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. 2021 May 21;11(6):283. doi: 10.1007/s13205-021-02846-0

Cytotoxic and antimicrobial activities of secondary metabolites isolated from the deep-sea-derived Actinoalloteichus cyanogriseus 12A22

Xiaoying Zhang 1, Chunfeng Song 1, Yan Bai 1, Jiangchun Hu 1,✉, Huaqi Pan 1,✉
PMCID: PMC8140039  PMID: 34094802

Abstract

A new deep-sea-derived actinomycete 12A22 was isolated from the sediment of the South China Sea which showed potential cytotoxic and antimicrobial activities. The actinomycete was identified as Actinoalloteichus cyanogriseus by investigating morphological characteristics and phylogenetic analyses based on its 16S rRNA gene sequence. Two compounds, cyclo-(L-Pro-D-Pro-L-Tyr-L-Tyr) (1) and 2-hydroxyethyl-3-methyl-1,4-naphthoquinone (2), were isolated and characterized from the fermentation broth of the strain 12A22. Compound 2 exhibited significant inhibitory activities against a variety of phytopathogenic fungi (Fusarium oxysporum f. sp. cucumerinum, Setosphaeria turcica, and Botrytis cinerea) and Gram-positive bacterium (Bacillus subtilis). In particular, this compound showed better antifungal activity against Botrytis cinerea than positive control amphotericin B. Besides, compound 2 showed moderate cytotoxic activity against human breast cancer MDA-MB-435 cells with IC50 10.59 µM, weaker than the positive control diaminedichloroplatinum with 5.91 μM. Our results suggested that this naphthoquinone could be used as a potential antimicrobial and antitumor agent.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-021-02846-0.

Keywords: Actinoalloteichus, Cyclotetrapeptide, Naphthoquinone, Antimicrobial activity, Cytotoxic activity

Introduction

The discovery of novel microorganisms and bioactive metabolites plays an important role in resisting diseases and pathogens (Berdy 2012). For a long time, actinomycetes were considered as the most economic value microorganisms which could produce fascinating antimicrobial and antitumor agents (Mitousis et al. 2020; Prudence et al. 2020; Sharma et al. 2019). The increasing numbers of literatures indicated that marine environments were important resources for the discovery of multifarious actinomycetes and bioactive metabolites (Kamjam et al. 2017; Lu et al. 2020). In addition, deep-sea environment provides a source of the discovery of the bioactive secondary metabolites due to its unique and extreme environment (Kamjam et al. 2017; Yi et al. 2020). It was reported that about 75% of deep-sea natural products showed significant biological activities (Pilkington 2019). In the recent years, a series of bioactive metabolites had been isolated from the deep-sea-derived actinomycetes. For example, two dimeric neoabyssomicins were isolated from the deep-sea actinomycete Streptomyces koyangensis SCSIO 5802, which displayed selective activities against vesicular stomatitis virus and methicillin-resistant Staphylococcus aureus (Huang et al. 2018). Four novel sealutomicins were isolated from deep-sea actinomycete Nonomuraea sp. MM565M-173N2, and one of them revealed strong antibacterial activity against carbapenem-resistant Enterobacteriaceae (Igarashi et al. 2021). Novel polyene macrolactams were isolated deep-sea Streptomyces sp. OUCMDZ-3159, which displayed antifungal activity against Candida albicans ATCC 10231 (Wang et al. 2021).

Continuing the course of screening for antimicrobial and antitumor agents, we had characterized many potent antimicrobial metabolites from a few extremophilic actinomycetes from sediments of the South China Sea (Pan et al. 2013, 2015b). In this study, a new deep-sea actinomycete 12A22 was screened with promising cytotoxic and antimicrobial activities. And it was determined to be Actinoalloteichus cyanogriseus based on its phenotypic and genotypic data. In addition, two metabolites were isolated and identified from the fermentation broth of the strain 12A22. The chemical structures of two metabolites were elucidated by NMR spectroscopic data. Finally, the antimicrobial and cytotoxic activities of two metabolites were assayed by paper agar disk diffusion assay and MTT method, respectively.

Materials and methods

Strain and culturing conditions

The strain 12A22 was isolated from a sediment collected at the depth of 2134 m from the South China Sea (94.3364° E, 1.4256° N). It was routinely cultured and maintained on ISP3 medium at 28 °C. ISP3 medium consists of oat flour 20 g, trace element solution 1 mL (MgSO4·7H2O 0.001 g, ZnSO4·7H2O 0.001 g, MnCl2·4H2O 0.001 g, FeSO4·7H2O 0.001 g, CoCl2·6H2O 0.001 g, CaCl2 0.001 g, H3BO3 0.001 g and distilled water 1 L), agar 18 g, distilled water 1 L, pH 7.2.

Strain identification

The identification of the strain 12A22 based on its phenotypic and genotypic properties. In particular, the strain 12A22 was inoculated on potato sucrose agar medium with 2.5% (w/v) sea salt (2.5% PSA) (potato 200 g, sucrose 20 g, sea salt 25 g, agar 18 g, distilled water 1 L, pH 7.0), Gauze′s synthetic medium NO.1 (Gauze′s 1) (soluble starch 20 g, KNO3 1 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, sea salt 25 g, agar 18 g, distilled water 1 L, pH 7.0–7.2) and ISP3 medium for 14 days at 28 °C. Colors of the aerial and substrate mycelia were determined by color chips from the ISCC-NBS color charts. Morphological properties were observed by a light microscope (BX41, Olympus) and a scanning electron microscope (QuantaTM250, FEI) (Pan et al. 2015a).

The total genomic DNA of the strain 12A22 was extracted by Takara genomic DNA kit. The 16S region was amplified by polymerase chain reaction (PCR) combination of the primer 27F (5′-AGRGTTYGATYMTGGCTCAG-3′) and 1492R (5′-GGYTACCTTGTTACGACTT-3′). The PCR reaction system in a total of 50 µL volume, which contained 5 μL 10 × PCR buffer, 6 μL dNTP (2.5 mmol/μL), 1 μL of each primer (25 pmol/μL), rTaq 0.5 μL, 2 µL genomic DNA, 34.5 μL double distilled water. The PCR was performed at an initial annealing temperature of 95 °C for 5 min; followed by 35 cycles of 95 °C for 30 s denaturation, 56 °C for 40 s annealing and 72 °C for 1.5 min extension; a final elongation at 72 °C for 10 min. Subsequently, the PCR products were verified by 2% agarose gel electrophoresis and sequenced by Dalian Takara company. In the end, the 16S rRNA gene sequences were analyzed by BLAST multiple sequence alignment on the NCBI website, along with a phylogenetic tree was built by the MEGA 6.0 software, and the topologies of the resultant trees were evaluated by bootstrap analysis with 1000 replicates.

Fermentation conditions

The strain 12A22 was inoculated on ISP3 solid medium by scribing technique and cultured at 28 °C for 5 days, then the agar was cut into pieces (0.5 × 0.5 cm) and transferred to a 500 mL flask containing 150 mL of VM medium (10 g glucose, 10 g soluble starch, 10 mL glycerin, 5 g corn syrup, 2 g peptone, 1 g yeast extract, 0.5 g NaCl, 3 g CaCO3, 1 L distilled water, pH 7.4) and incubated in a rotary incubator for 3 days (28 °C, 180 rpm). Subsequently, the fermentation broth in the flask was inoculated into a 50 L fermenter (Zhenjiang East Biotech Equipment and Technology Co., Ltd., China) containing 30 L of VM medium, incubated for 7 days as following condition: tank pressure 0.05 MPa, ventilation 0.50–1.0 v/v min, 250 rpm, cultivation temperature 28 ± 0.5 °C.

Isolation and purification of bioactive metabolites

At first, the fermentation broth was centrifuged (4000 rpm for 20 min), then the supernatant was extracted with macroporous adsorbent resin HP20 (Mitsubishi Chemical Co., Ltd., Japan.) and placed in a rotary incubator for 2 h (28 °C, 180 rpm). Secondly, the macroporous resin HP20 was collected and washed in sequence with deionized water and 10% ethanol (EtOH)/H2O (v/v). And the solvent was discarded. Then, the macroporous resin HP20 was eluted three times with 95% EtOH/H2O (v/v), the solvent was collected and dried by a rotary evaporator to obtain crude extract A (yield: 12.48 g). The mycelia were ultrasonically extracted with acetone three times and dried using a rotary evaporator to obtain crude extract B (yield: 3.13 g).

Crude extracts A and B were merged together and dissolved in methanol, then the insoluble matter was discarded. Preliminarily the merged crude extract was subjected to silica gel column chromatography (500–600 mesh, Qingdao Ocean Chemical Co. Ltd, China) with a gradient of dichloromethane/methanol (CH2Cl2/MeOH) system (100/0, 95/5, 90/10, 80/20, 70/30, 60/40, 50/50 and 0/100 v/v) to obtain eight fractions (F1-F8). Then, the F3 fraction (yield: 2.601 g, eluted with 90/10 v/v) was separated on an ODS column chromatography with a gradient of MeOH/H2O (100/0, 60/40, 40/60, 30/70, 20/80, v/v) to obtain five subfractions (rF1–rF5). Subsequently, the rF5 subfraction (yield: 513.1 mg, eluted with 20/80 v/v) was separated by Sephadex LH-20 (H&E Co., Ltd., Beijing, China) column eluted with MeOH, and was divided into three subfractions (Fr. A, Fr. B, and Fr. C) by thin-layer chromatography. Fr. B (yield: 180 mg) was further separated by a semi-preparative HPLC system (Dionex, Sunnyvale, CA, USA) using a C18 YMC-Pack ODS-A column (5 μm, φ 10 × 250 mm) to obtain compound 1 (yield: 17.5 mg). The rF3 subfraction (yield: 100.9 mg, eluted with 40/60) was also subjected to the Sephadex LH-20 column with MeOH system to obtain the fraction Fr. D. The Fr. D was separated by preparative thin-layer chromatography (TLC) (solvent system: petroleum ether/acetone, 2/1 v/v) to obtain compound 2 (yield: 9.2 mg).

Nuclear magnetic resonance (NMR) analyses

Dimethylsulfoxide-d6 (DMSO-d6,) was used as solvent to dissolve the compounds 1 and 2. One-dimensional (1H NMR and 13C NMR) and two-dimensional NMR spectroscopies were recorded on a Bruker AV 600 spectrometer (Germany). Two-dimensional NMR spectroscopies, like correlation spectroscopy (COSY), heteronuclear single-quantum correlation spectroscopy (HSQC), and heteronuclear multiple-bond correlation spectroscopy (HMBC) were used to identify the complex structures of compounds.

Antimicrobial activities

Five fungal pathogens (Fusarium oxysporum f. sp. cucumerinum S19, Rhizoctonia solani H7, Setosphaeria turcica H1, Botrytis cinerea H6, and Candida albicans ATCC 10231) and three bacteria (Staphylococcus aureus ATCC 29213, Bacillus subtilis CMCC 63501, and Escherichia coli ATCC 25922) were used to evaluate the antimicrobial activities of compounds 1 and 2, the strains mentioned above were deposited at the Group of Microbial Biotechnology, Institute of Applied Ecology, Chinese Academy of Sciences. Antimicrobial activity was carried out by the paper-agar disk diffusion assay as described methods previously (Chen et al. 2010). Samples were dissolved in sterile water with 0.5% DMSO (v/v) and diluted to a concentration of 1 mg/mL. 10 μL of the sample solutions were dropped into 8 mm sterile filter paper disks, then the filter paper disks were placed evenly on the solid medium with eight test strains. Amphotericin B was used as the positive control, 0.5% (v/v) DMSO was used as the blank control, three replicates were done for each sample. Antimicrobial activity was calculated by the diameter of inhibitory zones in the solid medium after incubated more than 48 h at 28 °C.

Cytotoxic activity

The tumor breast carcinoma cells line MDA-MB-435 was purchased from the American Type Culture Collection (ATCC). The cytotoxic activity assay was carried out by the MTT method (Li et al. 2012). Diaminedichloroplatinum (DDP) was used as a positive control. Briefly, MDA-MB-435 cells were placed in 96-well plates and incubated for 12 h. Then, the cells were treated with various concentrations of compounds (2.5–40 µM), and incubated at 37 °C for 48 h. Then, the supernatant was removed and 20 μL of the MTT (2.5 mg/mL) was added to each well incubated at 37 °C for 4 h, then 100 μL of DMSO was added to 96-well plates and incubated for 20 min. Finally, the absorbance was measured at 570 nm using a Thermo scientific Multiskan FC multiplate photometer. The inhibition rate was calculated using the following formula: inhibition rate (%) = [(Acontrol − Asample)/(Acontrol − Ablank)] × 100% where Acontrol was absorbance of the control, Asample was absorbance of sample, and Ablank was absorbance of blank.

Statistical analysis

All measurements of antimicrobial and cytotoxic activities were repeated in triplicate and data were expressed as mean ± SD (standard deviations). 50% growth inhibition (IC50) was calculated from the regression equation using SPSS 17.0 (Statistical Program for Social Sciences, SPSS Inc., Chicago, IL, USA).

Results and discussion

The identification of the strain 12A22

The actinomycete 12A22 was a Gram-positive strain and grew well on 2.5% PSA, Gauze′s 1, and ISP3 media. The strain 12A22 showed good growth on 2.5% PSA and ISP3 media, while moderate growth on Gauze′s 1. Its aerial mycelium was extremely abundant on three media tested, pale blue on ISP3 and grayish-white on 2.5% PSA or Gauze′s 1. The substrate mycelium was grey and could produce a black diffusible pigment (Fig. 1). The strain 12A22 produced straight or rectiflexibile spore chains with a spore size of about 0.7–0.5 × 1.1–0.8 µm with a smooth surface (Fig. 1). The morphological characteristics of the strain 12A22 were similar to Actinoalloteichus cyanogriseus (Tamura et al. 2000). An almost complete 16S rRNA gene sequence (1344 bp) of the strain 12A22 was obtained and submitted in the GenBank Database with the accession number MK346266. Alignment of the 16S rRNA gene sequences revealed that the strain was closely related to Actinoalloteichus species, and had the highest similarity with Actinoalloteichus cyanogriseus IFO14455T (100.0%). The constructed phylogenetic tree further showed that the strain 12A22 clustered with the most closely related strain A. cyanogriseus IFO14455T and separated from other related representatives of the genus Actinoalloteichus (Fig. 2). Therefore, the strain 12A22 was identified as A. cyanogriseus based on the comprehensive phenotype and phylogenetic analysis.

Fig. 1.

Fig. 1

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The morphologic characteristic and scanning electron micrograph of the strain 12A22 on 2.5% PSA medium for 14 days at 28 °C. Bar, 4 µm

Fig. 2.

Fig. 2

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Neighbor-joining phylogenetic tree of 16S rRNA gene sequences of the strain 12A22 using MEGA 6.0. Number at nodes indicate levels of bootstrap support (%) based on a neighbor-joining analysis of 1000 resampled datasets; only values > 50% are given. NCBI accession numbers are given in parentheses

The rare actinomycete genus Actinoalloteichus was originally proposed in 2000 with the description of a single species Actinoalloteichus cyanogriseus (Tamura et al. 2000). So far, a total of six Actinoalloteichus species have been reported, including Actinoalloteichus cyanogriseus (Tamura et al. 2000), Actinoalloteichus spitiensis (Singla et al. 2005), Actinoalloteichus hymeniacidonis (Zhang et al. 2006), Actinoalloteichus nanshanensis (Xiang et al. 2011), Actinoalloteichus hoggarensis (Boudjelal et al. 2015), and Actinoalloteichus fjordicus (Nouioui et al. 2017). Members of genus Actinoalloteichus distributed in soil (Boudjelal et al. 2011; Fujita et al. 2016; Xiang et al. 2011), desert (Kumar et al. 2021; Singla et al. 2005), inland solar saltern (Jose and Jebakumar 2013), marine sediments (Manivasagan et al. 2014), and marine sponges (Karuppiah et al. 2015; Nouioui et al. 2017) etc.

It was worth noting that more and more intriguing bioactive secondary metabolites have been discovered from Actinoalloteichus species. For instance, neomaclafungins A–I were isolated from Actinoalloteichus sp. NPS702 and exhibited significant antifungal activity against Trichophyton mentagrophytes ATCC 9533 (Sato et al. 2012). In addition, novel cyclopentenone derivatives (Wang et al. 2014), novel cyanogramide (Fu et al. 2014), 1-isopentadecanoyl-3β-d-glucopyranosyl-X- glycerol (Duncan et al. 2015), caerulomycins (Fu et al. 2011; Mei et al. 2019), novel dokdolipids A-C (Choi et al. 2019), and novel marinacarbolines (Qin et al. 2020) were isolated from Actinoalloteichus spp., and showed various kinds of cytotoxic activities. Here, we described a new deep-sea-derived Actinoalloteichus cyanogriseus 12A22, which revealed potent antimicrobial and cytotoxic activities. Our research supported that rare actinomycete genus Actinoalloteichus could be a new source for the discovery potent antimicrobial and antitumor agents.

Isolation and identification of bioactive compounds

Two compounds 1 and 2 were isolated and purified from the fermentation culture of the strain 12A22 by HP20 macroporous adsorption resin, flash chromatography on silica gel, ODS column chromatography, Sephadex LH-20 column chromatography, thin-layer chromatography and semi-preparative HPLC. Compound 1 was obtained as a yellow amorphous powder. The 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6) spectra indicated a typical signal of tetrapeptide skeleton (Fig. S1 and S2). Four α-amino proton signals (δH 4.24, 4.03, 3.92, and 2.83) and four α-amino carbon signals (δC 58.43, 58.19, 57.23, and 56.05) showed four amino acid residues. Two AA′BB′ coupling system of aromatic protons at δH 7.04 (2H, d, J = 8.4 Hz), δH 6.89 (2H, d, J = 8.4 Hz), δH 6.66 (2H, d, J = 8.4 Hz), and δH 6.63 (2H, d, J = 8.4 Hz), two sp2 hybrid carbon connecting oxygen atoms (δC 156.56 and 156.03), two methylene carbon signals (δC 38.66 and 34.80), together with two NH proton signals (δH 8.12 and 7.86) indicated that compound 1 had two tyrosine residues. Moreover, four amino carbons at δC 58.43, 57.23, 44.59, and 44.61 and six methylene carbons at δC 28.59, 27.87, 21.86, 21.33, 44.61, and 44.59 indicated that compound 1 contained two proline units. The binding sequence and attribution of amino acids were further determined by 2D NMR (Figs. S3–S6). The COSY correlations and the HMBC correlations further confirmed the presence of 2 proline residues and 2 tyrosine residues. What′s more, HMBC spectrum revealed that δH 8.12 (NH-Tyr1) and δH 3.92 (α-H-Tyr1) were related to δC 168.43 (CO-Pro2), which indicated Pro2 was connected to Tyr1. δH 7.86 (NH-Tyr2) was related to δC 165.00 (CO-Tyr1), which demonstrated Tyr1 was connected to Tyr2. When combined the above data of 1D-NMR and 2D-NMR with 1D-NMR data of previous report (Table S1) (Mitova et al. 2003), the structure of compound 1 was further elucidated to be a known compound cyclotetrapeptide L-prolyl-D-prolyl-L-tyrosyl-L-tyrosine (Fig. 3).

Fig. 3.

Fig. 3

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Chemical structures of compounds 1 and 2 isolated from Actinoalloteichus cyanogriseus 12A22

Compound 2 was obtained as a yellow crystals (methanol). In the 1H NMR spectrum (Fig. S7), a typical ortho-disubstituted benzene ring was identified based on a group of aromatic protons at δH 7.98 (2H, m) and 7.82 (2H, m). In the 13C NMR spectrum (Fig. S8), ten sp2 hybrid carbon signals including six aromatic carbons, two olefinic carbons at δC 125.89 and 125.83, as well as two α, β unsaturated ketone carbon signals at δC 184.64 and 184.18 suggested that compound 2 contained a 1, 4-naphthoquinone framework. The key NMR data of this skeleton in the same solvent were consistent with previous report (Toma et al. 1975). A lower field signal (δH 3.53, 2H, m) for two protons on the oxygenated carbon and an oxygenated proton signal (δH 4.78, 1H, brt, J = 5.8 Hz) indicated the presence of a CH2OH group. A triplet signal (δH 2.77, 2H, t, J = 6.9 Hz) indicated a methylene group which attached to a sp2 hybrid quaternary carbon and connected to CH2OH. A singlet methyl signal (δH 2.13, 3H, s) suggested this methyl group connection in one sp2 hybrid quaternary carbon. The above NMR data suggested that the methyl group (δH 2.13) and CH2CH2OH group (δH 2.77–3.53–4.78) can only be connected to the carbons at the positions of C-2 and C-3 in 1, 4-naphthoquinone skeleton. Thus, the compound 2 was identified as a known compound 2-hydroxyethyl-3-methyl-1, 4-naphthoquinone (Fig. 3), which was supported by previous reports (Fukami et al. 2000; Jansen et al. 2014). And all the proton and carbon signals of compound 2 was assigned in Table S2.

The cyclo-(L-Pro-D-Pro-L-Tyr-L-Tyr) (1) was only isolated by Mitova et al. (2003) from Pseudomonas sp. associated with the sponge Ircinia muscarum. The 2-hydroxyethyl-3-methyl-1,4-naphthoquinone (2) was firstly reported in 2000 from a rare actinomycete Actinoplanes capillaceus K95-5561T (Fukami et al. 2000). Until 2014, the compound 2 was identified and reported from a novel myxobacterium for the second time (Jansen et al. 2014). To the best of our knowledge, compounds 1 and 2 have been identified in the genus Actinoalloteichus for the first time in our study. Our results suggested that these cyclotetrapeptide and naphthoquinone could be synthesized by different bacteria, and their biosynthetic gene clusters may be present in microbial genomes of different species.

Antimicrobial and cytotoxic activities

Antimicrobial activities and cytotoxicity revealed that compound 1 was inactive in these assays, which was consistent with previous report (Mitova et al. 2003). On the contrary, compound 2 exhibited significant inhibitory activities against a variety of pathogenic fungi (F. oxysporum f. sp. cucumerinum, S. turcica, and B. cinerea) and Gram-positive bacterium (Bacillus subtilis), while exhibited inactive against R. solani (Table 1). It was worth noting that compound 2 displayed better antifungal activity against B. cinerea (diameter of inhibition zone 16.83 ± 1.07 mm) when compared with reference amphotericin B (diameter of inhibition zone 10.72 ± 0.62 mm) (Table 1). Previous study also indicated compound 2 showed weak activities against Gram-positive bacteria (Bacillus subtilis DSM 10 and Staphylococcus aureus DSM 346), Gram-negative bacterium (Chromobacterium violaceum DSM 30191), yeasts (Rhodotorula glutinis DSM 10134 and Candida albicans DSM 1665), and filamentous fungi (Mucor hiemalis DSM 2656) (Jansen et al. 2014). In addition, the results of cytotoxicity revealed that compound 2 and positive control DPP exhibited a IC50 value of 10.59 ± 2.72 μM and 5.91 ± 0.12 μM against human breast cancer MDA-MB-435 cells, respectively. This suggested that compound 2 had moderate cytotoxic activity. To our knowledge, this is the first report on cytotoxic activity of 2-hydroxyethyl-3-methyl-1, 4-naphthoquinone (2).

Table 1.

Antimicrobial activity of compounds 1 and 2

Test strains Inhibitory zone (mm)
Compound 1 Compound 2 Amphotericin B Control
Fungi
 Fusarium oxysporum f. sp. cucumerinum S19 8 10.96 ± 0.74 11.86 ± 0.29 8
 Rhizoctonia solani H7 8 8 13.19 ± 0.25 8
 Botrytis cinerea H6 8 16.83 ± 1.07 10.72 ± 0.62 8
 Setosphaeria turcica H1 8 12.66 ± 1.36 16.45 ± 0.66 8
Yeast
 Candida albicans ATCC 10231 8 8 NA 8
Bacteria
 Bacillus subtilis CMCC 63501 8 21.86 ± 0.37 NA 8
 Staphylococcus aureus ATCC 29213 8 8 NA 8
 Escherichia coil ATCC 25922 8 8 NA 8
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The tested concentration was 1 mg/mL; inhibition zone values represent the means ± SD of three parallel measurements. The diameter of the filter paper was 8 mm. The 8 mm inhibition zone indicated no activity

NA not assayed

B. cinerea was one of the most common destructive airborne phytopathogens that infected over 200 plant hosts, and caused the substantial crop losses, such as strawberry (Petrasch et al. 2019), tomato (Liu et al. 2021), and chili pepper (Barra-Bucarei et al. 2020) etc. F. oxysporum f. sp. cucumerinum was a devastating vascular wilt disease in cucumber greenhouses worldwide, for which there were few effective control measures (Scarlett et al. 2015). S. turcica was an important foliar pathogen that caused leaf blight of maize and sorghum (Ma et al. 2020; Tong et al. 2017). These fungal pathogens resulted in huge economic losses of agricultural produce. In our research, compound 2 showed significant inhibitory activities against important phytopathogenic fungi, which allowed us to develop new biopesticide with agronomical potential.

Conclusion

In summary, we described a novel deep-sea-derived Actinoalloteichus cyanogriseus 12A22, which exhibited potential cytotoxic and antimicrobial activities. Two secondary metabolites, cyclo-(L-Pro-D-Pro-L-Tyr-L-Tyr) (1) and 2-hydroxyethyl-3-methyl-1, 4-naphthoquinone (2) were isolated from fermentation broth of the strain 12A22 and were structurally elucidated. Compound 2 showed significant inhibitory activities against various phytopathogenic fungi and moderate cytotoxic effect against cancer cell MDA-MB-435. For all we know, the cytotoxic activity of compound 2 was found for the first time. Furthermore, compounds 1 and 2 were discovered from a member of the genus Actinoalloteichus firstly. Our results suggested that compound 2 can be used for developing new antimicrobial and antitumor agents with diverse agronomical and pharmacological potentials.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 17465 kb) (17.1MB, docx)

Acknowledgements

This research was supported by the National Natural Science Foundation of China (31872036, 41776178, and 41576136), the LiaoNing Revitalization Talents Program (XLYC1807268), the Science and Technology Innovation Program for the Youth Talents of Shenyang (RC170266), and the Youth Innovation Promotion Association CAS (2018229).

Author contributions

HP designed and conducted the experiments, and analyzed the data. XZ and CS participated in the bioactivity assays and data analysis. XZ and HP wrote the manuscript. YB and JH revised the manuscript. All authors reviewed and approved the manuscript.

Funding

This research was supported by the National Natural Science Foundation of China (31872036, 41776178 and 41576136), the LiaoNing Revitalization Talents Program (XLYC1807268), the Science and Technology Innovation Program for the Youth Talents of Shenyang (RC170266), and the Youth Innovation Promotion Association CAS (2018229).

Availability of data and materials

All data generated or analysed during this study are included in this published article and its supplementary information.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Contributor Information

Xiaoying Zhang, Email: zhangxiaoying16@mails.ucas.ac.cn.

Chunfeng Song, Email: songchunfeng0101@163.com.

Yan Bai, Email: baiy@iae.ac.cn.

Jiangchun Hu, Email: hujc@iae.ac.cn.

Huaqi Pan, Email: panhq@iae.ac.cn.

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Supplementary Materials

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