Structural modification of herboxidiene by substrate ...

Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6431-6

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Structural modification of herboxidiene by substrate-flexible cytochrome 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 by Streptomyces chromofuscus exhibiting herbicidal activity as well as antitumor properties. Using different substrateflexible cytochrome P450s and glycosyltransferase, different novel derivatives of herboxidiene were generated with structural modifications by hydroxylation or epoxidation or conjugation with a glucose moiety. Moreover, two isomers of herboxidiene containing extra tetrahydrofuran or tetrahydropyran moiety in addition to the existing tetrahydropyran moiety were characterized. The hydroxylated products for both of these compounds were also isolated and characterized from S. chromofuscus PikC harboring pikC from the pikromycin gene cluster of Streptomyces venezuelae and S. chromofuscus EryF harboring eryF from the erythromycin gene cluster of Saccharopolyspora erythraea. The compounds generated were characterized by high-resolution quadrupole-time-of-flight electrospray ionization mass spectrometry (HR-QTOF-ESI/MS) and 1H- and 13C-nuclear magnetic resonance (NMR) analyses. The evaluation of antibacterial activity against three Gram-positive bacteria, Micrococcus luteus, Bacillus subtilis, and Staphylococcus aureus, indicated that modification resulted in a transition from anticancer to antibacterial potency. 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 of BT-Convergent Pharmaceutical Engineering, Sun Moon University, 70, Sunmoon-Ro221, Tangjeong-myeon, Asan, Chungnam 333-708, Republic of Korea e-mail: [email protected] Y. J. Yoon Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Republic of Korea

Keywords Streptomyces . Glycosylation . Hydroxylation . Epoxidation . Antibacterial

Introduction Herboxidiene, characterized structurally by its cis-substituted tetrahydropyran acetic acid moiety and side chain consisting of an E-E-conjugated diene (Edmunds et al. 1997), was isolated from Streptomyces chromofuscus A7847 (ATCC 49982) as a novel polyketide with effective herbicidal activity against several annual weeds (Wideman et al. 1992; Isaac et al. 1992). There was revised interest in the biological activities of this compound, because of its anticholesterol (Koguchi et al. 1997) and antitumor activities (Sakai et al. 2002; Hasegawa et al. 2011). Advanced studies at the molecular level have shown that it exhibits antitumor activity by targeting the spliceosome, particularly by inhibiting constitutive splicing targeting spliceosomal subunit SF3b (Gao et al. 2013; Effenberger et al. 2014; Hasegawa et al. 2011; Lagisetti et al. 2013). A flurry of reports revealed that genes encoding splicing factors, including the drug target splicing factor 3B subunit 1 (SF3B1), are among the most highly mutated in various malignancies. The drugs targeting the spliceosome generally alter gene expression, including alternative splicing of genes that are important in cancer progression (Bonnal et al. 2012). Due to its high potency, several successful chemical synthetic approaches have been attempted for preparing herboxidiene (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; Premraj et al. 2012; Yadav et al. 2014) and its totally synthetic biologically active analog, 6-norherboxidiene (Lagisetti et al. 2013).

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However, these multistep chemical processes may not be efficient or flexible enough for the synthesis of new herboxidiene analogs. Thus, to open up new avenues for biochemical characterization and a surge in the possibilities of generating new analogs of herboxidiene, the 53-kb biosynthesis gene cluster for herboxidiene was examined by genome sequencing and gene inactivation studies (Shao et al. 2012). Moreover, characterizations of some key steps in biosynthetic pathway were attempted and new analogs were generated through gene inactivation (Yu et al. 2013, 2014). Similarly, enhanced production of herboxidiene itself by a metabolic engineering approach was also accomplished (Jha et al. 2014). Developing a Bgood^ drug from a natural product is a great challenge, because there are stringent requirements for improving properties such as absorption, distribution, metabolism, excretion, and toxicity, while maintaining acceptable potency, which can be time consuming and expensive (Menzella and Reeves 2007). Thus, some new emerging techniques such as Bcombinatorial biosynthesis,^ based on precise genetic manipulation of the enzymes for natural products, may help in developing products with better pharmacodynamic and pharmacokinetic properties. The basic concept of this approach uses combining and recombining the metabolic pathways of different organisms at a genetic level to create a desired genetic scaffold. These technologies mostly rely on enzymes with broad substrate tolerances, such as glycosyltransferases, to achieve combinatorial attachment of sugars to natural product aglycons (Blanchard and Thorson 2006; Wohlert et al. 1998; Sanchez et al. 2005; Salas and Méndez 2009) or combinatorial assembly of complex multi-modular enzymes, such as polyketide synthase (PKS) and NRPS (Menzella and Reeves 2007; Wong and Khosla 2012). The cytochrome P450 genes, integrated in biosynthetic pathways, are capable of aliphatic and aromatic bond hydroxylation, double-bond epoxidation, heterocyclization, aryl and phenolic ring coupling, oxidative rearrangements of carbon skeletons, and C–C bond cleavage in different natural products, with high chemo-, regio-, and stereo-selectivity (Podust and Sherman 2012). Glycosylation has also been an effective tool for the diversification of natural products (Simkhada et al. 2010) by enhancing the solubility and stability, broadening the biological potency and applications (Singh et al. 2012), and sometimes even altering the biological properties of the compounds (Kren and Martinkova 2001). Thus, manipulations using these genes may provide a platform for introducing the desired structural variability and creating designer molecules with new biological activities. Here, we explored the generation of new herboxidiene analogs by applying combinatorial biosynthesis strategies to S. chromofuscus, where the host strain was crafted in terms of its in vivo catalytic machinery for modifying herboxidiene using selected substrate-flexible cytochrome P450 from different biosynthetic gene clusters prevalent in different

actinomycetes and a flexible glycosyltransferase. Here, the native producer strain was engineered metabolically for hydroxylation using a dedicated hydroxylase pikC from the pikromycin gene cluster of Streptomyces venezuelae ATCC 15439 and eryF from the erythromycin gene cluster of Saccharopolyspora erythraea NRRL 2338 or epoxidation using epoxidase, epoF, from the epothilone gene cluster of Sorangium cellulosum strain So ce90 and glycosylation using YjiC, a flexible glycosyltransferase from Bacillus licheniformis DSM 13 (ATCC 14580). From these approaches, we were able to generate different derivatives of herboxidiene. Among them, some compounds (11, 4, 12, and 8) had remarkable antibacterial activity against Gram-positive bacteria, whereas other derivatives showed mild or no activity in comparison to the parent compound. The assessment of cytotoxicity induced by compounds indicated that there was a loss of cytotoxicity in all compounds, while the parent compound retained effective cytotoxic potency.

Materials and methods Microorganisms and vectors All the plasmids and bacterial strains used in this study are listed in Table S1. The pGEM-T Easy Vector (Promega, Madison, WI, USA) was used to clone the polymerase chain reaction (PCR) products. pGEM-3Zf + (Promega) was used as a subcloning vector. Similarly, pIBR25 (Sthapit et al. 2004) was used as the expression vector and pSET152 (Bierman et al. 1992) was used as the integration vector for the genetic engineering of S. chromofuscus. DNA manipulations were carried out in Escherichia coli XL1 Blue MRF (Stratagene, La Jolla, CA, USA). E. coli JM110 was used for the propagation of nonmethylated DNA. E. coli strains were cultivated at 37 °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 genetically engineered hosts were cultured in different media for different purposes. ISP medium 2 (yeast extract 0.4 %, malt extract 1 %, and glucose 0.4 %) was used as a seed medium and R2YE as a regeneration medium for recombinants. The regeneration medium was supplemented with apramycin or thiostrepton, as required. Previously optimized high production 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, supplementation with 4 % glucose was used to increase the pool of glucose 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 instructions. PCR was performed in a Thermal Cycler Dice (Takara). The amplification conditions for PCR were as follows: an initial denaturation at 94 °C for 7 min, 30 cycles of denaturation at 94 °C for 1 min, annealing at 55–65 °C for 1 min, extension at 72 °C for 1 min, and a final extension at 72 °C for 7 min. The PCR products were purified and cloned into the pGEM-T Easy Vector for DNA amplification and sequencing. DNA preparation, digestion, ligation, and other DNA manipulations were performed using standard techniques for E. coli. The chemicals and enzymes used in this study were purchased from Sigma (St. Louis, MO, USA). In silico analyses and comparisons of nucleotide and protein sequences were performed using BLAST, FASTA, and ClustalW. Construction of the recombinant vector The pIBR25 and pSET152, under the strong ermE* promoter, were used for the construction of recombinants. The primers used for amplification of specific DNA fragments are listed in Table S2. The PCR products of yjiC (AE017333.1) and galU (NC_007779.1) were isolated from the genomic DNA of B. 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) were isolated from the genomic DNA of S. venezuelae ATCC 15439, S. erythraea NRRL 2338, and S. cellulosum So ce90, respectively. The PCR products were purified and cloned into the pGEM-T Easy Vector and sequenced prior to cloning in the expression vector and integrative vector to verify that no mutation had been introduced during the PCR amplification. After the sequence was analyzed, pikC and eryF were cloned into the BamHI and XbaI sites of pSET152 to form the recombinant plasmids pPikC152 and pEryF152, respectively. The epoF along with orf6 was cloned into PacI and HindIII sites of pSET152 to form the recombinant plasmid pEpoF152. Similarly, yjiC was cloned in BamHI/EcoRI and galU in EcoRI/HindIII sites, respectively, for expression of both genes from a single vector, pIBR25, to construct pGYIBR. Transformation and generation of recombinant strains The preparation of protoplasts, transformation, and selection of S. chromofuscus transformants were carried out according to standard protocols (Kieser et al. 2000). All the recombinants pGYIBR, pEryF152, pPikC152, and pEpoF152 including empty vectors pIBR25 and pSET152

were propagated in E. coli JM110 to obtain demethylated DNA for transformation in the Streptomyces strain. After demethylation of all the recombinant vectors, they were introduced into S. chromofuscus by polyethylene glycol (PEG)mediated protoplast transformation. For protoplast transformation, the native producer strain S. chromofuscus was cultured in 50 mL of seed medium. After 60 h, the mycelium was harvested by centrifugation (3200 rpm, 12 min, 4 °C) and washed with 15 mL of sucrose solution (10.3 %) and further washed with 15 mL of P buffer. Finally, 10 mL of lysozyme solution (2 mg/mL in P buffer) was added to the cell pellets and the content was incubated for 50 min at 37 °C. After incubation, the mix was filtered and centrifuged (6000 rpm, 12 min), then washed with P buffer twice, and mixed with 1 mL of P buffer. Next, 100 μL of the resulting mix was added to 20 μL of plasmid DNA and 200 μL of 40 % PEG 1000 and centrifuged for 1 min. The supernatant was partially discarded and then mixed with 100 μL of P buffer. Finally, it was plated on R2YE plates. The plates were incubated at 28 °C for 24 h and then overlaid with 0.3 % agar solution containing 10 μg/ mL thiostrepton or 60 μg/mL apramycin for selecting recombinants containing the expression or integrative vector, respectively. After 1 week, thiostrepton- or apramycinresistant colonies, as appropriate, were selected and cultured in liquid ISP medium 2. Transformation of each strain was confirmed by isolation of the plasmid, PCR, and restriction enzyme digestion. The transformants w e r e d e s i g n at e d a s S. c h romo fu sc u s p G Y I B R , S . c h ro m o f u s c u s E r y F, S . c h ro m o f u s c u s P i k C , S. chromofuscus EpoF, S. chromofuscus IBR25, and S. chromofuscus SET152, respectively (Table S1). Morphological analysis of S. chromofuscus and transformant S. chromofuscus, S. chromofuscus IBR25, S. chromofuscus pGYIBR, S. chromofuscus SET152, S. chromofuscus EryF, S. chromofuscus PikC, and S. chromofuscus EpoF were streaked on a R2YE agar plate and incubated at 28 °C for 8 days under continuous observation. The growth pattern and morphological attributes were analyzed by visual observation by the naked eye. Production, extraction, and purification of novel herboxidiene To analyze the novel herboxidiene production, 5 % of the seed of S. chromofuscus pGYIBR, S. chromofuscus EryF, S. chromofuscus PikC, and S. chromofuscus EpoF were grown in 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 extracted with a double volume of ethyl acetate, and the extract was dried under reduced pressure using a rotary evaporator and 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 a UV detector (236 nm) with a solvent system consisting of water (0.025 % trifluoroacetic acid) and 100 % methanol with a flow rate of 1 mL/min for 71 min. The methanol concentrations 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) connected to a UV detector (236 nm) over 81 min using binary conditions with water (0.025 % trifluoroacetic acid) and methanol of 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), and 10 % (75–80 min). Detection and mass analysis Ultra-pressure liquid chromatography (UPLC)-photodiode array (PDA) analysis was performed using a reversed-phase UPLC-PDA with a C18 column (ACQUITY UPLC BEH, C18, 1.7 μm) connected to a PDA (UPLC LG 500 nm) at an absorbance of 236 nm. The binary mobile phases were composed of same solvents as described previously. Total flow was maintained at 0.3 μL/min for 15 min. The flow of methanol 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 then stopped at 15 min. For exact mass analysis, a high-resolution quadrupole-time-of-flight electrospray ionization mass spectrometry (HR-QTOF-ESI/MS) analysis was performed in positive ion mode using an ACQUITY (Billerica, USA) column coupled with a SYNAPT G2-S (Waters Corp.) column.

Evaluation of anticancer activities of compounds To evaluate the effects of the different novel analogs of herboxidiene on the proliferation and viability of B16F10 melanoma and HeLa cervical cancer cells, cells were grown in a Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum (FBS; Invitrogen). All cells were maintained at 37 °C in a humidified 5 % CO2 incubator. For cell growth assays, cells seeded at 2000 cells/well in 96-well plates (SPL Life Sciences, Gyeonggi, Korea) were treated with each compound at various concentrations for 72 h. Cell growth 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 of herboxidiene purified from different transformants of S. chromofuscus was assayed for antibacterial activity against three different pathogenic Gram-positive bacteria— Staphylococcus aureus subsp. aureus KCTC 1916, Bacillus subtilis KACC 17047, and Micrococcus luteus KACC 13377—using a paper disc diffusion method on MuellerHinton agar (MHA) plates. The inocula containing 107 CFU/mL were spread on MHA plates for the bioassay. The sterile filter paper discs (6 mm in diameter) containing 5 μL of 100 mM compounds were placed on the surface of the inoculated agar plates. The plates were incubated at 37 °C for up to 2 days under continuous observation. Each compound was tested in triplicate, and the zone of inhibition was measured in millimeters in diameter.

Result Construction of recombinant vectors and recombinant strains of S. chromofuscus

Nuclear magnetic resonance analysis To characterize the novel structure of the herboxidienes, each purified product was dried under reduced pressure using a rotary evaporator followed by freeze-drying. To remove the trace of water, the samples were treated with deuterium oxide repeatedly, followed by freeze-drying. Finally, the freezedried samples were resuspended in 500 μL dimethyl sulfoxide-d6 (DMSO-d6) and assessed in a 700-MHz spectrometer to determine 1H-nuclear magnetic resonance (NMR), 13CNMR, two-dimensional NMR correlation spectroscopy (COSY), nuclear Overhauser effect spectroscopy (NOESY), rotational frame NOE spectroscopy (ROESY), heteronuclear single quantum correlation (HSQC), and heteronuclear multiple-bond correlation (HMBC).

The glucose-1-phosphate uridylyltransferase (galU) from E. coli K12 and the UDP-glucosyltransferase (yjiC) from B. licheniformis DSM 13 were cloned together into pIBR25 (high copy expression vector) under the control of a strong promoter, ermE*, to construct the recombinant plasmid pGYIBR. The pikC, cytochrome P450 monooxygenase in the pikromycin biosynthetic gene cluster from S. venezuelae, was cloned into pSET152 (integration vector) under the control of a strong promoter, ermE*, to construct pPikC152. Similarly, eryF, a cytochrome P450, characterized as having hydroxylation activity in the biosynthetic pathway of erythromycin to yield erythronolide B, was also cloned into pSET152 under the control of the strong promoter, ermE*, to construct pEryF152. 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 promoter ermE* to construct the pEpoF152 recombinant plasmid (Park et al. 2008). All the recombinant vectors were confirmed by restriction digestion analysis and transformed into S. chromofuscus. The resulting recombinants of S. chromofuscus were designated as S. chromofuscus pGYIBR, S. chromofuscus IBR25, S. chromofuscus PikC, S. chromofuscus EryF, S. chromofuscus EpoF, and S. chromofuscus SET152. Effect of recombinant plasmids on growth of recombinant strains of S. chromofuscus Surprisingly, we observed that the morphologies of S. chromofuscus PikC, S. chromofuscus EryF, and S. chromofuscus EpoF were markedly different from S. chromofuscus wild type and S. chromofuscus SET152 on R2YE plates and were found to grow very slowly, whereas S. chromofuscus SET152 exhibited similar growth to that of S. chromofuscus (Fig. S1A). Similarly, S. chromofuscus pGYIBR was significantly different and grew slowly on the plate, whereas S. chromofuscus wild type and S. chromofuscus IBR25 exhibited similar growth (Fig. S1B). HR-QTOF-ESI/MS analysis of derivatives of herboxidiene To analyze the novel herboxidienes, S. chromofuscus PikC and S. chromofuscus EryF were cultured in 3 L of production media No. 6A6. The culture broths were harvested, centrifuged, extracted, and injected into a HPLC system. The HPLC, coupled with HR-QTOF-ESI/MS analysis, showed different novel hydroxylated herboxidiene products represented by herboxidiene A1 (11) (Edmunds et al. 1997), 5hydroxy-herboxidiene A1 (4), 5-hydroxy-25-demethylherboxidiene A1 (3), 5,6-dihydroxy-herboxidiene A1 (6), 5, 6-dihydroxy-25-demethyl-herboxidiene A1 (5), herboxidiene B1 (12), 5-hydroxy-herboxidiene B1 (8), 5-hydroxy-25demethyl-herboxidiene B1 (7), 5,6-dihydroxy-herboxidiene B1 (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 and S. chromofuscus EryF showed the same HPLC profile and mass spectrum. Finally, compounds 11 (~40 mg), 4 (~35 mg), 12 (~42 mg), and 8 (~34 mg) were obtained after purification and characterized by NMR analysis. Compounds 3, 6, 5, 7, 10, and 9 were not produced in sufficient amounts

for a detailed analysis by NMR. On the basis of these results, a pathway was proposed for hydroxylation (Fig. 1). Analysis of extracted compound from S. chromofuscus EpoF showed different novel herboxidiene products, represented 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 14 were not produced in sufficient quantities for analysis by NMR, whereas compound 1 was reported previously (Jha et al. 2014). These results supported the proposed pathway of epoxidation (Fig. 2). Analysis of compounds extracted from S. chromofuscus pGYIBR showed different novel herboxidiene products, represented by 18-O-β-D-glucopyranoside herboxidiene (16) and 18-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 pathway of glycosylation (Fig. 3). Structural elucidation of novel herboxidiene analogs by NMR Purified herboxidiene A1 (11) and 5-hydroxy-herboxidiene A1 (4) were subjected to NMR analyses at 700 MHz in DMSO-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 further structural determination, two-dimensional NMR analyses of 11 and 4 were performed, which included COSY (Figs. S6C and S7C), ROESY (Figs. S6D and S7D), HMQC-DEPT (Figs. S6E and S7E), HMBC (Figs. S6F and S7F), and HMBC/HMQC-DEPT (Figs. S6G and S7G) overlapping studies. By these analyses, herboxidiene A1 (11) was identified, which has been synthesized previously by chemical methods (Edmunds et al. 1997). Similarly, 5-hydroxyherboxidiene A1 (4) (2-((2R,4R,5S,6S)-4-hydroxy-6-((2E,4E, 6S,8R)-8-hydroxy-8-((4R,5R)-4-methoxy-5methyltetrahydrofuran-2-yl)-6-methylnona-2,4-dien-2-yl)-5methyltetrahydro-2H-pyran-2-yl) acetic acid) was identified as a 5-OH derivative of herboxidiene A1 (11). Structural elucidation of compounds 12 and 8 Purified herboxidiene B1 (12) and 5-hydroxy-herboxidiene B1 (8) were subjected to NMR analyses at 700 MHz in DMSO-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) and 13 C-NMR of 8 (Fig. S9B). Furthermore, two-dimensional

Appl Microbiol Biotechnol OH

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Fig. 1 Pathway approaches for hydroxylation in herboxidiene from S. 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 are shown based on mass spectrometry analysis. AC acidic condition

NMR analyses of 12 and 8 were performed, which included COSY (Figs. S8C and S9C), ROESY (Figs. S8D and S9D), HMQC-DEPT (Figs. S8E and S9E), HMBC (Figs. S8F and S9F), and HMBC/HMQC-DEPT (Figs. S8G and S9G) overlapping 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-pyran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro2H-pyran-2-yl) acetic acid. Similarly, 5-hydroxyherboxidiene B1 (8) was identified as a 5-OH derivative of

Fig. 2 Pathway approaches for epoxidation in herboxidiene from S. chromofuscus EpoF. Expected structures of different analogs (13 and 14) of herboxidiene are shown based on mass spectrometry analysis

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

12, represented by 2-((2R,4R,5S,6S)-4-hydroxy-6-((S,2E,4E)7-((2S,3R,4S,5R,6R)-3-hydroxy-5-methoxy-2,4,6trimethyltetrahydro-2H-pyran-2-yl)-6-methylhepta-2,4-dien2-yl)-5-methyltetrahydro-2H-pyran-2-yl) acetic acid. Anticancer activities of compounds Previously, herboxidiene has been reported with anticancer activity (cytotoxicity) against various cell lines. The effects of herboxidiene, and its novel analogs, on cancer cell proliferation were evaluated. Only herboxidiene (1) showed effective cytotoxic activity against both B16F10 and HeLa cells, but none of the other compounds exhibited any significant cytotoxic activity (Fig. 4) The cytotoxicity of 1 against HeLa (cervical adenocarcinoma) has been already reported, but in our study, we observed that 1 had activity against B16F10 (murine metastatic melanoma) cells, indicating that herboxidiene is able to inhibit cell proliferation efficiently. This is the first report of activity of herboxidiene against metastatic melanoma cells as B16F10 (Fig. 4). Antibacterial activity of compounds The antibacterial activity for all compounds was investigated by disc diffusion assays against five different human pathogens including three Gram-positive (M. luteus, B. subtilis, and S. aureus) and two Gram-negative (Pseudomonas aeruginosa and Enterobacter cloacae) bacteria. About 5 μL from 100 mM concentrated solutions of each compound dissolved in DMSO were loaded on a sterile disc with the same concentration of herboxidiene (1) as control and placed on a plate containing an equal number of pathogens spread on MHA plates, and the zone of inhibition was observed. Some of the derivatives of herboxidiene exhibited antibacterial activity at different levels (Table 1). Herboxidiene A1 (11) exhibited the Bbest^ activity against S. aureus and M. luteus. Compounds 4

and 8 exhibited activity only against Bacillus, where 4 was superior. However, the parental herboxidiene (1) showed no antibacterial activity over the range, indicating that the modification in the core structure was responsible for the transition of anticancer to antibacterial activity. The results are summarized in Table 1.

Discussion Natural products produced by living organisms including plants and microorganisms are used in the pharmaceutical drug discovery, agriculture, and food industry. They are commonly called Bsecondary metabolites,^ and Streptomyces spp. are major producers of such compounds with versatile activities, such as antibiotics, antitumor agents, immunosuppressant, and herbicides (Chaudhary et al. 2013). Organic chemistry methods are routinely used for synthesizing or diversifying natural products, but harvesting the product (or a modifiable precursor) from natural sources is often the most costeffective way of production. Moreover, chemically synthetic methods can cause substantial production of hazardous byproducts or chemicals (Sanchez et al. 2005). Thus, biological processes, especially harnessing rational metabolic engineering approaches, are becoming useful tools for the discovery, development, and scale-up of useful compounds (Khosla and Keasling 2003). Combinatorial biosynthesis is one recent metabolic engineering approach where the genetic constitution of an organism is altered with a designed genetic circuit for reconstituting a production profile for compounds of interest or benefit. Previously, regioselective hydroxylation of different membered ring macrolactones with predicted structural alterations has been used with PikC (Yoon et al. 2002; Lee et al. 2005) and EryF (Lee et al. 2005), although no concrete data about the activities of novel derivatives has been reported. In this

Appl Microbiol Biotechnol Fig. 4 Anticancer activities of novel herboxidiene

study, the substrate flexibility of this cytochrome P450 was assessed over herboxidiene to generate novel hydroxylated derivatives of herboxidiene. We were also able to isolate two novel isomers of herboxidiene: one of them containing an extra tetrahydrofuran ring (11) and the other containing an extra tetrahydropyran ring (12) in addition to the single tetrahydropyran ring in the parental structure. Subsequently, both of the products were achieved with hydroxyl group attached at the fifth position in the native tetrahydropyran ring.

Table 1

A possible reason for the formation of this unusual ring structure may be attributable to the cleavage of epoxide bond and subsequent ring closing at a different center under acidic conditions with methanol and water, favoring a nucleophilic attack from 18-OH on epoxide and an intramolecular rearrangement (Johnson 1999; Hunt 2014). Previously, 11 was reported using a chemo-synthetic approach (Edmunds et al. 1997). Moreover, we were able to generate a novel hydroxylated derivative of 11. However, the formation of 12 with the

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

Bacteria strains

Staphylococcus aureus Bacillus subtilis Micrococcus luteus

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)

13 ND 15

R 10 R

R ND ND

R 6 R

R R R

13 ND 15

R ND R

R ND R

R ND 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

Appl Microbiol Biotechnol

additional tetrahydropyran was unique and has not been reported by any biological or chemical process before. Compound 8, which is the hydroxylated product of 12, is also a novel structure. Evaluation of the anticancer activities of all these compounds indicated that there was significant loss of cytotoxic activity against the cell lines tested in comparison with the parent molecule. This is consistent with a previous report where significant loss in activity (>200-fold) was observed for a triene compound as compared to its epoxide derivative (Lagisetti et al. 2013). Similar result was observed for another structurally related compound, pladienolide B, where removal of the epoxy group reduces splicing inhibition by more than fivefold, supporting the notion that the epoxy group makes a major contribution to splicing inhibition activity (Effenberger et al. 2014). These results are also consistent with early reports of the dependence of activity on an intact epoxide group in several semisynthetic analogs of herboxidiene (Isaac et al. 1992). In another instance, 18-OH, which acts as hydrogen bond donor represents an important pharmacophore in herboxidiene and is strictly required to effectively interact with SF3B1. In our case, 11, 4, 12, and 8 obtain additional ring structures with a loss of both of the crucial pharmacophore features—i.e., the 18-OH and epoxy group—which presumably results in a loss of anticancer activity (Lagisetti et al. 2013). However, all of these compounds exhibit differing levels of antibacterial activity against the different Gram-positive bacteria tested. The structure-activity relationships for diverse compounds showed that compounds containing tetrahydrofuran were more active than the tetrahydropyran derivative. Tetrahydrofuran analogs are generally expected to exhibit superior biological activity relative to tetrahydropyran derivative counterpart, based on favorable entropic factors (Yu et al. 2005). Similarly, using the substrate-flexible glycosyltransferase, YjiC, which has been characterized for its promiscuous activity towards various small molecules (Pandey et al. 2013, 2014) and macrolides (Wu et al. 2012), glycoconjugated derivatives of 1 and 2 were generated. Similarly, using the dedicated epoxidase EpoF involved in the formation of the epoxide ring between C12 and C13 in epothilone, an anticancer compound (Park et al. 2008), epoxidated derivatives 14 and 13 were generated successfully. The key challenge in combinatorial biosynthesis involving the modular PKS is the significantly reduced productivity of the hybrid system, where the yield of new compounds may be 100-fold lower in comparison with other products, complicating the processes of purification and precise structural characterization (Yoon et al. 2002). In our study as well, we were not able to generate all of these compounds in significant amounts, whereas the largescale fermentation for hydroxylation yielded enough titer at the same conditions. Thus, we were unable to characterize all the compounds by NMR. Glycosylation affects the major drug properties, such as pharmacokinetics, pharmacodynamics,

solubility, and potency (Gantt et al. 2011). Furthermore, the sugar residues can enhance the in vitro uptake through the sugar transporter GLUT, which may be overexpressed in tumors, improving the oral bioavailability and in some cases enhancing the biological activity of the compounds (Le et al. 2014). The prevalence of epoxide groups has been closely associated with the biological potency of different natural products by direct interaction with the target through covalent interaction (Piggott and Karuso 2004), and in herboxidiene as well, a loss of the epoxide moiety causes a significant loss in activity (Lagisetti et al. 2013). Thus, it can be presumed with adequate confidence that the novel glycoconjugates or epoxidated derivatives of herboxidiene may possess better activities, which can be harnessed further by enhancing the production by using knowledge on metabolic flux and host engineering. In conclusion, the present study shows that combinatorial biosynthesis can be a rational tool for redesigning the structural aspects of herboxidiene. S. chromofuscus was used as a host for the expression of genes from various sources, including Gram-negative bacteria, such as E. coli, and Grampositive bacteria, such as Bacillus sp. and other Actinomycetes strains. Thus, we believe that S. chromofuscus can be used as efficient catalytic machinery for harnessing the substrate flexibility of genes from other gene clusters that will adopt herboxidiene as a substrate. Moreover, the feasibility of easy genetic manipulation and optimized medium parameters makes it a good target for host engineering for characterizing the novel metabolites to a greater extent. We believe that similar approaches can be applied to structurally related or diverse compounds produced by other Actinomycetes. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (NRF-2014R1A2A2A01002875).

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