Inducing Secondary Metabolite Production by Combined Culture of

Article pubs.acs.org/jnp

Inducing Secondary Metabolite Production by Combined Culture of Talaromyces aculeatus and Penicillium variabile Zhenzhen Zhang,† Xueqian He,† Guojian Zhang,†,‡ Qian Che,† Tianjiao Zhu,† Qianqun Gu,† and Dehai Li*,†,‡ †

Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, P.R. China ‡ Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, P.R. China S Supporting Information *

ABSTRACT: Four new polyketides, penitalarins A−C (1−3) and nafuredin B (4), together with the known biogenetically related nafuredin A (5) were isolated from a mixed culture of a deep-sea-derived fungus Talaromyces aculeatus and a mangrove-derived fungus Penicillium variabile. Liquid chromatography/mass spectrometry analysis showed that none of compounds 1−5 was produced by either of the two fungi when cultured alone under the same condition. The structures of 1−4, including absolute configurations, were deduced based on the interpretation of MS, NMR data, and time-dependent density functional theory calculations of specific electronic circular dichroism spectra. Compounds 1−3 possess a 3,6-dioxabicyclo[3.1.0]hexane ring, and 4 showed cytotoxicity with IC50 values ranging from 1.2 to 9.8 μM against a panel of human cancer cell lines.

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together, new metabolites were detected by HPLC-UV analysis. A follow-up examination of the cocultivation system resulted in the isolation of four new polyketides, penitalarins A−C (1−3) and nafuredin B (4), together with one known biogenetically related compound nafuredin A (5).14 The cytotoxicity of compounds 1−5 was evaluated, and compound 4 was shown to be cytotoxic against HeLa, K562, HCT-116, HL-60, A549, and MCF-7 cell lines with IC50 values ranging from 1.2 to 9.8 μM.

icroorganisms have been proven to be important sources of drug leads with unique structures and pronounced biological activities.1 However, biosynthetic pathways are often not activated (also referred to as cryptic or silent) in single strain culture, which leaves the whole metabolic potential untapped.2 This deficiency could be partially balanced by using a microbial consortia production system that involves cocultivation of two or more microbes.3 In the coculture system, microbial strains would grow together either symbiotically or in competition for nutrients and space, finally establishing an equilibrium state in population and lifestyle.4,5 Those microbial interactions allow the metabolism of each cell type to be specifically tuned to the situation and may exhibit sophisticated metabolic capabilities not achieved by individual strains.6,7 This method of production has resulted in the discovery of a number of compounds with diverse structures and promising bioactivities.8−10 So far, most cocultivation research has focused on strains from the same or similar habitats,7,11,12 whereas the cocultivation of microorganisms from completely unrelated environments remains an intriguing topic with unforeseen implications. With the purpose of releasing the metabolic potential of microorganisms to produce novel bioactive molecules, a panel of strains isolated from diverse marine environments such as deep sea, Antarctic, and mangrove zones were selected from our library to perform cocultivation. We observed that when the deep-sea-derived fungus Talaromyces aculeatus (from a depth of 3386 m, Indian Ocean) and the mangrove-derived fungus Penicillium variabile13 (Fujian Province, China) were cultured © 2017 American Chemical Society and American Society of Pharmacognosy

Received: May 13, 2017 Published: November 16, 2017 3167

DOI: 10.1021/acs.jnatprod.7b00417 J. Nat. Prod. 2017, 80, 3167−3171

Journal of Natural Products

Article

Table 1. 1H (500 MHz) and 13C (125 MHZ) NMR Data for Compounds 1−4 in DMSO-d6 1 position

δC, type

1 2 3 4 5 6

76.6, CH 63.6, CH 65.8, C 83.0, CH 128.3, CH 134.4, CH

7

127.7, CH

8

142.0, CH

9 10

34.7, CH 47.3, CH2

11

134.5, C

12 13

126.9, CH 125.2, CH

14

138.4, CH

15 16 17 18 19 20 21 22 23 23-OH

38.4, CH 29.7, CH2 12.1, CH3 170.9, C 52.5, CH3 14.1, CH3 20.2, CH3 16.7, CH3 20.5, CH3

2 δH (J in Hz)

4.48, s 3.95, s 4.39, d (9.0) 5.57, dd (9.0, 15.2) 6.23, dd (10.6, 15.2) 6.01, dd (10.6, 15.2) 5.64, dd (7.8, 15.2) 2.40, m 2.03, m, 1.93, dd (7.5, 13.3)

δC, type 76.7, CH 63.6, CH 65.8, C 83.0, CH 128.4, CH 134.4, CH

3

δH (J in Hz)

δC, type

4.50, s 3.96, s

δH (J in Hz)

4 δC, type

4.48, s 3.95, s

4.40, d (9.0) 5.59, dd (9.0, 15.2) 6.25, dd (10.5, 15.2)

76.6, CH 63.6, CH 65.8, C 83.0, CH 128.4, CH 134.5, CH

4.39, d (9.0) 5.57, dd (9.0, 15.2) 6.24, dd (10.5, 15.2)

163.3, C 118.0, CH 156.3, CH 68.0, C 84.4, CH 124.5, CH

127.7, CH 6.03, dd (10.3, 15.2)

127.7, CH

6.01, dd (10.5, 15.2)

134.7, CH

142.0, CH 5.66, dd (7.8, 15.2)

142.0, CH

5.64, dd (7.8, 15.2)

127.7, CH

34.7, CH 47.3, CH2

34.7, CH 47.3, CH2

2.40, m 2.04, dd (7.1, 13.3), 1.93, dd (7.6, 13.4)

142.1, CH 34.7, CH

2.41, m 2.05, dd (7.3, 13.3), 1.93, dd (8.2, 13.7)

134.6, C

134.5, C

5.73, d (10.8) 6.15, dd (10.9, 15.2) 5.41, dd (7.8, 15.2)

126.9, CH 5.74, d (10) 125.1, CH 6.17, m

127.0, CH 127.4, CH

5.74, d (10) 6.19, dd (10.5, 15.2)

134.5, C 126.9, CH

138.6, CH 5.43, dd (7.8, 15.2)

134.7, CH

5.35, dd (7.8, 15.2)

125.2, CH

2.04, m 1.26, m 0.79, t (7.4)



1.99, m 1.48, m, 1.16, m 0.78, t (7.5)

138.4, CH 38.4, CH 29.7, CH2 12.1, CH3 21.4, CH3 20.1, CH3 16.7, CH3 20.5, CH3

3.67, s 1.31, s 0.90, d (6.6) 1.65, s 0.93, d (6.6)

2.28, m 1.40, m 3.36, m 3.69, s 1.33, s 0.91, d (6.3) 1.66, s 0.95, m

47.3, CH2

3.67, s 1.31, s 0.90, d (6.6) 1.65, s 3.28, m 4.41, t (5.4)

δH (J in Hz) 5.83, d (9.8) 6.88, d (9.8) 4.73, d (6.7) 5.64, m 6.30, dd (10.5, 14.8) 6.08, dd (10.5, 15.3) 5.68, m 2.41, m 2.04, m, 1.95, dd (7.6, 13.3) 5.73, dd (10.8, 14.8) 6.16, dd (10.8, 15.1) 5.43, dd (7.8, 15.1) 2.07, m 1.27, m 0.81, d (7.4) 1.09, s 0.92, d (6.7) 1.67, s 0.94, d (6.7)

correlation (HMBC) from H3-21 to C-8/C-9/C-10, from H3-22 to C-10/C-11/C-12 and from H3-23 to C-14/C-15/C-16 (Figure 1). The hydrogenated furan moiety was indicated by the HMBC from H-1 and H3-20 to C-2, C-3, and C-4, from H-2 to C-1, from H-4 to C-1, C-2, and C-3, as well as the chemical shifts of CH-1 (δC 76.6, δH 4.48) and CH-4 (δC 83.0, δH 4.39). The epoxy ring was suggested by the chemical shifts of CH-2 (δC 63.6, δH 3.95) and C-3 (δC 65.8). The COSY correlations between H-4 and H-5 and HMBC from H-4 to C-6 attached the olefinic side chain to C-4. The ester carbonyl carbon (δC 170.9, C-18) was assigned at C-1 on the basis of HMBC from H-1 to C-18. Finally, the planar structure of 1 was established by connecting the methoxy group to C-18 as evidenced by the HMBC from H3-19 (δH 3.67) to C-18. Penitalarins B and C (2 and 3) were both purified as pale powders. The 1H and 13C NMR spectroscopic data of 2 and 3 (Table 1) indicate that both compounds possess the same skeleton as 1 but differed by the substituted groups. The chemical shifts of CH2-17 (δC 59.2, δH 3.36) of 2 and CH2-23 (δC 64.9, δH 3.28) of 3 indicated that 2 and 3 are the oxidation products of 1 with the CH3-17 and CH3-23 changed to hydroxymethyl groups, respectively, which were also in agreement with the 2D NMR data (Figure 1). The relative configuration of the 3,6-dioxabicyclo[3.1.0]hexane moiety in 1 was deduced based on the NOESY correlations (Figure 1) and coupling constants. The NOEs between H-2 and H-20 indicated that they were on the same face of the furan ring. The coupling constant of 3JH‑1,H‑2 (0 Hz) suggested a trans configuration of H-1 and H-2, indicating that the dihedral

Herein, we reported the details of the isolation, structure elucidation, and biological activities of these new compounds.

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RESULTS AND DISCUSSION The T. aculeatus and P. variabile were individually grown on potato dextrose agar medium at 28 °C for 3 days and then cocultured by inoculating their spores aseptically into Erlenmeyer flasks (500 mL) each containing 100 mL of maltose-based medium. The mixture of T. aculeatus and P. variabile was cultured at 28 °C and agitated at 180 rpm for 9 days (45 L). The EtOAc extract (40 g) of the fermentation was fractionated by vacuum-liquid chromatography (VLC) on silica gel, Sephadex LH-20 column chromatography, medium-pressure liquid chromatography (MPLC, ODS), and semipreparative high-performance liquid chromatography (HPLC) to afford compounds 1−5 (guided by liquid chromatography/mass spectrometry (LC-MS) analysis). Penitalarin A (1) was obtained as a pale powder. The molecular formula was determined as C23H34O4 based on the highresolution electrospray ionization mass spectrometry (HRESIMS), indicating seven degrees of unsaturation. The 1D NMR data (Table 1) of 1 suggested the presence of six methyls with one methoxy group (δC 52.5, δH 3.67), two methylenes, seven sp2 methines, five sp3 methines, and three nonprotonated carbons including one carbonyl. The planar structure of 1 was determined by interpretation of 1D and 2D NMR spectroscopic data. A methylated olefinic hydrocarbon chain (from C-4 to C-17) was deduced from the correlated spectroscopy (COSY) crosspeaks (H-4/H-5/H-6/H-7/H-8/H-9/H2-10, H-12/H-13/H14/H-15/H2-16/H3-17) and heteronuclear multiple bond 3168



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Figure 2. Experimental ECD spectrum of 1 (black curve) and the calculated one of the truncated model 1a (red curve) (σ = 0.40 eV).

Figure 1. Key COSY, HMBC, and NOESY correlations of 1−4.

angles ω1‑H,C‑1,C‑2,2‑H are nearly 90°.15 With the NOESY correlations between H-4 and H-19, the methoxy group and H-4 were assigned at the same face of the furan ring. Thus, the relative configuration of the 3,6-dioxabicyclo[3.1.0]hexane was determined to be 1R*,2S*,3R*,4S*. The absolute configuration of the 3,6-dioxabicyclo[3.1.0]hexane moiety in 1 was determined by comparison of the experimental electronic circular dichroism (ECD) curve and the calculated ECD curve for the truncated model (1R,2S,3R,4S)-1a using time-dependent density functional theory (DFT). The DFT reoptimization of the initial Merck molecular force field (MMFF) minima was performed at the B3LYP/6-31+g(d) level with a polarizable continuum model (PCM) solvent model for MeOH. The good agreement of the calculated ECD spectra of (1R,2S,3R,4S)-1a with the experimental data suggested the absolute configuration of the 3,6-dioxabicyclo[3.1.0]hexane moiety as 1R,2S,3R,4S (Figure 2). The geometry of double bonds in the olefinic chain was deduced to be E by the NOESY correlations (H-5/H-7, H-6/H-8, H-10/H-12, H-13/H3-22, and H-12/H-14). With the consideration of biogenetic origin and coisolation of nafuredin A (5), whose absolute configuration was determined based on asymmetric synthesis,16 the chirality of C-9 and C-15 was indicated to be 9R and 15S, same as those in 5, which also was supported by the NMR data. Finally, the absolute configuration of 1 was determined as 1R,2S,3R,4S,9R,15S. The similar ECD spectra (Figure 3) and close NMR data of 1−3 indicated that they share the same absolute configurations. Nafuredin B (4) was isolated as a pale powder with the molecular formula of C22H32O3. The 1D NMR data (Table 1) of 4 suggested the presence of five methyls, two methylenes, 12 methines including nine sp2 methines, and three nonprotonated carbons with one carbonyl. The 1H and 13C NMR data of 4 are close to those of nafuredin A (5).14 The major differences

Figure 3. Experimental ECD spectra of 1 (red), 2 (black), and 3 (green).

were the replacement of the two oxidized sp3 methines in 5 with a double bond in 4, consistent with the downfield shifts of C-2/C-3 (68.0 ppm/58.5 ppm in 5 vs 118.0 ppm/156.3 ppm in 4), supported by the COSY correlations of H-2/H-3, and the key HMBC from H-2 to C-1/C-4, from H-3 to C-1/C-5, from H-5 to C-3/C-4, and from H3-19 to C-3/C-4/C-5 (Figure 1). The stereochemistry of the methylated olefinic chain (from C-6 to C-18) in compound 4 was deduced to be the same as that in compounds 1−3 and 5, supported by the almost identical NMR data and biogenetic consideration. The relative configuration of the δ-lactone ring of 4 was deduced from the NOESY experiment (Figure 1). The NOEs between H-19 and H-6 suggested that the Me-19 faced the same side of the pyrone ring as C-6. To determine the absolute configurations of C-4 and C-5, the solution conformers and ECD spectra of a truncated model (4S,5R)-4a (Figure 4) were calculated. The good agreement of the calculated ECD spectra of (4S,5R)-4a with the experimental data of 4 suggest the 4S,5R absolute configuration. The cytotoxicities of compounds 1−5 were evaluated against HeLa, HCT-116, MCF-7, and A549 cell lines by sulforhodamine B (SRB) method17 and K562 and HL-60 cell lines by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.18 Compound 4 showed cytotoxicity against HeLa, 3169



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K562, HCT-116, HL-60, A549, and MCF-7 cell lines with IC50 values of 5.5, 2.9, 1.4, 1.2, 5.1, and 9.8 μM, respectively, whereas others were not active (IC50 > 50.0 μM) (Table 2). Table 2. Cytotoxicity of Compounds 1−5 against Six Cancer Cell Lines (IC50 μM) HeLa

K562

HCT-116

HL-60

A549

MCF-7

4 1−3 and 5 ADMa

5.5 >50.0 0.5

2.9 >50.0 0.6

1.4 >50.0 0.2

1.2 >50.0 0.2

5.1 >50.0 0.8

9.8 >50.0 0.6

a

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on a JASCOP-1020 digital polarimeter. UV spectra were recorded on Waters 2487, whereas the ECD spectrum was measured on a JASCO J-815 spectropolarimeter. 1H NMR, 13C NMR, DEPT, and 2D NMR spectra were recorded on an Agilent 500 MHz DD2 spectrometer. HRESIMS and ESIMS data were obtained using a Thermo Scientific LTQ Orbitrap XL mass spectrometer. Column chromatography was performed with Sephadex LH-20 (Amersham Biosciences). VLC was carried out over silica gel (Qingdao Marine Chemical Factory). MPLC was performed using a C18 column (Agela Technologies, YMC-Pack ODS-A, 3 × 40 cm, 5 μm, 20 mL/min). Preparative HPLC collection used a C18 column (Waters, YMC-Pack ODS-A, 10 × 250 mm, 5 μm, 3 mL/min). LC-MS was perfomed using Acquity UPLC H-Class coupled to a SQ Detector 2 mass spectrometer (Waters) using a BEH C18 column (1.7 μm, 2.1 × 50 mm). Fungal Material. The fungal strain Talaromyces aculeatus, identified based on sequencing of the ITS region (Genbank no. KY76541), was isolated from deep-sea sediment from the Indian Ocean (depth 3386 m, E 88.73°, N 10.00°, collected in April 2008). The isolation and identification of Penicillium variabile has been previously described.13 These two fungi were deposited at the Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, People’s Republic of China. Fermentation. Erlenmeyer flasks (500 mL) containing 100 mL of media were directly inoculated with spores of two fungi. The media contained maltose (20 g), mannitol (20 g), glucose (10 g), sodium glutamate (10 g), yeast extract (3 g), corn syrup (1 g), KH2PO4 (0.5 g), and MgSO4·7H2O (0.3 g) dissolved in 1 L naturally collected seawater (Huiquan Bay, Yellow Sea). The culture flasks were shaken at 180 rpm and 28 °C for 9 days. Extraction and Purification. The whole fermentation broth (45 L) was filtered through cheese cloth to separate the supernatant from the mycelia. The supernatant was extracted with EtOAc (3 × 45 L), and the mycelia were macerated and extracted with acetone (3 × 15 L). All extracts were evaporated under reduced pressure to give a crude gum (40.0 g). The supernatant and mycelia extracts were combined after evaporation to dryness based on their similar HPLC-UV profiles and used for chemical workup. The extract was separated by VLC on silica gel using a stepped gradient elution of petroleum ether/EtOAc (from 10:0 to 0:10) and EtOAc/MeOH (from 10:0 to 0:10) to give seven fractions (Fr.1 to Fr.7). Fr.4 was further separated by MPLC (ODS) using a stepped gradient elution of MeOH−H2O (30:70 to 100:0) to yield 12 subfractions (Fr.4-1 to Fr.4-12). Fr.4-6 was then separated by semipreparative HPLC eluted with MeOH−H2O (75:25) to obtain the 1 (4.0 mg, tR = 20 min), 2 (6.2 mg, tR = 14 min), and 3 (3.2 mg, tR = 16 min). Fr.6 was separated on a Sephadex LH-20 column with MeOH to provide six subfractions (Fr.6-1 to Fr.6-6). Fr.6-3 was then separated by semipreparative HPLC eluted with MeOH−H2O (65:35) to obtain the 4 (2.5 mg, tR = 28 min) and 5 (15.0 mg, tR = 20 min). Penitalarin A (1): pale powder; [α]D20 +40 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 240 (4.23) nm; CD (1.3 mM MeOH) λmax (Δε) 220 (−15.86) and 245 (+31.92) nm; for 1H and 13C NMR data, see Table 1; HRESIMS m/z 397.2344 [M + Na]+ (calcd for C23H34O4Na, 397.2349). Penitalarin B (2): pale powder; [α]D20 +25 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 240 (3.88) nm; CD (1.3 mM MeOH) λmax (Δε) 220 (−16.38) and 245 (+37.45) nm; for 1H and 13C NMR data, see Table 1; HRESIMS m/z 391.2470 [M + H]+ (calcd for C23H35O5, 391.2479). Penitalarin C (3): pale powder; [α]D20 +28 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 240 (3.64) nm; CD (0.7 mM MeOH) λmax (Δε) 220 (−5.20) and 245 (+23.44) nm; for 1H and 13C NMR data, see Table 1; HRESIMS m/z 413.2295 [M + Na]+ (calcd for C23H34O5Na, 413.2298). Nafuredin B (4): pale powder; [α]D20 +19 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 240 (3.55) nm; CD (1.5 mM MeOH) λmax (Δε) 220 (−30.40) and 245 (+37.59) nm; for 1H and 13C NMR data, see Table 1; HRESIMS m/z 367.2238 [M + Na] + (calcd for C22H32O3Na, 367.2244).

Figure 4. Experimental ECD spectrum of 4 (black curve) and the calculated one of the truncated model 4a (red curve) (σ = 0.40 eV).

compounds

Article

ADM = doxorubicin (positive control).

Cocultivation attracts great interest from both chemists and biologists as the activation of silent biosynthetic pathways can be utilized for the production of new bioactive molecules. During the mixed culture process, possible interaction routes of the two microorganisms are proposed including the following: (a) one organism stimulates assembly of natural products in another organism via secretion of chemical signals; (b) one organism produces intermediates that are further modified by another organism; (c) cell−cell contact triggers more complex processes during cocultivation, etc.4−6 Nafuredin (5, nafuredin A in this paper) was first discovered from the fungus Aspergillus niger in 200114 and then isolated from Trichoderma citrinoviride19 and Phoma sp. nov.20 Nafuredin had been reported to potently inhibit NADH-fumarate reductase (complexes I + II) activity at nanomolar level.21 Penitalarins A−C (1−3) are the first naturally occurring compounds reported to contain 3,6-dioxabicyclo[3.1.0]hexane instead of the δ-lactone ring. Biogenetically, compounds 1−5 are proposed to be formed from the key epoxy containing intermediate (Figure S1, Supporting Information). Compounds 4 and 5 were generated by intramolecular ester condensation, dehydration, and oxidation. Different from the formation of 4 and 5, the 3,6-dioxabicyclo[3.1.0]hexane moiety in 1−3 was generated by a vital step of intramolecular SN2 inversion. With further dehydration, methylation, and oxidation, compounds 1−3 were formed. Although the details for the process of generating compounds 1−5 are currently unknown, none of these compounds could be detected by LC-MS when the two fungi were cultivated alone under the same condition (Figures S2 and S3, Supporting Information). 3170



Article

Cytotoxicity Assay. Cytotoxic activities of 1−4 were evaluated using HeLa cell line (human cervical cancer), HCT-116 cell line (human colon cancer), MCF-7/ADM cell line (doxorubicin-resistant breast cancer cell), and A549 cell line (lung cancer) by SRB method and K562 cell line (human acute myelocytic leukemia) and HL-60 cell line (human promyelocytic leukemia) by the MTT method. Doxorubicin hydrochloride was used as the positive control. The detailed methodologies for biological testing have been described in our previous report.22 Computation Section. Conformational searches were run by employing the “systematic” procedure implemented in Spartan’1423 using MMFF. All MMFF minima were reoptimized with DFT calculations at the B3LYP/6-31+G(d) level using the Gaussian09 program.24 The geometry was optimized starting from various initial conformations, with vibrational frequency calculations confirming the presence of minima. Time-dependent DFT calculations were performed on four lowest-energy conformations for (1R,2S,3R,4S)-1a and three lowestenergy conformations for (4S,5R)-4a (>3% population) using 30 excited states and using a PCM for MeOH. ECD spectra were generated using the program SpecDis25 by applying a Gaussian band shape with 0.40 eV width for 1a and 4a, from dipole length rotational strengths. The dipole velocity forms yielded negligible differences. The spectra of the conformers were combined using Boltzmann weighting, with the lowest-energy conformations accounting for about 97% of the weights. The calculated spectra were shifted by 10 nm for 1a and 15 nm for 4a to facilitate comparison to the experimental data.

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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/acs.jnatprod.7b00417. Possible biosynthetic pathway of compounds 1−5, LC/MS and HPLC-UV analysis of the metabolic extracts, MS, 1D and 2D NMR spectra for compounds 1−4, and details for ECD calculations (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 0086-532-82031619. Fax: 0086-532-82033054. E-mail: [email protected]. ORCID

Qian Che: 0000-0003-0610-1593 Dehai Li: 0000-0002-7191-2002 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21372208, 21542001, and 41676127), the Shandong Provincial Natural Science Fund for Distinguished Young Scholars (JQ201422), AoShan Talents Program Supported by Qingdao National Laboratory for Marine Science and Technology (2015ASTP-ES09), NSFC-Shandong Joint Fund for Marine Science Research Centers (U1606403), Scientific and Technological Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology (2015ASKJ02), Shandong province key research and development program (2016GSF201204), and Fundamental Research Funds for the Central Universities (201564026). We also thank Dr. N. Moscatello (State University of New York at Buffalo) for helping with the manuscript preparation.

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REFERENCES

(1) Demain, A. L. J. Ind. Microbiol. Biotechnol. 2014, 41, 185−201. (2) Challis, G. L. J. Med. Chem. 2008, 51, 2618−2628. 3171


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