-2-yl)-6-methylnona-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl) acetic acid) was identifiedas a 5-OH derivative of herboxidiene A1 (11).

Structural elucidation of compounds 12 and 8

Purified herboxidiene B1 (12) and 5-hydroxy-herboxidieneB1 (8) were subjected to NMR analyses at 700 MHz inDMSO-d6. These included 1H-NMR of herboxidiene B1(12) (Fig. S8A) and 1H-NMR of 5-hydroxy-herboxidiene B1(8) (Fig. S9A), followed by 13C-NMR of 12 (Fig. S8B) and13C-NMR of 8 (Fig. S9B). Furthermore, two-dimensional

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NMR analyses of 12 and 8 were performed, which includedCOSY (Figs. S8C and S9C), ROESY (Figs. S8D and S9D),HMQC-DEPT (Figs. S8E and S9E), HMBC (Figs. S8F andS9F), and HMBC/HMQC-DEPT (Figs. S8G and S9G) over-lapping studies. By these analyses, herboxidiene B1 (12) was

identified as 2-((2R,5S,6S)-6-((S,2E,4E)-7-((2S,3R,4S,5R,6R)-3-hydroxy-5-methoxy-2,4,6-trimethyltetrahydro-2H-py-ran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl) acetic acid. Similarly, 5-hydroxy-herboxidiene B1 (8) was identified as a 5-OH derivative of

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Fig. 1 Pathway approaches for hydroxylation in herboxidiene fromS. chromofuscus PikC and S. chromofuscus EryF. The compounds 4, 8,11, and 12 were confirmed by NMR analysis, whereas expected

structures of different analogs (3, 5, 6, 7, 9, and 10) of herboxidiene areshown based on mass spectrometry analysis. AC acidic condition

Fig. 2 Pathway approaches forepoxidation in herboxidiene fromS. chromofuscus EpoF. Expectedstructures of different analogs(13 and 14) of herboxidiene areshown based on massspectrometry analysis

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12, represented by 2-((2R,4R,5S,6S)-4-hydroxy-6-((S,2E,4E)-7-((2S ,3R ,4S ,5R ,6R)-3-hydroxy-5-methoxy-2,4,6-trimethyltetrahydro-2H-pyran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl) acetic acid.

Anticancer activities of compounds

Previously, herboxidiene has been reported with anticanceractivity (cytotoxicity) against various cell lines. The effectsof herboxidiene, and its novel analogs, on cancer cell prolif-eration were evaluated. Only herboxidiene (1) showed effec-tive cytotoxic activity against both B16F10 and HeLa cells,but none of the other compounds exhibited any significantcytotoxic activity (Fig. 4) The cytotoxicity of 1 againstHeLa (cervical adenocarcinoma) has been already reported,but in our study, we observed that 1 had activity againstB16F10 (murine metastatic melanoma) cells, indicating thatherboxidiene is able to inhibit cell proliferation efficiently.This is the first report of activity of herboxidiene against met-astatic melanoma cells as B16F10 (Fig. 4).

Antibacterial activity of compounds

The antibacterial activity for all compounds was investigatedby disc diffusion assays against five different human patho-gens including three Gram-positive (M. luteus, B. subtilis, andS. aureus) and two Gram-negative (Pseudomonas aeruginosaand Enterobacter cloacae) bacteria. About 5 μL from100 mM concentrated solutions of each compound dissolvedin DMSO were loaded on a sterile disc with the same concen-tration of herboxidiene (1) as control and placed on a platecontaining an equal number of pathogens spread on MHAplates, and the zone of inhibition was observed. Some of thederivatives of herboxidiene exhibited antibacterial activity atdifferent levels (Table 1). Herboxidiene A1 (11) exhibited theBbest^ activity against S. aureus andM. luteus. Compounds 4

and 8 exhibited activity only against Bacillus, where 4 wassuperior. However, the parental herboxidiene (1) showedno antibacterial activity over the range, indicating that themodification in the core structure was responsible for thetransition of anticancer to antibacterial activity. The re-sults are summarized in Table 1.

Discussion

Natural products produced by living organisms includingplants and microorganisms are used in the pharmaceuticaldrug discovery, agriculture, and food industry. They are com-monly called Bsecondary metabolites,^ and Streptomyces spp.are major producers of such compounds with versatile activ-ities, such as antibiotics, antitumor agents, immunosuppres-sant, and herbicides (Chaudhary et al. 2013). Organic chem-istry methods are routinely used for synthesizing or diversify-ing natural products, but harvesting the product (or a modifi-able precursor) from natural sources is often the most cost-effective way of production. Moreover, chemically syntheticmethods can cause substantial production of hazardous by-products or chemicals (Sanchez et al. 2005). Thus, biologicalprocesses, especially harnessing rational metabolic engineer-ing approaches, are becoming useful tools for the discovery,development, and scale-up of useful compounds (Khosla andKeasling 2003). Combinatorial biosynthesis is one recent met-abolic engineering approach where the genetic constitution ofan organism is altered with a designed genetic circuit forreconstituting a production profile for compounds of interestor benefit.

Previously, regioselective hydroxylation of different mem-bered ring macrolactones with predicted structural alterationshas been used with PikC (Yoon et al. 2002; Lee et al. 2005)and EryF (Lee et al. 2005), although no concrete data aboutthe activities of novel derivatives has been reported. In this

Fig. 3 Pathway approaches for glycosylation in herboxidiene from S. chromofuscus PGYIBR. Expected structures of different analogs (15 and 16) ofherboxidiene are shown based on mass spectrometry analysis

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study, the substrate flexibility of this cytochrome P450 wasassessed over herboxidiene to generate novel hydroxylatedderivatives of herboxidiene. We were also able to isolate twonovel isomers of herboxidiene: one of them containing anextra tetrahydrofuran ring (11) and the other containing anextra tetrahydropyran ring (12) in addition to the singletetrahydropyran ring in the parental structure. Subsequently,both of the products were achieved with hydroxyl group at-tached at the fifth position in the native tetrahydropyran ring.

A possible reason for the formation of this unusual ring struc-ture may be attributable to the cleavage of epoxide bond andsubsequent ring closing at a different center under acidic con-ditions with methanol and water, favoring a nucleophilic at-tack from 18-OH on epoxide and an intramolecular rearrange-ment (Johnson 1999; Hunt 2014). Previously, 11 was reportedusing a chemo-synthetic approach (Edmunds et al. 1997).Moreover, we were able to generate a novel hydroxylatedderivative of 11. However, the formation of 12 with the

Table 1 Antibacterial susceptibility test of novel herboxidiene. Used disc with 5 μl of 100 mM of compound. Assays were done in triplicate

Bacteria strains Compound/inhibition zone (mm, ±1) in diameter at 8 h Compound/inhibition zone (mm, ±1) in diameter at 16 h

(11) (4) (12) (8) (1) (11) (4) (12) (8) (1)

Staphylococcus aureus 13 R R R R 13 R R R R

Bacillus subtilis ND 10 ND 6 R ND ND ND ND R

Micrococcus luteus 15 R ND R R 15 R R R R

(11) = herboxidiene A1, (4) = 5-hydroxy-herboxidiene A1, (12) = herboxidiene B1, (8) = 5-hydroxy-herboxidiene B1, and (1) = herboxidiene

ND not determined, R resistant

Fig. 4 Anticancer activities ofnovel herboxidiene

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additional tetrahydropyran was unique and has not been re-ported by any biological or chemical process before.Compound 8, which is the hydroxylated product of 12, is alsoa novel structure. Evaluation of the anticancer activities of allthese compounds indicated that there was significant loss ofcytotoxic activity against the cell lines tested in comparisonwith the parent molecule. This is consistent with a previousreport where significant loss in activity (>200-fold) was ob-served for a triene compound as compared to its epoxide de-rivative (Lagisetti et al. 2013). Similar result was observed foranother structurally related compound, pladienolide B, whereremoval of the epoxy group reduces splicing inhibition bymore than fivefold, supporting the notion that the epoxy groupmakes a major contribution to splicing inhibition activity(Effenberger et al. 2014). These results are also consistent withearly reports of the dependence of activity on an intact epoxidegroup in several semisynthetic analogs of herboxidiene (Isaacet al. 1992). In another instance, 18-OH, which acts as hydro-gen bond donor represents an important pharmacophore inherboxidiene and is strictly required to effectively interactwith SF3B1. In our case, 11, 4, 12, and 8 obtain additionalring structures with a loss of both of the crucialpharmacophore features—i.e., the 18-OH and epoxygroup—which presumably results in a loss of anticancer ac-tivity (Lagisetti et al. 2013). However, all of these compoundsexhibit differing levels of antibacterial activity against the dif-ferent Gram-positive bacteria tested. The structure-activity re-lationships for diverse compounds showed that compoundscontaining tetrahydrofuran were more active than thetetrahydropyran derivative. Tetrahydrofuran analogs are gen-erally expected to exhibit superior biological activity relativeto tetrahydropyran derivative counterpart, based on favorableentropic factors (Yu et al. 2005).

Similarly, using the substrate-flexible glycosyltransferase,YjiC, which has been characterized for its promiscuous activ-ity towards various small molecules (Pandey et al. 2013,2014) and macrolides (Wu et al. 2012), glycoconjugated de-rivatives of 1 and 2 were generated. Similarly, using the ded-icated epoxidase EpoF involved in the formation of the epox-ide ring between C12 and C13 in epothilone, an anticancercompound (Park et al. 2008), epoxidated derivatives 14 and13 were generated successfully. The key challenge in combi-natorial biosynthesis involving the modular PKS is the signif-icantly reduced productivity of the hybrid system, where theyield of new compounds may be 100-fold lower in compari-son with other products, complicating the processes of purifi-cation and precise structural characterization (Yoon et al.2002). In our study as well, we were not able to generate allof these compounds in significant amounts, whereas the large-scale fermentation for hydroxylation yielded enough titer atthe same conditions. Thus, we were unable to characterize allthe compounds byNMR.Glycosylation affects themajor drugproperties, such as pharmacokinetics, pharmacodynamics,

solubility, and potency (Gantt et al. 2011). Furthermore, thesugar residues can enhance the in vitro uptake through thesugar transporter GLUT, which may be overexpressed in tu-mors, improving the oral bioavailability and in some casesenhancing the biological activity of the compounds (Le et al.2014). The prevalence of epoxide groups has been closelyassociated with the biological potency of different naturalproducts by direct interaction with the target through covalentinteraction (Piggott and Karuso 2004), and in herboxidiene aswell, a loss of the epoxide moiety causes a significant loss inactivity (Lagisetti et al. 2013). Thus, it can be presumed withadequate confidence that the novel glycoconjugates orepoxidated derivatives of herboxidiene may possess betteractivities, which can be harnessed further by enhancing theproduction by using knowledge on metabolic flux and hostengineering.

In conclusion, the present study shows that combinatorialbiosynthesis can be a rational tool for redesigning the struc-tural aspects of herboxidiene. S. chromofuscus was used as ahost for the expression of genes from various sources, includ-ing Gram-negative bacteria, such as E. coli, and Gram-posit ive bacteria, such as Bacillus sp. and otherAc t i nomyce t e s s t r a i n s . Thu s , we be l i e v e t h a tS. chromofuscus can be used as efficient catalytic machineryfor harnessing the substrate flexibility of genes from othergene clusters that will adopt herboxidiene as a substrate.Moreover, the feasibility of easy genetic manipulation andoptimized medium parameters makes it a good target for hostengineering for characterizing the novel metabolites to a great-er extent. We believe that similar approaches can be applied tostructurally related or diverse compounds produced by otherActinomycetes.

Acknowledgments This work was supported by the National ResearchFoundation of Korea (NRF) grant funded by the Korean government(MEST) (NRF-2014R1A2A2A01002875).

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