Micromonospora zhangzhouensis sp. nov., a Novel Actinobacterium Isolated from Mangrove Soil, Exerts a Cytotoxic Activity in vitro

A new bacterial strain, designated HM134T, was isolated from a sample of soil collected from a Chinese mangrove Avicennia marina forest. Assessed by a polyphasic approach, the taxonomy of strain HM134T was found to be associated with a range of phylogenetic and chemotaxonomic properties consistent with the genus Micromonospora. Phylogenetic analysis based on the 16s rRNA gene sequence indicated that strain HM134T formed a distinct lineage with the most closely related species, including M. rifamycinica AM105T, M. wenchangensis CCTCC AA 2012002T and M. mangrovi 2803GPT1-18T. The ANI values between strain HM134T and the reference strains ranged from 82.6% to 95.2%, which was below the standard criteria for classifying strains as the same species (96.5%). Strain HM134T and related species shared in silico dDDH similarities values below the recommended 70% cut-off for the delineation of species (range from 25.7–62.6%). The DNA G+C content of strain HM134T was 73.2 mol%. Analysis of phylogenetic, genomic, phenotypic and chemotaxonomic characteristics revealed that strain HM134T is considered to represent a novel species of the genus Micromonospora, for which the name M. zhangzhouensis sp. nov. is proposed. The extract of strain HM134T was demonstrated to exhibit cytotoxic activity against the human cancer cell lines HepG2, HCT-116 and A549. Active substance presented in the fermentation broth of strain HM134T was isolated by bioassay-guided analysis and purified afterwards. A new derivative of diterpenoid was identified through electrospray ionizing mass spectrometry (MS) and nuclear magnetic resonance (NMR). The compound showed different cytotoxic activities against cancer cells, with the highest cytotoxicity against HCT-116, corresponding to IC50 value of 38.4 μg/mL.

chemotaxonomic analysis. The major respiratory quinone of strain HM134 T was identified as MK-10(H6). The absence of MK-10(H4), MK-9(H6) and MK-9(H4) could distinguish HM134 T from its close neighbor strains. The cell wall of strain HM134 T contained meso-diaminopimelic. Whole-cell hydrolysates predominantly contained xylose, arabinose and glucose. The detailed fatty acid and polar lipid profiles of strain HM134 T are shown in Supplementary Table S2 and Fig. S4, respectively. The most abundant fatty acids (>10%) detected in strain HM134 T included iso-C 16:0 (30.3%), iso-C 15:0 (14.1%) and 10-methyl C 18:0 (TBSA, 12.4%), consistent with the previous findings that iso-C 16:0 and iso-C 15:0 were the predominant cellular fatty acids of the genus Micromonospora 33 and reference strains (Supplementary Table S2). The polar lipid profile of strain HM134 T comprised diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), an unidentified phospholipid (PL1), three unidentified glycolipids (GL1, GL2 and GL3) and two unidentified lipids (L1 and L2). DPG and PE were also detected in the three reference strains (Table 1) and other Micromonospora species. The presence of GL1-3 and the absence of PI, PIM and PS could differentiate strain HM134 T from the reference strains.
Phenotypic Characterization of the HM134 Isolate. Strain HM134 T grew well on ISP 1, ISP 2, ISP 3 34 agars, tryptone soy agar and nutrient agar after 7-14 days at 28 °C, moderately on ISP 4, ISP 5 34 and Streptomyces agar, and poor on ISP 7 34 and calcium malate agar 35 . The colors and substrate mycelia were dependent on the culture medium used (Table 2). Aerial hyphae were absent and no soluble pigment was produced in any of the culture media. Morphological observation of strain HM134 T revealed that single spores were formed on the end of substrate hyphae (Supplementary Fig. S5). Growth was observed at pH 4.5-9.5 (optimum pH 5.5-8.5), with 0-7% NaCl tolerance (optimum 0-1%) and at 14-37 °C (optimum 20-28 °C). Nitrate was weakly reduced to nitrite but not to N 2 . Cells were found to be positive for catalase but negative for melanoid pigment production. Liquefaction of gelatin, milk coagulation, hydrolysis of esculin and soluble starch were found to be positive, but negative for hydrolysis of cellulose, H 2 S production, Voges-Proskauer test and methyl red test. According to Table 1, strain HM134 T and all the reference strains could hydrolyze esclin and gelatin, utilize N-acetyl-glucosamine and potassium gluconate, meanwhile esterase(C4) in strain HM134 T is weakly positive, which could be distinguished from other reference strains. Also the ability to hydrolyze starch revealed discrepancy between strain HM134 T and the most close strain M. rifamycinica AM105 T . In addition, thirteen strains were chosen as representatives of adjacent clusters, the phenotypic characteristics were collected from literatures and compared with strain HM134 T (See Supplementary Table S3). The detailed physiological and biochemical properties are presented in the species description.
Cytotoxic activity of strain HM134 t extract. The cytotoxic potential of HM134 T extract was tested against several human-derived cancer cell lines (HCT-116, HepG2 and A549) and the results are summarized in Fig. 3. All cancer cell lines showed susceptibility to the extract of stain HM134 T with inhibition ratios range from 88.84-98.5% (100 μg/mL extract was tested). The extract exhibited the highest toxicity against HCT-116 cells with the inhibition ratios of 98.50 ± 4.8% and 48.73 ± 2.5% when tested at the concentration of 100 μg/mL and 20 μg/mL, respectively. As indicated, the inhibition ratio of A549 was significantly reduced at lower extract concentration (decreased to 14.94 ± 2.3%). The correspongding inhibition ratios on HepG2 cells were 95.34 ± 5.7% and 26.84 ± 4.2%, respectively. Furthermore, we observed a dose-dependent effect when the extract was tested against human-derived cancer cell lines. Overall, the results suggested that the HM134 T extract has a higher cytotoxic effect against the HCT-116 cell lines than the HepG2 and A549 cell lines. Based on the results of the cytotoxicity, we further characterized the bioactive metabolites of strain HM134 T against HCT-116, HepG2 and A549 cells. Identification of active compounds. Bioassay-guided isolation of the active components of HM134 T was carried out as described in the Materials and Methods. The active metabolite was characterized by spectroscopic analyses and by comparison with the data available from literature. This compound was obtained as a yellow

Enzyme activity
Alkaline phosphatase

Hydrolysis of:
Starch       H-13 C long-range correlations from H-7 to C-9, C-21 and from H-9 to C-7 and C-21 showed that C-7 and C-9 was connected through C-8 and a carboxyl group was situated at C-8. Similarly, HMBC correlations from H-3 to C-5 and C-18, from H 2 -5 to C-3 and C-18, from H 3 -19 to C-18 indicated that C-3 and C-5 was connected via C-4 and one acetate was attached to C-4. As a result, the structure of the compound was shown in Fig. 4. According to the NOESY data (See Supplementary Fig. S14), the correlation of H-2/H-5 and H-7/H-10 revealed that both the double bonds could be assigned to be of E-configuration. Compound 1 was named as (7E,11E)-6-hydroxy-1-isopropyl-11-(methoxycarbonyl)-4-methylene-1,2,3,4,4a,5,6,9,10,12a-decahydrobenzo [10]annulene-7-carboxylic acid. Genotypic characterization of the HM134 t isolate and screening for antibiotic biosynthetic gene clusters. The genome of strain HM134 T consists of one circular chromosome (7,565,212 bp, 73.2% G+C), with the absence of plasmid. A total of 6853 protein-coding sequences (CDS) and 109 RNA genes were predicted. The genomic features of strain HM134 T were summarized in Table 3.

Cytotoxicity of bioactive metabolite from
Among the 6853 CDSs, only 4688 CDSs were classified into COG categories (See Supplementary Table S5). The major categories included transcription (9.8%), carbonhydrate and amino acid transport and metabolism (8.2% and 7.7%, respectively), signal transduction mechanisms (5.9%), coenzyme, lipid and inorganic ion transport and metabolism (5.7%, 5.6% and 5.4% respectively). Noteworthy, the poorly characterized category General function prediction only (11.85%) included many secondary metabolites synthesis clusters, indicating that there were lots of functional genes were unclear in strain HM134 T . A total of 32 secondary metabolite gene clusters were detected using antiSMASH. There were 4 terpene clusters, 2 type I PKS clusters, 1 type II PKS cluster, 1 type III PKS cluster, 5 NRPS clusters, 1 siderophore, 3 bacteriocins and 11 heterozygous PKS-NRPS clusters found in strain HM134 T . These results highlighted the genomic potential of the inspected isolates for natural products discovery. Furthermore, the presence of terpene clusters is primarily responsible for the synthesis of the newly terpene derivate.

Discussion
Based on the polyphasic approach analysis, strain HM134 T was markedly different from the most closely related type strains of the genus Micromonospora. Therefore, strain HM134 T merits assignment to a novel species in the genus Micromonospora, for which the name M. zhangzhouensis sp. nov. is proposed. The type strain is HM134 T .
The extract from strain HM134 T demonstrated 88.8-98.5% inhibition ratio against human cancer cell lines (HepG2, HCT-116 and A549) when tested at a concentration at 100 μg/mL. Since the strain HM134 T is a novel Micromonospora species, it would be a potential reservoir of natural products with cytotoxic activity. Bioassay-guided separation and purification by multiple methods were successfully applied to identify the active fractions. The active compound 1 identified as a novel diterpenoid derivative that exhibited cytotoxic activity against cancer cells.
Terpenes are one of the major secondary metabolites with different compound types, including monoterpenenes, diterpenes, sesquiterpenes, triterpenes, sesterterpenes and norterpenes 36,37 . Although actinomycetes harbor the genetic potential to produce terpenes, terpenoid natural products are rarely observed when cultured in fermentation broths. The carbon skeleton of compound 1 is similar to cembrane-type diterpenoids, which form a large and structurally different group of natural products that can be isolated from both terrestrial and marine organisms. Cyclisation of a geranylgeraniol derived precursor between carbon 1 and 14 generates a 14-membered diterpenoid, named cembrane or thumbergane 38 . As previously reported, coelenterates are recognized as the  www.nature.com/scientificreports www.nature.com/scientificreports/ most prominent source of cembranoids 39,40 . From a biomedical perspective, cytotoxicity is the most remarkable characteristic of cembranoids. In addition, cembranoids possess multiple biological activities such as neuroprotective, anti-inflammatory, antimicrobial, antiarthritic effects. In previous study, Luo, et al. 41 reported that eight cembrane-type diterpenoids were isolated from Macaranga pustulat, a including three new compounds that exert cytotoxicity (IC 50 > 20 μM) towards human cancer cell lines (CNE1, CNE2 and HCT116). In another study, researchers isolated two new cembrane diterpenes from the flowers of Nicotiana tabacum L. with anti-tumor activities against human tumor cell lines (HepG2, A549 and HCT-116) 42 . In addition, four unknown cembrane-type diterpenoids exhibiting hepatoprotective activity at 10 μM against paracetamol-induced HepG2 cell damage were isolated from the gum resin of Boswellia sacra Flueck 43 . Another similar skeleton structure of compound 1 is cladiellane-type diterpenoid, one of the class of metabolites from gorgonians with the skeletons containing an ether bridge across C-2 and C-9 44 . These metabolites also displayed a wide range of bioactivities. For example, Ru, et al. 45 reported three cladiellane-type diterpenoids exhibiting moderate anti-inflammatory activity with the IC 50 values range from 15.8 to 43.7 μM. Previous studies demonstrated that hydroxylated derivatives showed improved anti-cancer activity 46 , suggesting that the hydroxyl group of compound 1 may contribute to the suppressive effect on human cancer cells.
In summary, the strain HM134 T , a novel species of the genus Micromonospora was successfully isolated from the mangrove soil of Zhangzhou, China. The findings of this study demonstrated that the strain HM134 T exhibited significant cytotoxic activity against human cancer cell lines (HepG2, HCT-116 and A549) and a new diterpenoid derivative was found. This study provides a comprehensive description of the novel strain Micromonospora zhangzhouensis HM134 T and elucidated the potential of the strain as a resource for anticancer or drug discovery. Hence, further studies to provide in-depth research on the cytotoxic property of this strain are highly valuable.

Materials and Methods
Sample collection and isolation of actinomycetes. Strain HM134 T was isolated from a soil sample DNA extraction and purification. Genomic DNA was extracted using a Quick Bacteria Genomic DNA Extraction kit (DongSheng Biotech). The 16S rRNA gene of strain HM134 T was amplified using the universal primers 27 F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492 R (5′-TACGGYTACCTTGTTACGACTT -3′) 48 . The amplification products were cloned into the pMD19-T vector (TaKaRa) and then sequenced. The obtained 16S rRNA gene sequence (1480 nt) was analyzed by performing pairwise sequence alignments using the NCBI nr database (http://www.ncbi.nlm.nih.gov) and the EzTaxon-e server (https://www.ezbiocloud.net) 49 . Multiple sequence alignments based on the 16S rRNA gene sequences of strain HM134 T and related taxa were performed using the CLUSTAL X program of the MEGA 5 software package 50 . Phylogenetic trees were reconstructed from 1000 replicates using the neighbor-joining 51 , maximum-likelihood 52 and maximum-parsimony 53 Scientific RepoRtS | (2020) 10:3889 | https://doi.org/10.1038/s41598-020-60677-0 www.nature.com/scientificreports www.nature.com/scientificreports/ methods based on 1000 replications and bootstrap analysis. To testify the phylogenetic tree reconstructed by MEGA 5, All-Species Living Tree LTPs123 and database arb-6.0.6 were used as the reference, SINA webserver 54 and ARB software 55 were used for alignment of 16S rRNA gene sequence into LTPs123 and generation of a new maximum-likehood phylogenetic tree, respectively.
The complete genome was sequenced at the Beijing Genome Institute (BGI, Shenzhen, China) using a PacBio RS II platform and Illumina HiSeq. 4000 platformand. The genome was assembled as described previously 56 . The DNA G+C content was determined by Rapid Annotation System Technology (RAST) 57 . The genome sequences of ten reference strains were retrieved from the GenBank database (Project accession numbers were listed in Supplementary Table S1). The average nucleotide identity (ANI) was calculated using the OrthoANI algorithm of the Chun lab's online Average Nucleotide Identity calculator 58 . The in silico dDDH value was calculated using the GGDC web server 59 available at https://ggdc.dsmz.de/ggdc.php#. Glimmer version 3.02 (Delcher et al., 2007) was used to predict open reading frames (ORFs) according to the manufacturers' instructions. Following this, ORFs were annotated using the NCBI NR, SwissProt 60 , KEGG 61 , GO 62 and COG 63 databases. The tRNA and rRNA genes were predicted using tRNAscan-SE 64 and RNAmmer 65 , respectively. CRISPR repeats were predicted using CRISPR finder 66 . Biosynthetic gene clusters of secondary metabolites were predicted using the antiSMASH 3.0 web server 67 . The gene sequence of gyrB was identified by RAST of the genome sequence of strain HM134 T . The phylogenetic tree based on the housekeep gene (gyrB) of strain HM134 T and other strains was constructed using neighbor-joining method, with the Tamura Phenotypic characterization of the HM134 t isolate. The temperature range for growth was determined in ISP2 (pH 7.0) at 4-45 °C (4, 10, 15, 20, 28, 30, 35, 37 and 45 °C). The tolerance to NaCl concentrations was tested in ISP2 (pH 7.0) with the concentrations of NaCl at 0-10.0% (w/v), with an increment of 0.5%. The pH range for growth was tested with an interval of 0.5 pH unit, by supplementation of ISP 2 medium with 30 mM buffering agents at 28 °C: 2-(N-morpholino) ethanesulfonic acid for pH 5.5-6.5, 3-(N-morpholino) propanesulfonic acid for pH 6.5-8.0, tricine for pH 8.0-9.0, and bis-Tris propane for pH 9.0-9.5. The optimal growth was determined after 7 days of incubation, and the growth limits were determined after 14 days of incubation. HM134 T was incubated at 28 °C for 21 days on ISP 2 medium, and cell morphology was examined and observed using an optical microscopy (BX40; Olympus) after Gram staining, a transmission electron microscopy (80 kV, JEM-1230; Jeol) after uranyl acetate (0.5%, w/v) staining and a scan electron microscope (3.0 kV, SU8010, Hitachi) after fixation by osmium tetroxide vapor (4%, w/v). The culture characteristics were determined following growth on tryptone-yeast extract agar (ISP 1), yeast extract-malt extract agar (ISP 2), oatmeal agar (ISP 3), inorganic salts-starch agar (ISP 4), glycerol-asparagine agar (ISP 5), and tyrosine agar (ISP 7) agars 34 ; Gauze's No.1 agar, nutrient agar, tryptone soya agar 68 and calcium malate agar 35 for 14 days at 28 °C. The colors of substrate and aerial mycelia were determined based on comparison with the ISCC-NBS color system 69 . Catalase and oxidase activity were tested following the method described by Sun, et al. 70 . Hydrolysis tests were performed with different substrates supplemented with gelatin, skimmed milk, starch (5 g/L); CM-cellulose (2 g/L); Tweens 20, 40, 60 and 80 (1%, v/v) and adenine, guanine, xanthine and hypoxanthine (0.5%, v/v). Anaerobic growth was determined in an anaerobic system (Anaero Pack-Micro Aero, 2.5-L, MGC, Japan) on ISP 2 supplemented with various electron acceptors as described by Chen, et al. 71 . Nitrate reduction was tested according to the protocol of Dong and Cai 72 . The methyl red and Voges-Proskauer tests were examined as described by Lányi 73 . Other biochemical properties and enzyme activities were tested using API ZYM and API 20NE kits (bioMérieux) according to the manufacturer's instructions.
Chemotaxonomic characterization. Cells used for the analysis of fatty acids were harvested from the third quadrants of ISP 2 agar plates. Fatty acid methyl esters (FAMEs) were extracted as described by Kuykendall, et al. 74 and analyzed according to the instructions of the Microbial Identification System (MIDI; Microbial ID). Isoprenoid quinones were extracted using a CHCl 3 /MeOH mixture (2:1, v/v) from freeze-dried cells (500 mg) and analyzed using an HPLC-MS system (Agilent) 75 . Polar lipids were extracted and separated by two-dimensional thin-layer chromatography on silica gel 60 F 254 plates (Merck). Molybdophosphoric acid, ninhydrin reagent, molybdenum blue, and α-naphthol/H 2 SO 4 reagents were used for the detection of total lipids, lipids containing free aminolipids, phosphorus-containing lipids and glycolipids, respectively 76 . The analyses of sugars and amino acids in whole cell hydrolysates were performed following previous methods 77 .
Preparation of the HM134 t fermented broth and extract. Strain HM134 T was inoculated into a 500-mL Erlenmeyer flask containing 200 mL of GYM medium (containing malt extract 10.0 g, yeast extract 4.0 g, glucose 4.0 g, CoCl 2 ·6H 2 O 0.005 g in 1.0 L tap water at pH 7.0-7.2) as seed medium prior to fermentation process. Afterwards, 1% (v/v) of the starting stock culture was transferred to a 1 L Erlenmeyer flask containing 25% volume of the fermentation medium and incubated at 28 °C for 7 days on a rotary shaker at 250 rpm. The fermentation medium, H9A, consisted of soluble starch 20.0 g, glucose 20.0 g, soybean powder 10.0 g, yeast extract 5.0 g, malt extract 4.0 g, CaCO 3 2.0 g, MgSO 4 ·7H 2 O 2.0 g, NaCl 3.0 g, in 1.0 l tap water at pH 7.0-7.2. All culture media were sterilized at 121 °C for 30 min. The cell-free supernatant was collected by centrifugation at 4,000 × rpm for 10 min then subjected to freeze drying process. The freeze-dried sample was repeatedly extracted with methanol and the final extract concentrated using a rotary evaporator at 40 °C. The final concentrate was suspended in dimethyl sulfoxide prior to bioactivity assays. (2020) 10:3889 | https://doi.org/10.1038/s41598-020-60677-0 www.nature.com/scientificreports www.nature.com/scientificreports/ In vitro anti-tumor cytotoxicity. HCT-116 (humancolorectal carcinoma), A549 (human lung carcinoma) and HepG2 (human hepatocellular carcinoma) cell lines were obtained from the Department of New Drug Screening, Zhejiang Hisun Pharmaceutical Co., Ltd.(Taizhou, China). Cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (w/v) fetal bovine serum in a humidified incubator (5% CO 2 in air at 37 °C). The antitumor activities of different concentrations of HM134 T extracts (20 and 100 μg/mL) were evaluated by the CCK-8 colorimetric method. The cell lines were cultured in DMEM containing 10% calf serum at 37 °C for 4 h in a 5% CO 2 incubator. The adherent cells in the logarithmic growth phase were digested and seeded on a 96-well culture plate at a density of 1 × 10 4 cells per/well. Test samples and controls were added to the medium and incubated for 48 h. Then, cell counting kit-8 (CCK-8, Dojindo) was added to the medium and incubated for 3 h. Cell viability was determined by measuring the absorbance at 450 nm using a SpectraMax M5 microplate reader (Molecular Devices Inc., Sunnyvale, CA, USA) 78 . The inhibitory rate of cell proliferation was expressed as IC 50 values. Doxorubicin was used as a positive control while cells containing 0.5% DMSO were tested as negative control.
Isolation and characterization of bioactive metabolites. The cultivation procedure described in section Preparation of the HM134 T Fermented Broth and Extract was repeated and the filtrate (30 L) of the culture broth was collected. The filtrate was separated and purified in an HP-20 macroporous resin (Mitsubishi, Japan) column, and then eluted with absolute ethyl alcohol. After concentrating to dryness using a rotary evaporator at 40 °C, the residue (30.0 g) was resolved by chromatography on a silica gel column eluted with n-heptane/ethyl acetate mixtures that were run with a growing polarity (100:0 to 30:70, v/v) to obtain six fractions (Fr1-6). Results of bioactivity assays (in vitro antitumor toxicity) indicated that the Fr1 (n-heptane/ethyl acetate, 95:5) fractions was cytotoxic in vitro. The active fraction was repeatedly purified, and separated on Sephadex LH-20 gel column (GE Healthcare, Glies, UK). Semi-preparative HPLC (Shimadzu LC-8A, Shimadzu-C18, 5 μm, 250×20 mm Shimadzu, Kyoto, Japan) were performed to obtain compounds 1 (8.4 mg).
Structural identification of the bioactive metabolite was conducted by spectroscopic analysis. 1 H nuclear magnetic resonance (NMR) and 13 C NMR spectra were acquired using a Bruker DRX-400 spectrometer (400 MHz for 1 H and 100 MHz for 13 C) (Bruker, Rheinstetten, Germany). Chemical shifts were reported in ppm. (δ). Residual CHCl 3 (δ H 7.26 ppm; δ C 77.0) was used as an internal standard, with coupling constants (J) expressed in Hz. 1 H and 13 C NMR assignments were supported by the results of the 1 H-1H COSY, HMQC, and HMBC experiments. The electrospray ionization MS data were recorded using a Time-of-Flight Mass Spectrometer X500R Q-TOF (Sciex, USA).