**3. Discussion**

Actinomycetia derived from mangroves is a promising source for exploring novel bioactive natural products. A multitude of bioactive compounds, including the promising compounds salinosporamide A, xiamycins, and indolocarbazoles, have been isolated from mangrove actinomycetia [13,14,62,63]. Salinosporamide A, a potent 20S proteasome inhibitor, is the first mangrove-derived compound that entered phase I clinical trials for the treatment of multiple myeloma only three years after its discovery [13,64,65]. Xiamycin exhibits selective anti-HIV activity and also represents one of the first examples of indolosesquiterpenes isolated from prokaryotes [66]. The Leizhou Peninsula, located in the southernmost end of mainland China, has 9284.3 ha of mangrove distributed in over 100 sites along the coastlines, comprising approximately 79% of the total mangroves area in Guangdong province and 33% in China [67,68]. However, the mangrove from the Leizhou Peninsula is underexplored, and only a few reports on the diversity and antibacterial activity of its inhabiting actinomycetia have been published to date. Our group started exploring the diversity and bioactivity of the mangrove plant endophytic actinomycetia in this region in 2015. In total, 159 strains in 19 genera affiliated with 12 families were isolated, and 64 out of 88 tested strains exhibited activity against at least one of the tested pathogens [18]. In order to maximize the harvest of actinomycetial strains in this study, 13 soil samples from different mangrove sites were collected, and 12 isolation media were applied, leading to the isolation of 521 strains in 40 genera from 23 families. Thus, this ecosystem is proven to provide a highly productive and rich diversity of actinomycetia. The genera *Micromonospora* and *Streptomyces* were still numerically dominant, which is consistent with previous studies [63]. Interestingly, the addition of kelp or coconut juice into the medium (M11 and M12) increased the diversity of actinomycetia compared with the blank control (M1 and M4), which might be caused by trace substances from kelp or coconut juice assisting some strains growth since both seaweed and coconut trees grow in or along the sea and live in similar environments as mangroves. Lastly, 179 strains affiliated

to 40 different genera with a unique colony morphology were selected to evaluate their antibacterial activities.

Bioactivity screening is, in general, the initial step to find novel antibiotics. To comprehensively analyze the antibacterial spectra of the extracts, 12 strains of drug-sensitive and drug-resistant pathogens were used as indicators in this study. In order to find the strains producing more potent antibacterial metabolites, the test volume of extracts used in the assay was reduced to 50% of the normally applied volume in the same concentration. In this assay, we found that *Streptomyces* was still the main genus producing bioactive secondary metabolites, accounting for 61.7% of all active strains. Because the ISP2 medium supports the growth of most actinomycetia and also is known to promote secondary metabolite production [24,69,70], it was the only medium used for fermentation. ISP2 medium components are also reported to reduce noise level and interference with secondary metabolites detection in the UPLC–HRMS analysis [24]. In addition, application of only one medium facilitates comparability between the different strains in metabolomics analysis [71].

Dereplication has become a key issue for the discovery of new antibiotics. An effective approach was required to maximize the detection of chemical diversity and minimize the redundancy of the samples after the bioactivity and phylogeny screening [72,73]. UPLC-HRMS/MS-based metabolomics could maximize the detection of chemical diversity among extracts in a high throughput manner [74]. UPLC-HRMS/MS gives rapid separation from the complex strain extracts and increases confidence in identifying metabolites based on mass accuracy and isotope fit. Metabolomic methods are combined with chemoinformatics approaches, such as PCA, OPLS-DA, etc. PCA, an unsupervised method without grouping, was applied to overview the differences among numerous samples, identifying strains with distinct metabolites, while excluding strains with common chemical profiles [75]. Hou Yangpeng et al. applied LC/MS-PCA to discover novel natural products from marinederived *Streptomyces* spp.; the result was that 37% of all isolates produced a number of unique and putative new natural products, indicating that this approach could greatly improve the discovery rate from *Streptomyces* spp. [21]. However, the application of PCA was limited to the number of samples, usually between 20 and 50 strains, practically [21,24]. Therefore, 23 strains with strong anti-MRSA activity were selected for PCA analysis. The result showed that six strains with unique chemical profiles could be candidates for prioritization. After dereplicating, two *Streptomyces* strains (M22, H37) with putative novel compounds were prioritized.

OPLS-DA, a supervised method, was an effective statistic model for comparing two different sample groups [20]. A total of 8 out of the above 23 strains showing bioactivity against drug-sensitive *A. baumannii* were prioritized using the OPLS-DA model to find the bioactive compounds produced in the strains. Finally, *Streptomyces* sp. H37 with putative new compounds responsible for inhibitory activity against *A. baumannii* were prioritized. The OPLS-DA analysis effectively assists the bioactivity screening to find potential new antibiotics with grea<sup>t</sup> bioactivity and avoid targeting inactive metabolites in the follow-up chemical isolation. Molecular networking was a new dereplication strategy to rapidly overview the chemical family in the extracts, identify known compounds, and find the analogs with novelty. In this study, it can act as a complement for the PCA and OPLS-DA analysis to claim the relationship of the significant metabolites in the outlier strains, find the potential new congeners, and predict the structure using their MS/MS similarity. As shown in Figure 10, the predicted structure of compound **16** was consistent with the structure that was identified by spectroscopic analysis, including HRESIMS, 1D, and 2D NMR.

NPAtlas and StreptomeDB are recommended as the databases used for the dereplication of metabolites produced in microbial extracts, especially in actinomycetial extracts. These databases are manually curated by the Linington group out of Simon Fraser and Stefan Günther group from Freiburg, respectively. Unlike Antibase, MarinLit, and Dictionary of Natural Products (DNP), NPAtlas and StreptomeDB are open access, updated, and ready to be downloaded. To date, NPAtlas v19\_12 contains over 25,000 microbial-produced natural products [33], and StreptomeDB v3.0 includes 6524 compounds produced by *Strep-*

*tomyces* [34]. As shown in Tables 2 and 3, the dereplication of metabolites from different databases produced different results sometimes. If two professional microbial databases are simultaneously applied to the dereplication process, it can increase accuracy and effectiveness in dereplication and avoid omissions caused by using one database. Hence, more than one database should be used, and loose parameters, such as precursor and fragments tolerances in 10 ppm, should be set in the search method. The limitation of dereplication for secondary metabolites is usually caused by the difficulty of obtaining an authentic standard for every "hit" from the database. Additional data, such as UV and MS/MS data, can be helpful to ensure that the identified hits in the mass ion peaks are correct.

The trioxacarcin family is a family of complex aromatic polyketides, which is produced by *Streptomyces* strains. In 1981, the trioxacarcins (trioxacarcins A–C) were first isolated from *Streptomyces bottropensis* DO-45 [76]. Subsequently, they were reisolated from a marine *Streptomyces* sp. B8652 with additional four new analogs, trioxacarcin D–F and gutingimycin, in 2004 [52,60]. The structure of the trioxacarcins was characterized as a rigid, highly oxygenated polycyclic skeleton with a fused spiro epoxide function, and one or more unusual glycosidic residues, named 'trioxacarcinoses' [77]. Trioxacarcin-type compounds display extraordinary antiproliferative effects, such as anticancer, antibacterial, and anti-malaria activities [60,76,78], which have attracted attention for chemical synthesis [61,77,79,80], mode of action, and biosynthesis studies [81–85]. It was reported that the notable biological activity of trioxacarcin A is due to its tight interaction with DNA [86,87]. In our study, several putative novel trioxacarcin-type compounds were identified by the metabolomicsbased dereplication approach, such as compound **12** (7.16\_1028.3600*m/z*), compound **14** (7.47\_1028.3592*m/z*), compound **16** (6.69\_1030.3751*m/z*), compound **17** (7.94\_1013.3486*m/z*), and so on. However, some of them were unstable in scale-up fermentation. The contents of compounds **14** and **17** were decreased, making them hard to be isolated and accumulated for structural identification. Meanwhile, a low-yield compound **20** (7.86\_896.3265n) was increased in the scale-up fermentation. A similar phenomenon was also reported by other researchers in metabolomics analysis of bacterial strains [88]. Finally, two new trioxacarcins, gutingimycin B (**16**) and trioxacarcin G (**20**), along with gutingimycin (**12**, 7.16\_1028.3600*m/z*) were isolated from the scale-up fermentation broth of *Streptomyces* sp. M22. To the best of our knowledge, it is the second report of finding trioxacarcins with keto-reduced trioxacarcinose B moiety, except for trioxacarcin C. The new trioxacarcin-type members, compound **16** and compound **20**, together with known compound **12**, were evaluated for cytotoxicity against the H460 lung cancer cell line in this study, but no prominent cytotoxic activity against this cell line was observed in these compounds (IC50 > 1000 nM). The antibacterial activity of the new analogs and their cytotoxicity against other cell lines will be tested in the future. The successful isolation of novel trioxacarcin-type compounds from *Streptomyces* sp. M22 has demonstrated that our integrative strategies are effective, efficient, and suitable for seeking new antibiotics from those ecosystems inhabiting a large amount of actinomycetial strains.

#### **4. Materials and Methods**

## *4.1. Samples Collection*

Soil samples were collected in August 2019 at different mangrove sites in Leizhou Peninsular, Guangdong province, China. The locations where samples were collected and their information are shown in Figure 13 and Table S6. All samples were collected from depth of 5–10 cm with a sterile spatula, then packed in sterile bags, and brought to the laboratory at the earliest time. Each sample was air-dried in a laminar flow hood before grinding with a mortar and pestle.

**Figure 13.** Locations of the sampling sites (red star) in Leizhou Peninsula, Guangdong, China.

#### *4.2. Isolation of Actinomycetial Strains*

A total of 12 cultural media were used to isolate actinomycetial strains. All media were supplemented with nalidixic acid (25 mg/L), cycloheximide (50 mg/L), and potassium dichromate (50 mg/L) to inhibit the growth of Gram-negative bacteria and fungi. The recipes for 12 media are shown in Table S1. It should be mentioned that M11 is a modified version of M1 with the addition of 15 mL kelp solution instead of 15 mL distilled water in the recipe. The kelp solution was prepared as follows: 200 g fresh kelp was cut into small pieces, added into 200 mL distilled water, and boiled for 30 min. After cooling down, the kelp soup was filtered by absorbent cotton to obtain the kelp solution. Meanwhile, M12 is a modified version of M4 with the addition of 10 mL natural fresh coconut juice instead of 10 mL distilled water in the recipe.

Strains were isolated by using the dilution plating technique. A total of 5 g of each soil sample was diluted with 45 mL of sterile 0.1% Na4P2O7 solution, then mixed, homogenized, and shaken for 1 h at 180 rpm to release actinomycetia cells attached to the soil. Subsequently, the pretreated samples were prepared for ten-fold serial dilutions up to 10−4. A total of 0.2 mL of diluted sample (10−2, <sup>10</sup>−3, and <sup>10</sup>−4) from each soil sample was spread onto isolation agar plates, and plates were incubated for 7–14 days at 28 ◦C. Actinomycetia-like colonies depending on their morphological characters, pigment diffusion, and coloration of their mycelia were picked and streaked several times on ISP2 agar plates until pure actinomycetial colonies were isolated. The pure strains were maintained on ISP2 agar slants at 4 ◦C and preserved in 20% glycerol (*v*/*v*) at −80 ◦C.
