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Article Cite This: J. Am. Chem. Soc. 2018, 140, 18009−18015

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Design and Biosynthesis of Dimeric Alboflavusins with Biaryl Linkages via Regiospecific C−C Bond Coupling Zhengyan Guo,†,⊗ Pengwei Li,†,⊗ Guozhu Chen,‡,⊗ Chao Li,†,§ Zhenju Cao,†,§ Yuwei Zhang,†,§ Jinwei Ren,∥ Hua Xiang,†,§ Shuangjun Lin,⊥ Jianhua Ju,# and Yihua Chen*,†,§ †

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China Institute of Military Cognitive and Brain Sciences, Academy of Military Medical Sciences, Beijing 100850, China § University of Chinese Academy of Sciences, Beijing 100049, China ∥ State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China ⊥ State Key Laboratory of Microbial Metabolism, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China # CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China

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‡

S Supporting Information *

ABSTRACT: Alboflavusins (AFNs) are a group of cyclohexapeptides with moderate antibacterial and antitumor activities from Streptomyces albof lavus sp. 313. In vivo and in vitro studies proposed that AFNs are biosynthesized by a nonribosomal peptide synthetase machinery, and the 6-Cl-LTrp precursor is supplied by a tryptophan halogenase gene located outside the afn gene cluster. Guided by the structure− activity relationship knowledge about the AFN-like cyclohexapeptides, two dimeric AFNs (di-AFNs) with regiospecific biaryl linkages were designed and generated biotechnologically by expressing the P450 gene hmtS or clpS in S. albof lavus wild-type and mutant strains. The di-AFNs displayed much better antibacterial and antitumor activities than their monomers as anticipated, exemplifying a rational strategy to generate natural product congeners with improved bioactivities.

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acid and PIC residues resulted in kutznerides5 and dimeric cyclopeptides, including himastatin (3)6,7 and chloptosin (4),8 that are coupled by biaryl linkages between C-5 and C-5′ of the PIC residues (Figure 1C and Figure S1). Chloptosin monomer is structurally very similar to 1 with only two different residues. Compared to 1, chloptosin (4) has much higher inhibitory activities against Gram-positive bacteria (Table S1),8 implying that dimerization via the C−C bond to form a biaryl linkage is a potential way to increase AFNs’ bioactivities. This has also been supported by the SAR studies on 3. The dimeric cyclohexadepsipeptide 3 is a good antibiotic against Grampositive bacteria with MIC around 0.3−1.3 μM, while the himastatin monomer does not exhibit considerable antibacterial activity (>250 μM), emphasizing the influence of dimerization on its bioactivity.9 Although chemical synthesis of biaryls has a history of more than 100 years, it is still a great challenge to synthesize bicyclopeptides with biaryl linkages like 4.10,11 Chemical synthesis of 4 was accomplished via more than 30 steps in 3−4% overall yield;12,13 synthesis of 3 also needed a long route

INTRODUCTION Alboflavusins (AFNs), originally named as the NW-G series compounds,1−4 are antimicrobial and antitumor cyclohexapeptides isolated from Streptomyces albof lavus sp. 313. All nine AFNs (Figure 1 and Figure S1), represented by AFN A1 (1) and B1 (2), are composed of three L- or D-piperazic acids, an unusual tricyclic (2S,3aR,8aS)-3a-hydroxyhexahydropyrrolo[2,3-b]indole-2-carboxylic acid (PIC), an L-alanine, and a Dvaline (or an L-leucine for AFN A5) but differing in the extent of substitution and oxidation of constituent residues. The A and B series of AFNs were distinguished by their different configurations at C-27.2,3 Most AFNs displayed moderate inhibitory activities against Gram-positive bacteria such as Staphylococcus and Bacillus.1−4 Compounds 1 and AFN A4 also exhibited moderate cytotoxicity against human colon cancer, lung cancer, and hepatocellular carcinoma cell lines.2 Overall, the bioactivities of AFNs are attractive but need to be further enhanced for being considered as promising drug leads. In order to generate AFN derivatives with improved biological activities, we collected the information on AFN analogues and analyzed their structure−activity relationships (SARs) for clues. A search for cyclohexapeptides with piperazic © 2018 American Chemical Society

Received: September 19, 2018 Published: December 5, 2018 18009

DOI: 10.1021/jacs.8b10136 J. Am. Chem. Soc. 2018, 140, 18009−18015

Article

Journal of the American Chemical Society

Figure 1. Gene cluster and biosynthetic pathway of AFNs and di-AFNs. (A) Genetic organization of the afn gene cluster. (B) Proposed pathway for AFNs biosynthesis. Di-AFNs were generated by heterologous expression of the P450 coupling enzyme ClpS or HmtS in different S. albof lavus strains. (C) Structures of the dimeric cyclohexapeptides 3 and 4. The residues different from 1 were marked with blue in the monomers of 3 and 4.

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and had a similar low yield.14 In the successful chemical syntheses of 3 and 4, the dimeric core structures connected by biaryl linkages were built first, and the peptide chains were then added to form the bicyclopeptide skeletons.12,13 An alternative synthesis logic, in which the cyclohexapeptide monomer was assembled first and then coupled via biaryl linkage regiospecifically, was attempted at various reaction conditions but failed in chemical synthesis of 4.15 Interestingly, the latter synthetic logic was discovered in the biosynthesis of 3 recently; and a P450 enzyme HmtS was proposed to catalyze the coupling reaction based on the fact that only himastatin monomer was accumulated in the hmtS gene inactivated mutant.16,17 This discovery inspired us to utilize the P450 oxidative coupling enzymes like HmtS to generate dimeric AFNs with biaryl linkages biotechnologically. In this work, we partially elucidated the biosynthetic mechanism of AFNs and generated two dimeric AFNs with regiospecific biaryl linkages by expressing P450 coupling enzymes in S. albof lavus wild-type and mutant strains. Both of the two dimeric AFNs exhibited much better antibacterial and antitumor activities than their monomers as anticipated, exemplifying a rational way to increase the bioactivities of natural products biotechnologically.

RESULTS Generation of Di-AFN A1 (5). The key to successful generation of dimeric AFNs is to find HmtS-like P450 coupling enzymes that can accept AFNs as substrates. At first, ClpS, the orthologue of HmtS from 4, was tested as a catalyst. We envisaged that ClpS has a better chance to take AFNs as substrates than HmtS, since there are only two different amino acid residues between 1 and the chloptosin monomer, while the number between 1 and the himastatin monomer is five (Figure 1C). To clone the clpS gene, we analyzed the genome of Streptomyces sp. MK498-98 F1418 and localized the chloptosin biosynthetic gene cluster by analyzing the nonribosomal peptide synthetase (NRPS) genes carefully. A gene that encodes a P450 enzyme sharing a significant similarity with HmtS (61% identity) was assigned as the clpS gene (Figure S2). When clpS was overexpressed in S. albof lavus sp. 313, a new compound, which was proposed to be dimeric AFN A1 (di-AFN A1, 5) by HR-MS (C70H96Cl2N20O14, m/z [M + H] + 1511.687, calcd 1511.686), was detected in the recombinant strain S. albof lavus 313_clpS (Figure 2). An MS/MS analysis of 5 suggested that the dimerization occurred between the two PIC residues (Figure S3). The positive results of ClpS encouraged us to test its homologue HmtS. To our surprise, when gene hmtS was 18010

DOI: 10.1021/jacs.8b10136 J. Am. Chem. Soc. 2018, 140, 18009−18015

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Journal of the American Chemical Society

Figure 2. HPLC profiles of S. albof lavus mutants. (i−iii) authentic standards of 1, 2, and 7; (iv) S. albof lavus sp. 313; (v) S. albof lavus ΔafnG; (vi) S. alboflavus 313_clpS; (vii) S. alboflavus 313_hmtS; (viii) S. alboflavus ΔafnD; (ix) S. alboflavus ΔafnD_hmtS; (x) S. alboflavus ΔafnD_clpS; (xi) S. albof lavus Δaf nX; (xii) S. alboflavus ΔafnX + 6-Cl-L-Trp; (xiii) S. alboflavus ΔafnX_hmtS; (xiv) S. alboflavus ΔafnX_clpS.

the putative regulatory gene afnI was inactivated, production of both 1 and 2 was decreased dramatically, indicating that AfnI regulates the production of AFNs positively, and (ii) single gene inactivation of af n1 or afn2 had no obvious influence on production of AFNs, implying that they should be outside of the afn gene cluster (Figure S6 and S7). The left boundary of the afn gene cluster was determined to be gene af nA since the Δafn-1 mutant could yield AFNs as S. albof lavus sp. 313, while the ΔafnA mutant could produce only 1 but lost the ability to produce 2, suggesting that AfnA is involved in the formation of the AFN Bs. AfnA displays 59% identity with HmtN, a P450 monooxygenase decorating the Dpiperazic acid residue of himastatin (Figure S2),16 implying that AfnA catalyzes a similar monooxygenation at C-22 during the conversion from 1 to 2. Characteriztion of AfnD in Vivo. Besides the afnA gene, the af n gene cluster contains another P450 enzyme encoding gene afnD. AfnD shares a high similarity with HmtT (57% identity), which catalyzes the conversion from the L-Trp residue to the PIC residue in the biosynthesis of himastatin monomer (Figure S2).16,19 When the af nD gene was inactivated (Figure S8A), production of both 1 and 2 was abolished, and a new compound pre-AFN A1 (6) was accumulated in S. alboflavus ΔafnD (Figure 2). The chemical formula of 6 was determined to be C35H49ClN10O6 (m/z [M + H]+ 741.3599, calcd 741.3598) by HR-MS analysis. Careful NMR analyses revealed that 6 has an almost identical structure as 1, except the 6-Cl-PIC residue is substituted by a 6-Cl-L-Trp group, implying that AfnD is responsible for the conversion from 6 to 1 by catalyzing the C-3a hydroxylation and the

overexpressed in S. albof lavus sp. 313, 5 was produced at an even higher efficiency in S. albof lavus 313_hmtS than in S. albof lavus 313_clpS (Figure 2), revealing that HmtS possesses a significantly high degree of substrate promiscuity. Compound 5 was then purified and determined to be di-AFN A1 by NMR analyses (Table S2 and Figure S4). The C−C coupling bond was assigned between C-5 and C-5′ because C-4 and C-7 were excluded by the fact that the 1H NMR signals of H-4, H-4′, H7, and H-7′ are all singlets and by the HMBC correlations from H-4 to C-3a and C-7a and from H-7 to C-3b. Identification of the AFN Biosynthetic Gene Cluster. The considerable substrate promiscuities of the two P450 coupling enzymes, especially that of HmtS, implied that they could be used to generate more dimeric AFNs if we could supply diverse AFN monomers. Therefore, we set out to identify the AFN biosynthetic gene cluster and then to manipulate the afn gene cluster to generate different AFN analogues as potential substrates of HmtS and ClpS. After the genome of S. alboflavus sp. 313 was sequenced, an NRPS gene cluster, which contains many genes encoding proteins sharing high similarities with the enzymes involved in the biosynthesis of kutznerides5 and himastatin,16 was proposed to be the AFN biosynthetic gene cluster (Table S3). The NRPS gene afnG in this cluster was then inactivated by the insertion of an apramycin resistance gene cassette (Figure S5). Production of AFNs was totally abolished in the Δaf nG mutant, confirming the identity of the afn gene cluster (Figure 2). The boundaries of the afn gene cluster was predicted by bioinformatic analyses and then determined by sequential inactivation of the flanking genes. The afnI gene was assigned as the cluster’s right boundary based on the facts that (i) when 18011

DOI: 10.1021/jacs.8b10136 J. Am. Chem. Soc. 2018, 140, 18009−18015

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Journal of the American Chemical Society

Table 1. Antibacterial Activities of AFNs and Di-AFNsa

pyrroloindole ring formation to generate the 6-Cl-PIC residue (Table S4 and Figure S9). A Separated L-Trp Halogenase Gene afnX Is Involved in AFN Biosynthesis. It was conceived that HmtS should be able to accept AFN A2 (7), the dechloro form of 1, as a substrate, since the PIC residue of its native substrate is not halogenated. However, dimeric 7 was not detected in S. albof lavus sp. 313 overexpressing the hmtS gene, probably due to the very low titer of 7 in the wild-type strain. There is no halogenase gene in the afn gene cluster, implying that the chlorine in 1 was incorporated by a 6-Cl-L-Trp precursor. To construct a mutant that can produce a larger amount of 7, we searched the genome of S. albof lavus sp. 313 for L-Trp 6halogenase encoding genes and found only one candidate, afnX, which encoded a protein displaying 83% identity with SttH, a well-characterized L-Trp 6-halogenase from Streptomyces toxytricini.20 The afnX gene was about 1.1 Mb away from the af n gene cluster (Figure 1A). AfnX was expressed in Escherichia coli BL21 as an N-His6 tagged protein, purified to homogeneity, and verified as an L-Trp 6-halogenase converting L-Trp to 6-Cl-L-Trp with a Km of 64.74 ± 9.69 μM and a kcat of 0.098 ± 0.005 min−1(Figure S10 and S11). The ΔafnX inframe deletion mutant S. albof lavus Δaf nX was then constructed and fermented at the AFNs production conditions (Figure S8B). As anticipated, S. albof lavus ΔafnX could not produce 1 and 2 and accumulated a large amount of 7 (Figure 2). The production of 1 and 2 could be restored in the Δaf nX mutant by feeding 6-Cl-L-Trp as a precursor, further confirming that afnX encodes the L-Trp 6-halogenase involved in the biosynthesis of AFNs (Figure 2). Generation of Di-AFN A2 (8). To check whether HmtS and ClpS could accept compounds 6 or 7 as a substrate, the two enzymes were overexpressed in the Δaf nD or Δaf nX mutant, respectively. In the case of compound 6, no dimeric AFNs were detected in either S. albof lavus ΔafnD_hmtS or ΔafnD_clpS (Figure 2), indicating that the PIC structure is critical for both ClpS and HmtS. When the hmtS gene was overexpressed in the Δaf nX mutant, a new compound with the anticipated molecular weight as dimeric 7 (C71H98Cl2N20O14, HR-MS m/z [M + H]+ 1443.764, calcd 1443.764) was produced in S. albof lavus ΔafnX_hmtS as expected. It was determined to be di-AFN A2 (8) subsequently by careful MS/MS and NMR analyses (Table S5 and Figures S12 and S13). The biaryl linkage was assigned between C-5 and C-5′ according to the 1H−1H COSY correlation between H-6/H-7 and the HMBC correlations from H-4 to C-3a and C-7a. It was also supported by the ROESY correlations between H-6/H-4′ and between H-7 and the proton of 8-NH. Meanwhile, the catalytic activity of ClpS toward 7 was tested by overexpressing clpS in the Δaf nX mutant to generate S. alboflavus ΔafnX_clpS, which could also produce 8, suggesting that ClpS can also tolerate substrates without chlorine at the PIC residue (Figure 2). Antibacterial and Antitumor Activities of 5 and 8. With the two dimeric AFNs (5 and 8) and their corresponding monomers (1 and 7) in hand, we chose a series of antibacterial and cytotoxicity assays to test whether this dimerization can increase AFNs’ bioactivities as anticipated. When they were tested against various Gram-positive bacteria21 and cancer cell lines,22 both 5 and 8 exhibited much better inhibition activities than their corresponding monomers (Table 1 and 2). The IC50 values of 5 against human epitheloid cervix carcinoma (Hela), Caucasian ovary adenocarcinoma (SKOV3), and lung

MIC values (μM) samples

BS

BC

SA

MRSA1

MRSA2

MRSA3

1 5 7 8 Dap

12.5 0.32 100 1.56 0.32

25 0.63 50 0.78 0.32

12.5 0.78 50 1.56 0.2

12.5 0.78 100 1.56 0.2

25 2.5 100 0.78 0.16

25 0.63 100 0.78 0.32

a

Notes: BS, Bacillus subtilis BS 168; BC, Bacillus cereus CGMCC 1.0230; SA, Staphylococcus aureus ATCC 6538; MRSA 1, 2, and 3, methicillin-resistant Staphylococcus aureus strains 113, 1.2386, and 09L098; Dap, daptamycin.

carcinoma (A549) cell lines were 2 orders of magnitude lower than its monomer 1, revealing that the regiospecific dimerization can improve the bioactivities of AFNs significantly (Table 2 and Figures S14 and S15). In addition, compound 5 displayed better antibacterial and antitumor activities than 8 in most cases, and 1 also showed better activity than 7, indicating that halogenations at the PIC residues can influence AFNs’ bioactivity considerably. Notably, compound 8 exhibited a higher inhibition activity against methicillin-resistant Staphylococcus aureus strain 1.2386 than 5, although 1 inhibited this strain better than 7, which implied possible changes on the inhibition mechanism after the cyclohexapeptides are dimerized (Table 1).

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DISCUSSION Natural products are Mother Nature’s generous gifts in medical chemistry.23−25 For many natural products, whose biological activities are not good enough to be considered as promising drug leads, construction and screening of banks of natural product derivatives is a common way to obtain congeners with improved activities.23,26 Design and biosynthesis of di-AFNs in this work exemplify a rational strategy to generate natural product derivatives with better activities. Guided by the SAR knowledge about the AFN-like cyclohexapeptides, we designed and generated two di-AFNs by coupling AFNs via regiospecific biaryl linkages biotechnologically. The di-AFNs displayed much better antibacterial and antitumor activities than their monomers as anticipated. Along with increasing knowledge about SAR and natural product biosynthesis, more natural product congeners with improved activities may be generated in such a rational way. Biaryl moieties connected by C−C bond are found in many natural products, including ellagitannin from plants and diverse antibiotics from microbes (e.g., vancomycin, staurosporine, arylomycin, kotanin, and so on).10,17,27 The cross-coupling between aryl structures is catalyzed by laccase-like phenol oxidases in ellagitannin,10 while the C−C bond biaryl linkages in microbial natural products are mostly formed by P450 enzymes.17,27 As mentioned above, although the “classical” Ullmann reaction was reported as early as 1901, and different chemical strategies have been developed to couple aryls in the past century,11 it is still challenging to synthesize complicated natural products with regiospecific biaryl linkages. Taking advantage of the previous studies on the P450 aryl coupling enzymes,28−30 the regiospecific biaryl linkages between AFN monomers were formed by two P450 enzymes, HmtS and ClpS, in this study, showing that the biotechnological method can be used as powerful alternative ways in certain cases. 18012

DOI: 10.1021/jacs.8b10136 J. Am. Chem. Soc. 2018, 140, 18009−18015

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Journal of the American Chemical Society Table 2. Antititumor Activities of AFNs and Di-AFNsa IC50 values (μM) samples 1 5 7 8 5-FU

Hela 22.73 0.18 51.97 2.41 333.26

± ± ± ± ±

SKOV3 0.01 0.01 0.12 0.02 0.01

22.56 0.15 92.54 2.77 286.90

± ± ± ± ±

0.03 0.02 0.18 0.04 0.03

MCF-7 14.72 0.19 79.97 1.84 321.68

± ± ± ± ±

0.01 0.03 0.16 0.02 0.03

A549 29.16 0.19 85.82 1.39 446.90

± ± ± ± ±

HepG2 0.01 0.01 0.17 0.01 0.10

13.59 0.29 99.06 2.13 198.94

± ± ± ± ±

0.04 0.05 0.19 0.03 0.02

U251 9.39 0.28 82.07 0.95 200.97

± ± ± ± ±

SGC-7901 0.01 0.06 0.16 0.02 0.05

16.66 0.26 55.40 1.81 462.39

± ± ± ± ±

0.02 0.02 0.11 0.05 0.07

GES-1 31.66 0.41 112.50 1.12 747.80

± ± ± ± ±

0.23 0.07 0.68 0.06 0.85

a

Notes: Hela, human epitheloid cervix carcinoma cell; SKOV3, human Caucasian ovary adenocarcinoma cell; MCF-7, human breast adenocarcinoma cell; A549, human lung carcinoma; HepG2, human liver hepatocellular carcinoma cell; U251, human glioma cell; SGC-7901, human gastric cancer cell, GES-1, human normal gastric epithelial cell; 5-FU, 5-fluorouracil.

SAR knowledge of the AFNs analogues, we predicted that dimerization of AFNs at the C-5 position of the PIC residue with C−C bond will enhance the bioactivities of AFNs. The dimeric AFNs with regiospecific biaryl linkages were then obtained biotechnologically using two P450 coupling enzymes, HmtS and ClpS. A proposed biosynthetic pathway of AFNs was delineated during the process of making different AFN analogues to be tested as substrates of the two coupling enzymes. As anticipated, both 5 and 8 displayed much better antibacterial and antitumor activities than their monomers. The high degrees of substrate promiscuities of HmtS and ClpS indicate that they have potential applications in generation of varied dimeric cyclohexapeptides with regiospecific biaryl linkages.

For the purpose of supplying different AFN congeners tested as the substrates of HmtS and ClpS, we sequenced the genome of S. albof lavus sp. 313, identified the afn gene cluster, and inactivated several af n genes to generate varied AFN analogues. During this process, a putative AFN biosynthetic pathway was proposed as Figure 1B on the basis of bioinformatic analyses and the results of afn gene inactivation. The nonproteinogenic amino acid precursors, 2(S)-piperazic acid and 6-Cl-L-Trp, were proposed to be synthesized by genes outside the af n gene cluster. The former one may be derived from L-ornithine by an FAD-dependent N-hydroxylase and a heme-dependent dehydratase consecutively as described;31 the latter one is formed by the L-Trp 6-halogenase AfnX. After the hexapeptide chain is fully installed, it is released and cyclized by AfnG-TE, and the P450 enzyme AfnD closes the pyrroloindole ring of 6-Cl-PIC to shape 1. We proposed that compound 2, the representative of the AFN B series compounds, is converted from 1 by an epimerization at C-27 based on the observations that (i) compound 1 was yielded earlier than 2 during the fermentation of S. albof lavus sp. 313 and (ii) along with the accumulation of 2, compound 1 was decreased dramatically, indicating that 2 is derived from 1 (Figure S16). It was also supported by the accumulation of a significant amount of 1 in S. albof lavus ΔafnA, which lost the capacity to produce 2 (Figure S7). Dimeric AFNs, 5 and 8, have similar structures as 3 and 4. All four dimeric cyclohexapeptides with biaryl linkages display considerable inhibition activities against Gram-positive bacteria and certain human cancer cell lines. Compound 4 is an attractive target for drug development in that it can induce apoptosis in the apoptosis-resistant human pancreatic adenocarcinoma cell line AsPC-1 as well as in several apoptosis-sensitive cancer cell lines.8 Compound 3 was observed to lack distal site antitumor activity in vivo. The decrease of bioactivities of 3 in vivo was found to be related to certain fatty acid sodium salts, and it was shown that 3 could be trapped in the micelles formed by the fatty acid salts, which indicated that 3 might execute its inhibition effects by interacting with cell membrane.32 Compound 1 was also proposed to execute its inhibition effects by damaging cell membrane according to the scanning electron microscope data of cells treated with 1.33 The dimeric AFNs may inherit the functional mechanism of AFNs. However, the unusual high inhibition activity of 8 against MRSA2 implied that the dimerization should introduce different functional styles, which need to be elucidated by further studies.

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EXPERIMENTAL SECTION

General Methods. DNA manipulations were performed using standard procedures for E. coli and Streptomyces.34,35 HPLC analyses were carried out with an Apollo C18 column (5 μ m, 4.6 mm × 250 mm, Alltech, Deerfield, IL) on a Shimadzu HPLC system (Shimadzu, Kyoto, Japan). LC-HR-MS was performed on an Agilent 1260 HPLC/6520 QTOF-MS instruments (Santa Clara, CA) with the electrospray ionization source. NMR spectra were recorded at room temperature on a Bruker Advance 500 and 800 M NMR spectrometer (Billerica, MA). Bacterial Strains, Plasmids, and DNA Sequences. Bacterial strains and plasmids used in this study were summarized in Table S6. All oligonucleotide primers used in this study (Table S7) were synthesized by Generay (Shanghai, China). Common DNA sequencing was performed by Biosune Co. (Shanghai, China). Genomic DNA of S. alboflavus sp. 313 was sequenced by Majorbio Co. (Shanghai, China). Open reading frame prediction was carried out using Prodigal (http://compbio.ornl.gov/prodigal/). The secondary metabolite gene clusters were analyzed with antiSMASH (http://antismash.secondarymetabolites.org/). The gene functional annotations were performed with BLAST (http://www.ncbi.nlm.nih. gov/blast). Multiple alignments were performed with CLUSTALW (https://www.ebi.ac.uk/Tools/msa/clustalw2/). The sequences of the afn gene cluster and the afnX gene from S. alboflavus sp. 313 as well as the clpS gene from S. sp. MK498-98 F14 were submitted to GenBank (accession numbers are MH497044, MH497045, and MH878939, respectively). The genome sequence of S. albof lavus sp. 313 was also deposited in GenBank (accession number is RHGC00000000). Construction of S. albof lavus Mutants. Three different strategies were used in the construction of S. albof lavus single gene mutants (the experimental details are showed in the Supporting Information). S. albof lavus Δaf nG was constructed via singlecrossover gene disruption (Figure S5); S. albof lavus mutant strains ΔafnA, ΔafnI, Δafn-1, Δaf n1, and Δafn2 were constructed by replacing each of the target genes with the apramycin resistance gene cassette aac(3)IV (Figure S6), while S. alboflavus Δaf nD and ΔafnX

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CONCLUSIONS In this work, we exemplified a rational way to generate natural product congeners with improved activities. Guided by the 18013

DOI: 10.1021/jacs.8b10136 J. Am. Chem. Soc. 2018, 140, 18009−18015

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Journal of the American Chemical Society

Laboratory.21 Daptomycin (Dap) (J&K scientific, Beijing, China) was used as a positive control. The tested bacteria were incubated in the Mueller−Hinton broth at 30 °C, 220 rpm for 12 h, and the cell concentrations were diluted to approximately 1 × 106 colony-forming units with Mueller−Hinton broth. After incubation for 24 h at 30 °C, the MIC values were calculated. Cytotoxicity Assays. The cancer cell lines (HepG2 (liver), MCF7 (breast), Hela (cervix), SKOV3 (ovary), SGC-7901 (gastric), U251 (glioma), and A549 (lung)) and the normal mammalian cells (GES1(gastric) and HL-7702 (hepatic)) were purchased from the Cell Culture Center, Beijing Institute of Basic Medical Science of Chinese Academy of Medical Science (Beijing, China). Tumor cells were cultured with Dulbecco’s modified Eagle medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS, Kangyuan Biology, Beijing, China) in a CO2 incubator (37 °C, 5.0% CO2 air humidified). The potential effects of chemical compounds on cell viability were investigated using the MTT assay as an indicator of metabolically active cells.22 The percentage of cell viability and IC50 were estimated using Prism Graphpad software.37 All experiments were performed in triplicate.

were gene in-frame deletion mutants constructed using a CRISPR/ Cas9 gene inactivation system (Figure S8).36 Construction of S. albof lavus Recombinant Strains Overexpressing clpS or hmtS. The 1.2-kb clpS gene was PCR amplified from the genomic DNA of S. sp. MK498-98 F1418 with primer pair clpS-F/clpS-R and cloned into the NdeI and EcoRI sites of plasmid pCIMt005 to generate pCIMt005/clpS. Similarly, plasmid pCIMt005/hmtS was constructed by insertion of the 1.2-kb hmtS gene, which was PCR cloned with primer pair hmtS-F/hmtS-R from the genomic DNA of the himastatin producer Streptomyces hygroscopicus ATCC 536535 into the NdeI and EcoRI sites of pCIMt005. Different S. alboflavus recombinant strains overexpressing clpS or hmtS were then obtained by introduction of pCIMt005/clpS or pCIMt005/hmtS into S. alboflavus wild-type and the af n gene inframe deleted mutant strains, respectively. Production and Detection of AFNs and Di-AFNs. For the production of AFNs and di-AFNs, S. alboflavus sp. 313, the afn gene inactivation mutants, and the P450 coupling enzyme overexpression strains were cultured and fermented in medium G (1% glucose, 0.3% peptone, 0.25% NaCl, 0.1% CaCO3, pH 7.0) as described.1 To obtain enough AFNs and di-AFNs for structure elucidation, a two-stage culture procedure was used in the large-scale fermentations. The seed cultures of different strains were prepared by inoculating a loop of spores into a 250 mL flask containing 50 mL medium G with 50 μ g/ mL apramycin and incubated at 28 °C, 220 rpm for 24 h. The seed cultures were then inoculated (2% v/v) into 1 L flasks containing 200 mL medium G with 50 μ g/mL apramycin and cultured at 28 °C, 220 rpm for 5 days. The supernatants were collected for AFNs and diAFNs isolations. For detection of AFNs and di-AFNs, the Apollo C18 column column was developed with acetonitrile and water containing 0.1% trifluoroacetic acid at a flow rate of 1.0 mL/min. Percentage of acetonitrile was changed linearly from 15% to 50% for 5 min, from 50% to 87% for 25 min, 87% for 5 min, and from 87% to 100% for 5 min. The detection wavelength was 220 nm. Isolation of compound 5. The cell pellet of 100 L S. albof lavus 313_hmtS culture broth was discarded after centrifugation, and the supernatant was extracted with an equal volume of ethyl acetate for triple times. After evaporation at 35 °C in vacuo, the extract was subjected to a silica gel column (600 g, 200−300 mesh, 6.5 cm × 110 cm) and eluted with 4 L of petroleum ether, EtOAc, EtOAc−MeOH (50:50, v/v), and MeOH, respectively. The EtOAc−MeOH (50:50, v/v) fractions containing 5 were concentrated in vacuo and then performed on a Sephadex LH20 column (3.0 cm × 120 cm). Each eluted fraction was detected by HPLC. The fractions containing 5 were concentrated and further purified by preparative HPLC (Zorbax SB-C18 PrepHT, 7 μ m, 21.2 mm × 250 mm, Agilent, Santa Clara, CA) eluted with a gradient of acetonitrile in water with 0.1% trifluoroacetic acid (40% for 5 min, 40−100% for 30 min, 100% for 10 min) at a flow rate of 10 mL/min to obtain a crude fraction. Finally, the crude fraction was refined by semipreparative HPLC (Zorbax SBC18, 5 μ m, 9.4 mm × 250 mm, Agilent, Santa Clara, CA) eluted with acetonitrile/water (82:18, v/v) containing 0.1% trifluoroacetic acid at a flow rate of 3 mL/min to obtain 6.5 mg compound 5. Isolation of Compound 6. Compound 6 was isolated using a similar protocol as that for 5, except that the preparative HPLC step was eliminated. After eluted from the Sephadex LH-20 column (3 cm × 120 cm), the targeted fractions containing 6 were directly purified by semipreparative HPLC (Zorbax SB-C18, 5 μ m, 9.4 mm × 250 mm, Agilent, Santa Clara, CA, USA) eluted with a constant gradient of methanol:water (72:28, v/v) containing 0.1% trifluoroacetic acid at a flow rate of 3 mL/min to obtain 7.5 mg 6 from 10 L S. albof lavus ΔafnX culture. Isolation of compound 8. Compound 8 was isolated using the same procedure as that for 5. After refined by semipreparative HPLC, 4.8 mg 8 was obtained from 100 L S. alboflavus ΔafnX_hmtS culture. Antibacterial Assays. Antibacterial activity was measured by the microbroth dilution method in 96-well culture plates using Mueller− Hinton broth (Qingdao Nissui Biotechnologies Co. Ltd., Shandong, China) according to the Standard of National Committee for Clinical

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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/jacs.8b10136. Experimental details for construction of the afn gene mutants and in vitro characterization of AfnX, spectroscopic data, and copies of NMR spectra (PDF)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Hua Xiang: 0000-0003-0369-1225 Shuangjun Lin: 0000-0001-9406-9233 Jianhua Ju: 0000-0001-7712-8027 Yihua Chen: 0000-0001-9362-512X Author Contributions ⊗

G. Z., P. L., and G. C. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Professor Ben Shen, The Scripps Research Institute, for the gift of plasmid pBS1043. We thank Dr. Wenzhao Wang, Institute of Microbiology, CAS, for MS data collection. This work was supported in part by the MOST of China (2015CB150600) and the NSFC (31522001). Y.C. is a scholar of “the 100 Talents Project” of CAS. Z.G. is an awardee for “the Youth Innovation Promotion Association” of CAS (2017124).

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REFERENCES

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