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Jan 22, 2018 - Characterization of a Prenyltransferase for Iso-A82775C Biosynthesis and Generation of New Congeners of Chloropestolides. Yuanyuan Pan,...
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Articles Cite This: ACS Chem. Biol. 2018, 13, 703−711

Characterization of a Prenyltransferase for Iso-A82775C Biosynthesis and Generation of New Congeners of Chloropestolides Yuanyuan Pan,†,∥ Ling Liu,†,∥ Feifei Guan,† Erwei Li,† Jin Jin,‡ Jinyang Li,† Yongsheng Che,*,‡ and Gang Liu*,†,§ †

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 300350, China § University of Chinese Academy of Sciences, Beijing 100049, China ‡

S Supporting Information *

ABSTRACT: Chloropupukeananin and chloropestolides are novel metabolites of the plant endophyte Pestalotiopsis f ici, showing antimicrobial, antitumor, and anti-HIV activities. Their highly complex and unique skeletons were generated from the coisolated pestheic acid (1) and iso-A82775C (10) based on our previous studies. Here, we identified the biosynthetic gene cluster iac of 10 and characterized an iacE encoded prenyltransferase. Deletion of iacE abolished iso-A82775C production, accumulated the prenyl group-lacking siccayne (2), and generated four new chloropestolides (3−6). Compounds 5 and 6 showed antibacterial effects against Staphylococcus aureus and Bacillus subtilis, and 5 was also cytotoxic to human tumor cell lines HeLa, MCF-7, and SW480. These results provided the first genetic and biochemical insights into the biosynthesis of natural prenylepoxycyclohexanes and demonstrated the feasibility for generation of diversified congeners by manipulating the biosynthetic genes of 10. α−β−β−α architecture,16 are found exclusively in secondary metabolism and can be divided into two discrete families of phenol/phenazine and indole prenyltransferases based on substrate specificity. The first family is mainly found in bacteria, catalyzing the prenylation of naphthoquinones, hydroxybenzoates, phenazines, or benzodiazepines, while the indole counterpart is found in both bacteria and fungi.17 The ABBA prenyltransferases generally catalyze prenylation of aromatics by electrophilic substitution and share promiscuity for different substrates. Although the aromatic prenyltransferases are welldocumented in primary and secondary metabolisms, a prenyltransferase using the epoxycyclohexane scaffold as a substrate has yet been reported. Considering that chloropupukeananin and chloropestolides were assembled from the coisolated 1 and 10, it is logical to generate congeners of these novel metabolites by hybridizing the gene-disruption products of the precursors. For this purpose, we first performed targeted deletion of the putative prenyltransferase gene in the biosynthesis of 10 and characterized a prenyltransferase IacE as the key enzyme to catalyze the prenylation step. By searching the DNA sequences surrounding iacE, 10 putative open reading frames (ORFs) were found in the cluster, four of which were identified as the key genes with proposed respective roles. In addition, new

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tructurally diverse bioactive natural products are frequently isolated from the plant endophytic fungi and have attracted increasing attention due to their pharmacological and ecological significance.1 Notable examples include anticancer drugs paclitaxel and podophyllotoxin,2,3 the antibiotics altenusin,4 and the phytohormones indoleacetic and abscisic acids.5,6 Chloropupukeananin and chloropestolides A−D (11−14), the first chlorinated tricyclo-[4.3.1.03,7]-decane and bicyclo[2.2.2]oct-2-en-5-one derived metabolites, were isolated from a plant endophytic Pestalotiopsis f ici with antimicrobial, antitumor, and anti-HIV effects, and could be generated from the coisolated pestheic acid (1) and iso-A82775C (10) based on the structures of the coisolated precursors, intermediates, and final products.7−9 We recently identified the biosynthetic gene cluster pta for 1 and proposed its biosynthetic pathway.10 Precursor 10 is a new stereoisomer of A82775C,11 both are epoxycyclohexanes incorporating the prenyl and exocyclic isopropenylallene moieties as found in jesterone and ambuic acid.12,13 Although the total synthesis of 10 has been achieved,14 its biosynthesis remained unknown at the genetic level. Prenylation and postmodifications significantly expand the diversity and pharmacological profiles of natural products.15,16 Aromatic prenyltransferases catalyze the transfer of isoprenyl unit(s) to aromatic substrates to form C−C, C−O, or C−N bond(s) via electrophilic alkylation, an important step often leading to the production of diverse structures in plants, fungi, and bacteria.17,18 The ABBA prenyltransferases, a new superfamily of aromatic prenyltransferases consisting of the typical © 2018 American Chemical Society

Received: December 12, 2017 Accepted: January 22, 2018 Published: January 31, 2018 703

DOI: 10.1021/acschembio.7b01059 ACS Chem. Biol. 2018, 13, 703−711

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Figure 1. Gene iacE, essential for iso-A82775C production in P. f ici. (A) Structures of representative prenylepoxycyclohexanes. Red rectangles indicate the prenyl group. (B) Screening diagram of prenyltransferase encoding genes by transcriptional analysis, gene knockout, and HPLC analysis. HPLC (C) and HPLC-MS (D) analysis of 10 in WT, ΔiacE, and the complemented strain iacEC. All strains were cultured on rice for 7 days.

Figure 2. Biosynthetic gene (iac) cluster for iso-A82775C. (A) Organization of iac. Genes are indicated by different arrowheads according to their putative roles. (B) Sequence similarities and predicted functions of the deduced proteins. (C) Transcriptional analysis of the genes in iac by RTPCR. Transcript of actin gene was used as the positive control (since iacD, iacF, and iacG each contain an intron, smaller and larger PCR products reflect amplification of the cDNA and genomic DNA, respectively; the primers are listed in Table S5).

disruption mutant of iacE (scaffold 1511−395) did not produce 10 (Figure 1C and D). The iacE gene of 1383 bp is interrupted by a 63 bp intron, and its deduced product comprises 439 amino acids with a calculated MW of 49.14 kDa, showing high sequence similarity to aromatic prenyltransferases. The complemented strain of ΔiacE restored iso-A82775C production (Figure 1C and D), indicating that iacE is essential for its biosynthesis in P. f ici. Identification of Biosynthetic Gene Cluster. A 43.43 kb contiguous DNA fragment surrounding iacE was analyzed (Figure 2A), and 11 ORFs were predicted besides iacE (Figure 2B). The gene iacA encodes a putative tyrosinase, while the deduced product of iacB showed 45% identity to the hypothetical protein BBA_01712 from Beauveria bassiana ARSEF 2860. Three genes, iacC, iacG, and iacJ, encode the short chain dehydrogenase/reductase (SDR) proteins.19,20 The deduced iacC product has the typical motifs for TGXXXGXG cofactor binding and the SYXXXK active site, showing 30 and 20% identity to Nor-1 in aflatoxin biosynthesis in Aspergillus parasiticus21 and BtrF in butirosin biosynthesis in Bacillus

congeners, named chloropestolides H−K (3−6), were generated by disrupting iacE, and two of them showed antimicrobial activity against the Staphylococcus aureus and Bacillus subtilis, while one of them was also cytotoxic to human tumor cells, HeLa, MCF-7, and SW480.

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RESULTS AND DISCUSSION A Prenyltransferase Encoding Gene Essential for IsoA82775C Biosynthesis. Metabolite 10 is structurally related to A82775C, spartinoxide, jesterone, and ambuic acid (Figure 1A), all are prenylepoxycyclohexanes with unknown biosynthetic gene clusters or pathways. By searching the genome of P. f ici, 24 putative prenyltransferase genes were identified, and 14 candidates were expressed, leading to the production of 10 (Figure 1B). To identify the gene(s) for prenylation, eight of the 14 genes were disrupted by homologous recombination (HR) and verified by PCR (Figures 1B and S1). The extracts of these mutants were analyzed by HPLC-MS using WT as the control, which showed a distinct peak of 10 with a retention time of 15.4 min. HPLC-MS analysis revealed that the 704

DOI: 10.1021/acschembio.7b01059 ACS Chem. Biol. 2018, 13, 703−711

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Figure 3. HPLC analyses of the extracts of WT and individual gene disruption mutants cultured on rice for 7 days, detected at 220 nm. a, isosulochrin; b1, ficipyrone A; b2, RES-1214-1; b3, pestheic acid (1); c, pestalodiol C; d, dibutyl phthalate. 7 was accumulated in ΔiacF.

Scheme 1. Proposed Biosynthetic Pathways of iso-A82775C and the Chloropestolides

and ΔiacH produced the same level of 10 as WT. Collectively, iacC, iacE, iacF, and iacG are the key genes for iso-A82775C biosynthesis. The major peaks in the HPLC chromatogram of the WT extract were identified by comparison of their retention times (tR’s) and HRESIMS data with those of authentic samples (Figure S3). Peak a (tR = 11.53 min; HRESIMS m/z [M + H]+ 333.0970) was readily identified as isosulochrin, an intermediate in the biosynthesis of 1.24 While peak b (tR = 14.25 min) is a cluster of three peaks (b1−b3) as revealed by HPLC analysis of a diluted sample, which were identified sequentially as ficipyrone A (HRESIMS m/z [M + H]+ 271.1548),25 RES1214-1 (HRESIMS m/z [M + H]+ 349.0928),26 and 1.24 Peak c (tR = 15.83 min; HRESIMS [M + Na]+ 327.1565) was attributed to pestalodiol C, a monoacetate of 10. While peak d (tR = 17.97 min; HRESIMS m/z [M + H]+ 279.1595) was dibutyl phthalate,27 a common contaminant encountered during purification processes. Except for pestalodiol C, all compounds are irrelevant to iso-A82775C biosynthesis, leading to the speculation that these compounds and other intermediates produced in the disruption mutants were beyond the limit of detection. Proposed Biosynthetic Pathway. HPLC analyses of the extracts for large-scale fermentation cultures of WT, ΔiacC, ΔiacE, ΔiacF, and ΔiacG showed significantly different chromatograms (Figures 3 and S4A). The intermediate accumulated in ΔiacE (Figure S4B) was identified as 2 by comparison of its HRESIMS (m/z [M + H]+ 175.0756) and NMR data with literature values,28 which lacks the prenyl group in the epoxyhexane moiety (Figures S4C and S5). The structure for the intermediate (7) accumulated in ΔiacF, was elucidated based on the MS and NMR data. Analysis of

circulans,22 respectively. Since both enzymes catalyzed the ketohydroxy reactions, IacC might play a similar role in isoA82775C biosynthesis. IacD carries an EthD domain, while iacF encodes a cupin family of protein. Despite the cupin superfamily of proteins exhibiting low sequence similarity, they share a common six-stranded β-barrel core architecture and show various functions by serving as dioxygenases, decarboxylases, isomerases, hydrolases, and epimerases.23 Although IacC and IacG belong to the SDR family, IacG has lost the motifs typical for cofactor binding and catalytic sites, indicating that it uses substrates different from those of IacC. A putative oxidase encoded by iacH possesses a FAD binding domain, showing 71% identity to a hypothetical protein from Rosellinia necatrix. Two putative regulatory genes, iacI and iacK, encode putative zinc-finger transcriptional factors. Transcription of the iacB−iacK genes showed similar patterns (Figure 2C), with iacA upstream of iacB remaining silent regardless of iso-A82775C production, but orf1 downstream of iacK was constitutively expressed even when 10 was not produced. To determine the boundary of iac, iacA and orf1 were disrupted, and the resulting mutants still produced the same level of 10 as WT (data not shown), indicating that they localize outside iac. Therefore, iac contains a 34.55 kb insert (GenBank accession no. KU963195) and 10 complete ORFs. HPLC Analysis of Gene Disruption Mutants. The remaining nine genes in iac were also disrupted by HR, and the resulting mutants were verified by PCR (Figure S2). HPLC analyses of the extracts from all disruption mutants except for two regulatory genes revealed abolishment of iso-A82775C production in ΔiacC or ΔiacF, but accumulation of a new metabolite in ΔiacF (Figure 3). In addition, ΔiacG produced much less, ΔiacD or ΔiacJ produced slightly less, while ΔiacB 705

DOI: 10.1021/acschembio.7b01059 ACS Chem. Biol. 2018, 13, 703−711

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Figure 4. IacE catalyzed prenylation of epoxycyclohexane in vitro. (A) Purification of IacE-His6 after expression in E. coli. (B) HPLC analysis of the products from the reaction of heat-inactivated or native IacE-His6 with 2. (C) HPLC-MS analysis of 7 produced in IacE-catalyzed reaction. (D) IacE catalyzed conversion from 2 to 7. (E) The activities of IacE with different metal ions added.

HRESIMS data (m/z [M + H]+ 243.1384; C16H18O2) and NMR data (Table S1) revealed the same (3-methylbut-3-en-1yn-1-yl)benzene moiety as found in 2. HMBC correlations from H3-1 and H3-2 to C-3 and C-4; from H2-5 to C-3, C-4, C6, and C-7; and from H-7 to C-5 completed the C-1−C-5 subunit. Correlations from OH-8 to C-7, C-8, and C-9 and from OH-16 to C-6, C-10, and C-16 located two hydroxy groups at C-8 and C-16, respectively, completing the gross structure of 7 (Figure S6). On the basis of their structures, 2 and 7 are the substrates for IacE and IacF, respectively. The intermediate accumulated in ΔiacG was identified as a diisoprenyl-cyclohexene-type meroterpenoid by comparison of its HRESIMS (m/z [M + Na]+ 283.1310) and NMR data with those reported,29 which was renamed pestalodiol F (8) for the convenience of description. Similarly, the intermediate accumulated in ΔiacC was identified as pestalofone A (9).30 On the basis of genetic and chemical analyses, we proposed the biosynthetic pathway of 10. Prenylation of 2 by IacE generates 7, which is epoxidized to 8 by IacF and other enzymes, from which 9 is generated via the reactions catalyzed by IacG and other enzymes, and 10 is subsequently generated by IacC and other enzymes (Scheme 1). Biochemical Assay of IacE. Some currently available aromatic prenyltransferases were phylogenetically analyzed (Figure S7), and the cladogram showed that IacE resembled 7-DMATS, which catalyzes the prenylation of L-tryptophan (LTrp) at C-7 of the indole moiety in the presence of dimethylallyl diphosphate (DMAPP). Therefore, IacE likely catalyzes the prenylation of aromatic compounds or their derivatives. After expression in E. coli BL21 (DE3), his-tagged IacE was purified to homogeneity (Figure 4A), and a new product was obtained using 2 as the substrate (Figure 4B). Further results showed that IacE accepted DMAPP, not geranyl diphosphate, as a prenyl group donor (data not shown). The enzymatic product was isolated and purified by HPLC, and its

structure was established as 7 by analysis of its MS (Figure 4C) and NMR data (Table S1). Since it was also isolated from ΔiacF, the C-6 prenyl group in 2 is regioselectively catalyzed by IacE in P. fici (Figure 4D). Enzymic Activity and Substrate Specificity of IacE. We investigated whether the enzymatic reaction was dependent on divalent metal ions. The activity was arbitrarily set to 100% when incubated without EDTA and metal ions. Our results showed that IacE was independent of divalent metal ions for catalysis, consistent with the fact that it lacks the canonical prenyl diphosphate binding motif DXXD. The addition of 10 mM of Ca2+ and Mg2+ increased product formation by 180 and 150%, respectively. The activity of IacE was comparable to those when incubating 10 mM metal ions (Cu2+, Fe2+, Mn2+, Ni2+, Co2+) with EDTA but was decreased to less than 30% of that measured in the control with Zn2+ added (Figure 4E), indicating that the activity of IacE is independent of metal cofactors. The kinetic parameters including Michaelis−Menten constants (Km) and turnover numbers (kcat) of IacE toward DMAPP and 2 were determined by Hanes−Woolf and Lineweaver−Burk plots. The reaction catalyzed by IacE apparently followed the Michaelis−Menten kinetics, with an average kcat value of 7.04 s−1 calculated from the kinetic data of 2 and DMAPP. The Km values were determined as 46.25 and 34.52 μM for 2 and DMAPP, respectively, and the observed maximum reaction velocity was 358 pmol·s−1 mg−1. Combining with metabolite profiling, 2 was identified as the optimal and natural substrate for IacE. When IacE was incubated with pyrocatechol, hydroquinone, or hydroxybenzoic acid in the presence of DMAPP, the expected product was not detected under various conditions (Figure S8). HPLC analysis of the incubation mixture of L-Trp and DMAPP showed a peak (tR = 8.5 min), which was absent in the reaction mixture containing heat-denaturated IacE 706

DOI: 10.1021/acschembio.7b01059 ACS Chem. Biol. 2018, 13, 703−711

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spectra generated by the time-dependent density functional theory (TDDFT)32 for enantiomers 3a−3d (Figure 6). MMFF94 conformational search and DFT reoptimization at the B3LYP/6-31G(d) level yielded the 8 and 11 lowest energy conformers for 3a and 3c, respectively (Figures S10 and S11). The overall calculated spectra of 3a−3d were generated by Boltzmann weighting of the conformers, and the experimental ECD curve of 3 was nearly identical to that calculated for 3a, suggesting the 1S,3R,7R,10R configuration. Interpretation of the NMR data for 4 (Table S2) revealed that 4 was a stereoisomer of 3, with the same relative configuration except for C-10 (Figure S12). The absolute configuration was similalrly deduced as 1R,3S,7S,10R by ECD calculations. Analysis of the NMR data for 5 and 6 (Table S3) revealed that 5 and 6 were stereoisomers of 3 and 4, with their relative configurations deduced based on NOESY data and by analogy to 11 and 12 (Figure S13).31 The absolute configurations of 5 and 6 were also established by comparison of the experimental and theoretical ECD spectra calculated using TDDFT for enantiomers 5a−5d (Figure 6). MMFF94 conformational search and DFT reoptimization yielded three lowest energy conformers for 5a and 5c, respectively (Figures S14 and S15). The experimental ECD spectra for 5 and 6 matched the overall spectra calculated for 5a and 5c, respectively, allowing assignment of the 1S,3S,7R,10R and 1R,3R,7S,10R configurations for 5 and 6, respectively (Figure S16). Antibacterial Assay. Compounds 3−6 were tested against Staphylococcus aureus (CGMCC 1.2465) and Bacillus subtilis (ATCC 6633). Compounds 5 and 6 showed MIC values of 7.3 and 6.1 μg mL−1, respectively, against S. aureus (0.16 μg mL−1 for the positive control ampicillin), and 6.8 and 6.4 μg mL−1, respectively, against B. subtilis (1.3 μg mL−1 for the positive control gentamicin). Compounds 3 and 4 did not show detectable activity at 30 μg mL−1. Cytotoxicity Assay. Compounds 3−6 were tested against four human tumor cell lines, HeLa (cervical carcinoma), MCF7 (breast adenocarcinoma), SW480 (human colon adenocarcinoma), and T24 (bladder carcinoma). Compound 5 was cytotoxic to HeLa, MCF-7 ,and SW480 cells, showing IC50 values of 42.4, 29.5, and 64.3 μM, respectively, while the positive control cisplatin showed IC50 values of 7.4, 6.4, and 12.1 μM, respectively. The remaining compounds did not show detectable activity at 40 μM. Fungal prenyltransferases, well-known to catalyze the generation of metabolites with diverse structures and profound activities,33 are generally divided into two discrete families. The first one includes the cation-dependent and membrane-bound proteins with the (D/N)DXXD signature sequence, mainly involved in primary metabolite biosyntheses.34 The second family lacks the DXXD signature sequence and transfers a prenyl unit independent of metal ions, mainly involved in secondary metabolite biosyntheses. 35 On the basis of phylogenetic analysis, IacE belongs to the second family, catalyzing the prenylation of C-6 in 2 during iso-A82775C biosynthesis and maintaining its activity in a metal-free buffer containing EDTA. The highly restricted in vitro substrate specificity shown by IacE is similar to those membrane-bound prenyltransferases involved in prenylated flavonoid biosyntheses including naringenin 8-prenyltransferase SfN8DT-1,36 isoflavone-specific prenyltransferase SfG6DT, and chalconespecific isoliquiritigenin dimethylallyltransferase SfiLDT.37 Structurally, a prenyltransferase gene other than iacE could be required to form 2. Results from feeding experiments for

(Figure S8). Using other intermediates isolated from different mutants as the substrates failed to produce expected products (unpublished data), suggesting that IacE was highly specific toward 2 and accepted only DMAPP under the circumstances. Identification of New Congeners. Chloropestolides H− K (3−6) were isolated from ΔiacE extract, and their structures were determined by analyses of HRESIMS (C28H23ClO10) and NMR data (Tables S2 and S3). Interpretation of the 2D NMR data for 3 defined the same 2-ethynylbenzene-1,4-diol unit as in 2, and HMBC correlations from OH-23 to C-22, C-23, and C24; H-24 to C-22, C-23, C-26, and C-28; H-26 to C-21, C-22, C-24, C-27, and C-28; and H3-28 to C-24, C-25, and C-26 established a dihydroxymethylbenzoic acid moiety as appeared in 11−14.8,31 Correlations from H2-2 and H-9 to C-1, C-10, and C-11 linked C-1 to C-2, C-9, C-10, and C-18. Those from H2-2 to C-3, C-4, C-7, and C-12, and from H-9 to C-7 and C-8, indicated that C-7 is allylic to the C-8/C-9 olefin, and C-2, C-7, and C-12 are all attached to C-3. Correlations from H3-12 to C2, C-3, C-4, and C-7 connected the 2-ethynylbenzene-1,4-diol to C-3 via C-3/C-4. Further correlations of H3-19 and H3-20 with C-11 and C-8, respectively, located these methoxys. The chemical shifts for C-21 (δC 163.4) and C-27 (δC 153.3) connected each carbon to one of the two C-10 linked oxygen atoms via an ester and an ether linkage, respectively, completing the 5-hydroxy-7-methyl-4H-benzo[d][1,3]dioxin4-one unit. The chemical shift for C-7 (δC 80.4) and the pentacyclic nature of 3 required that the C-6 ketone and the remaining chlorine atom be directly attached to C-7 to form the planar structure of 3 (Figures 5 and S9).

Figure 5. Key NOE correlations of 3−6.

The relative configuration of 3 was proposed based on NOESY data and by analogy to 13 and 14.35 The C-8/C-9 Eolefin was assigned based on NOESY correlations of H-9 with H3-20, while those of H-2a with H-9 and H3-12 established the relative configurations for C-1, C-3, and C-7, which are the same as their counterparts in 13 and 14.31 However, the lack of relevant NOESY correlations precluded assignment of the relative configuration of C-10. The absolute configuration of 3 was deduced by comparison of the experimental electronic circular dichroism (ECD) spectrum with the simulated ECD 707

DOI: 10.1021/acschembio.7b01059 ACS Chem. Biol. 2018, 13, 703−711

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Figure 6. Experimental ECD spectra of 3−6 in MeOH and the calculated ECD spectra of 3a−3d and 5a−5d.

ΔiacE using 13C sodium acetate showed that none of the carbon atoms in 2 was labeled by sodium [1-13C] or [1,2-13C] acetate, indicating that the prenyl unit is not involved in the formation of 2, and 2 is not originated from the polyketide pathway. During accumulation of 2 in ΔiacE, new congeners 3−6 derived from the pathways of both 1 and 10 were isolated. Theoretically, additional new congeners could be generated by deleting key genes in the clusters of 1 or 10, which will be the focus in a future study. Conclusion. Iso-A82775C is a unique prenylepoxycyclohexane metabolite incorporating a prenyl group, an epoxy unit, and an isopropenylallene moiety exocyclic to hydroxycyclohexane ring. From the sequenced genome of P. f ici, a prenyltransferase gene iacE was identified and verified as the key gene in isoA82775C biosynthesis. To our knowledge, this is the first demonstration of a natural prenylepoxycyclohexane biosynthesis. In addition, four new chloropestolides were generated by genetic manipulation of iacE. Our findings also suggested that various substrates could be used in the biosyntheses of these metabolites. This study provided the first genetic and biochemical insights into the biosynthesis of natural prenylepoxycyclohexanes and demonstrated the feasibility to generate diverse congeners by manipulating key biosynthetic genes, paving the way to production of structurally complex fungal metabolites for screening and discovery of new drug leads.

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Constructions of Disruption Mutants and Complemented Strain. To construct the disruption mutants (ΔiacA−ΔiacK, Δpt3, and Δpt5), the DNA fragments containing the upstream and downstream regions of targeted genes were amplified from the genomic DNA of P. f ici with appropriate primers (Table S5). The amplified DNA fragments were respectively recombined into the upstream KpnI or downstream PacI site of pAg1-H3 using a Fast PCR Clone kit (PUEX) according to the manufacturer’s protocol. The downstream region of pt1, pt2, pt4, pt7, or pt8 was digested with AscI/ PacI and cloned into corresponding sites of pAg1-H3,10 while the upstream region was recombined into the KpnI site of corresponding plasmids carrying respective downstream regions, and the resulting plasmids were introduced into P. f ici by ATMT.10 The hygromycin B resistant colonies were selected after culturing on PDA at 28 °C for 3 days, and the disruption mutants were verified by PCR with the primers inside and outside of the corresponding genes. For complementation, the entire iacE with its putative promoter and downstream regions was amplified and inserted into the AscI/PacI site of pAgHG to generate pAgHGEC, which was introduced into ΔiacE by ATMT, and the transformants were selected on TSA plates supplemented with 100 μg mL−1 G418 and 400 μg mL−1 cefotaxime. The complemented strain was verified by PCR with corresponding primers. RNA Extraction, PCR, and RT-PCR. All primers used are listed in Table S5. Isolation of total RNAs, synthesis of cDNA, and PCR and RT-PCR were performed as described previously.10 RT-PCR products were detected by 1.5% (w/v) agarose gel electrophoresis and visualized by staining with ethidium bromide. Heterologous Expression of iacE and Product Purification. The coding region of iacE was amplified from the cDNA of P. f ici with the primer iacEc-F/iacEc-R and ligated with pEASY-Blunt to generate pEBiacE. After verification by sequencing, pEBiacE was digested with NdeI/HindIII, and the DNA fragment containing iacE was inserted into corresponding sites of pET28a to generate pET28a::iacE, which was introduced into E. coli BL21 (DE3). The expressed His-tagged IacE was purified, concentrated, and stored as described.10 Assay of IacE Activity. A mixture containing 100 mM Tris/HCl (pH 7.5), 15% (v/v) glycerol, 10 mM CaCl2, 1 mM DMAPP, and 2.5 mg of purified IacE in 100 μL solution was incubated at 37 °C for 30 min, and the reaction was quenched by adding 100 μL of ethyl acetate

METHODS

Strains, Plasmids, Media, and Growth Conditions. Strains and plasmids used are listed in Table S4. The media used and cultural conditions of strains have been described previously.10 Sequence Analysis. The nucleotide sequence is available from the GenBank database (accession no. KU963195). The ORFs were predicted using the GENSCAN Web Server at MIT (http://genes.mit. edu/GENSCAN.html) and FGENESH (Softberry). The deduced proteins were aligned with those from the database using online BLAST methods (http://www.ncbi.nlm.nih.gov/blast/). 708

DOI: 10.1021/acschembio.7b01059 ACS Chem. Biol. 2018, 13, 703−711

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1034 cm−1. 1H, 13C NMR and HMBC data, see Table S2. NOESY correlations (acetone-d6; 500 MHz): H-2a ↔ H-9, H3-12; H-9 ↔ H2a, H3-20; H3-12 ↔ H-2a; H3-20 ↔ H-9. HRESIMS: m/z 555.1060 (calcd for C28H24ClO10, 555.1053). Chloropestolide I (4). White powder. [α]D25: −157 (c 0.59, MeOH). UV (MeOH), λmax (log ε) 206 (4.35), 252 (3.98), 320 (3.73) nm. CD (c 3.0 × 10−4 M, MeOH) λmax (Δε): 226 (+17.7), 271 (−22.7), 313 (+6.09), 348 (−10.2) nm. IR (neat), νmax: 3445 (br), 2951, 2842, 1765, 1745, 1717, 1648, 1498, 1467, 1149, 1024 cm−1. 1H, 13 C NMR and HMBC data, see Table S2. NOESY correlations (acetone-d6; 500 MHz): H-2a ↔ H-9, H3-12; H-9 ↔ H-2a, H3-20; H312 ↔ H-2a; H3-20 ↔ H-9. HRESIMS: m/z 555.1051 (calcd for C28H24ClO10, 555.1053). Chloropestolide J (5). White powder. [α]D25: −260 (c 0.17, MeOH). UV (MeOH), λmax (log ε): 206 (4.43), 251 (4.02), 323 (3.78) nm. CD (c 5.6 × 10−5 M, MeOH), λmax (Δε): 227 (+26.8), 272 (−27.7), 319 (+6.76), 355 (−10.1) nm. IR (neat), νmax: 3512 (br), 2946, 2345, 1774, 1744, 1711, 1641, 1581, 1491, 1021 cm−1. 1H, 13C NMR and HMBC data, see Table S3. NOESY correlations (acetoned6; 500 MHz): H-2a ↔ H-9; H-2b ↔H3-12; H-9 ↔ H-2a; H3-12 ↔ H-2b. HRESIMS: m/z 555.1056 (calcd for C28H24ClO10, 555.1053). Chloropestolide K (6). White powder. [α]D25: −90.9 (c 0.10, MeOH). UV (MeOH), λmax (log ε): 212 (4.34), 255 (3.90), 320 (3.53) nm. CD (c 3.0 × 10−4 M, MeOH), λmax (Δε): 237 (+8.06), 272 (−16.0), 318 (+5.18), 354 (−4.36) nm. IR (neat), νmax: 3437 (br), 2958, 2830, 2360, 1762, 1765, 1708, 1634, 1587, 1450, 1096 cm−1. 1H, 13 C NMR and HMBC data, see Table S3. NOESY correlations (acetone-d6; 500 MHz): H-2a ↔ H-9; H-2b ↔H3-12; H-9 ↔ H-2a; H3-12 ↔ H-2b. HRESIMS: m/z 555.1062 (calcd for C28H24ClO10, 555.1053). Computational Details. Conformational analyses for 3 and 4 were performed via the Molecular Operating Environment (MOE) ver. 2009.10. (Chemical Computing Group, Canada) software using the MMFF94 molecular mechanics force field calculation. MMFF94 analyses were optimized using TDDFT at the B3LYP/6-31G(d) basis set level. The 30 lowest electronic transitions were calculated, and the rotational strengths of each electronic excitation were given using both dipole length and dipole velocity representations. ECD spectra were stimulated using a Gaussian function with a half-bandwidth of 0.25 eV. Equilibrium populations of conformers at 298.15 K were calculated from their relative free energies (ΔG) using Boltzmann statistics. The overall ECD spectra were generated according to Boltzmann weighting of each conformer. The systematic errors in prediction of the wavelength and excited-state energies are compensated employing UV correction. Antibacterial Assay. Antibacterial assays were conducted in triplicate following the recommendations from the Clinical and Laboratory Standards Institute (CLSI).38 S. aureus (ATCC 6538) and B. subtilis (ATCC 6663) were obtained from China General Microbial Culture Collection (CGMCC). Detailed procedures have been reported previously.39 MTS Assay. Cytotoxicity of the compounds was evaluated according to a previously published procedure.40

(EtOAc). After protein removal by centrifugation at 12 000g for 10 min, the product was analyzed by HPLC. To determine substrate specificity, 100 μL of standard solution containing 2 mM L-Trp or aromatic substrates (p-hydroxybenzoic acid and pyrocatechol or hydroquinone), 1 mM DMAPP, and 2.5 mg of IacE was used. To determine the kinetic parameters of IacE using 2 as the substrate, 1 mM DMAPP; 2.5 mg of IacE; and 2 at final concentrations of 0, 0.12, 0.24, 0.36, 0.48, or 0.6 mM in 100 μL of solution were used. To determine the kinetic parameters of IacE with DMAPP as the substrate, 2.5 mg of IacE; 0.36 mM 2; and DMAPP at final concentrations of 0, 0.34, 0.68, 1.00, 1.34, or 1.68 mM in 100 μL of solution were used. The solutions were incubated at 37 °C for 30 min. HPLC Analysis of the Products of IacE. A total of 20 mL of solution containing 100 mM Tris/HCl (pH 7.5), 15% (v/v) glycerol, 10 mM CaCl2, 1 mM DMAPP, 0.36 mM 2, and enough IacE was incubated at 37 °C for 3 h and extracted with EtOAc. After solvent evaporation, the residues were redissolved in methanol (MeOH), and the resulting products were analyzed as described previously.10 General Procedures for Chemical Analysis. Optical rotations were measured on a Rudolph Research Analytical polarimeter. UV data were obtained on a Shimadzu Biospec-1601 spectrophotometer. CD spectra were recorded on a JASCO J-815 spectropolarimeter. IR data were recorded using a Nicolet Magna-IR 750 spectrophotometer. The 1H and 13C NMR data were acquired with Varian Mercury-500 and NMR system-600 spectrometers using solvent signals (acetone-d6: δH 2.05/δC 29.8, 206.1) as references. HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HRESIMS data were obtained using an Agilent Accurate-Mass-Q-TOF LC/MS 6520 instrument equipped with an ESI source. The fragmentor and capillary voltages were kept at 125 and 3500 V, respectively. Nitrogen was supplied as the nebulizing and drying gas. The flow rate of the drying gas and the pressure of the nebulizer were 10 L min−1 and 10 psi, respectively. All MS experiments were performed in positive ion mode. Full-scan spectra were acquired over a scan range of m/z 100−1000 at 1.03 spectra s−1. Extraction and Isolation. The cultures from ΔiacE and ΔiacF were extracted repeatedly with EtOAc (3 × 2.0 L), and the solvent was evaporated to dryness under a vacuum to afford the crude extract (5.3 and 6.7 g from ΔiacE and ΔiacF, respectively), which was fractionated by silica gel column chromatography (CC) using petroleum ether/ EtOAc gradient elution. The fraction eluted with 10% (v/v) EtOAc (439 mg) from the ΔiacE extract was purified by RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm) to afford 2 (20.0 mg, tR 14.90 min; 50−60% (v/v) MeOH in H2O for 20 min; 2 mL min−1). The fractions eluted with 5% (v/v) EtOAc (1.2 g) from ΔiacF extract were combined and separated by Sephadex LH-20 CC eluted with 1:1 CH2Cl2−MeOH. Purification of the resulted subfractions by RP HPLC afforded 7 (18 mg, tR 16.50 min; 70−80% (v/v) MeOH in H2O for 20 min; 2 mL min−1). The fraction eluted with 20% (v/v) EtOAc (1.3 g) was subjected to ODS CC eluting with a gradient of MeOH in H2O, and the resulting subfractions were purified by RP HPLC using the same column to afford 3 (2.1 mg, tR 28.50 min; 55− 75% (v/v) MeOH in H2O for 35 min; 2 mL min−1), 4 (6.4 mg, tR 32.70 min; the same conditions as 3), 5 (2.2 mg, tR 24.10 min; 65− 75% (v/v) MeOH in H2O for 30 min; 2 mL min−1), and 6 (1.6 mg, tR 21.83 min; the same conditions as 5). Structure Elucidation. Structure determination was performed as previously described.7 Siccayne (2). 1H, 13C NMR, and MS data were consistent with literature values.28 Pestalodiol E (7). Yellow oil. UV (MeOH), λmax (log ε): 210 (4.54), 266 (4.28), 326 (3.98) nm. IR (neat), νmax: 3409 (br), 2971, 2857, 2190, 1670, 1609, 1461, 1151, 1034 cm−1. 1H, 13C NMR, and HMBC data, see Table S1. HRESIMS: m/z 243.1384 (calcd for C16H19O2, 243.1380). Chloropestolide H (3). White powder. [α]D25: −115 (c 0.16, MeOH). UV (MeOH), λmax (log ε): 207 (4.49), 251 (4.05), 320 (3.82) nm. CD (c 2.3 × 10−4 M, MeOH), λmax (Δε): 221 (+16.1), 249 (−1.78), 270 (+5.52), 309 (+7.83), 342 (−9.28) nm. IR (neat), νmax: 3451 (br), 2962, 2852, 2126, 1762, 1750, 1711, 1645, 1584, 1498,

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b01059.

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Tables listing strains, plasmids and PCR primers, and 1H and 13C NMR data for 3−7 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. 709

DOI: 10.1021/acschembio.7b01059 ACS Chem. Biol. 2018, 13, 703−711

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Gang Liu: 0000-0003-3583-2985 Author Contributions ∥

Contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by grants from NSFC (31470177, 21372004, and 31770056), National Program of Drug Research and Development (2012ZX09301-003), and the Youth Innovation Promotion Association of CAS (2011083).

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DOI: 10.1021/acschembio.7b01059 ACS Chem. Biol. 2018, 13, 703−711