*2.1. Alkaloids*

The nitrogenous alkaloids represented the most abundant class of compounds that were produced by the co-cultures of marine microorganisms with diverse skeletons and biological activities [15,16]. Eighty alkaloidal metabolites were isolated and identified from different microbial environments (Figure 2B), and the co-cultures of marine fungi–bacteria represented 51% of the total isolates (Figure 3).

**Figure 3.** Alkaloids isolated from the co-cultures of marine fungi–fungi, fungi–bacteria and bacteria–bacteria.

#### 2.1.1. Alkaloids Derived from the Co-Cultures of Different Marine Fungi

Several studies of co-cultures of fungal–fungal interactions from different marine sources were summarized as follows; 26 alkaloids were isolated and identified (Figures 2C and 3). The mixed fermentation of marine-derived fungi *Aspergillus sulphureus* KMM 4640 from muddy sand of the eastern Sakhalin shelf (Sea of Okhotsk, 26 m depth) and *Isaria felina* KMM 4639 from sediments (South China Sea, Vietnam shores, 10 m depth), led to the production of five novel prenylated indole alkaloids, 17-hydroxynotoamide D (**1**), 17-O-ethylnotoamide M (**2**), 10-O-acetylsclerotiamide (**3**), 10-O-ethylsclerotiamide (**4**) and 10-O-ethylnotoamide R (**5**) together with known compounds (-)-notoamide B (**6**), notoamide C (**7**), dehydronotoamide C (**8**), notoamide D (**9**), notoamide F (**10**), notoamide Q (**11**), 17-epi-notoamide Q (**12**), notoamide M (**13**) and sclerotiamide (**14**) (Figure 4) [17]. Among them, compounds **1**–**5** were only produced in the co-culturingprocess.

Compounds **2**, **6**, **8**, **13** and **14** inhibited the proliferation of the human prostate cancer cells 22Rv1 at 100 μM. Notably, **2** and **13** drastically reduced the viability of 22Rv1 prostate cancer cells at 10 μM by 25% and 55%, respectively. 22Rv1 cancer cell lines were resistant to hormone therapy at conventional chemotherapy including two new 2nd generation drugs enzalutamide and abiraterone owing to the presence of the androgen receptor splice variant-7 (AR-V7). Therefore, the active NPs drugs in these cells might be further investigated in the treatment of different human drug-resistant prostate cancer. **6** and **7** displayed weak cytotoxicity against HeLa and L1210 cell lines with half maximal inhibitory concentration (IC50) in the range of 22–52 μg/mL [18]. Although **6** and **7** had the similar structure with **9**, compound **9** did not display the similar cytotoxic activity against HeLa and L1210 cell lines. The significant difference in cytotoxicity might be attributed to the possible existence of pyrroloindole system in **9** rather than the dihydroxypyrano-2-oxindole ring system of **6** and **7 [18,19]**. In addition, compounds **1**, **2**, **5**, **9**, **13** and **14** did not exhibit any cytotoxicity against human non-malignant (HEK 293 T and MRC-9) or malignant (PC-3, LNCaP, and 22Rv1) cell lines at concentrations up to 100 μM for 48 h [17].

The co-fermentation of marine mangrove epiphytic fungi *Aspergillus* sp. FSY-01 and FSW-02 collected from a rotten fruit of mangrove *Avicennia marina* in Zhanjiang, Guangdong Province, China, yielded a new alkaloid, aspergicin (**15**), together with two known secondary metabolites, neoaspergillic acid (**16**) and aspergicine (**17**) (Figure 5) [20,21]. Notably, compounds **17** and **15** are chemically isomers, and consequently aspergicine (**17**) may be the precursor of aspergicin (**15**) through a proton 1, 2-shift [22].

**Figure 4.** Chemical structures of **1**–**14**.

**Figure 5.** Chemical structures of **15**–**17**.

Compounds **15** and **16** showed potent inhibitory activities against three Gram-positive bacteria, *Bacillus subtilis* (MIC, minimum inhibitory concentration that inhibits the growth of microbes by 80%, 15.62 and 1.95 μg/mL), *Staphylococcus epidermidis* (MIC 31.25 and 0.49 μg/mL) and *Staphylococcus aureus* (MIC 62.50 and 0.98 μg/mL), and three Gram-negative bacteria, *Escherichia coli* (MIC 31.25 and 15.62 μg/mL), *Bacillus proteus* (MIC 62.50 and 7.80 μg/mL) and *Bacillus dysenteriae* (MIC 15.62 and 7.80 μg/mL), respectively [22].

Marine fungi *Aspergillus sclerotiorum* SCSGAF 0053 and *Penicillium citrinum* SCSGAF 0052 were isolated from the gorgonian corals *Muricella flexuosa* collected from South China Sea, Sanya (18◦11 N, 109◦25 E), Hainan Province, China [23]. Due to the mixed fermentation of marine fungi, a red pigment appeared in the mixed fermentation broth could not be observed in any strain cultured separately. This special phenomenon suggested that a novel biosynthesis route was activated. Four novel alkaloids were obtained, including one oxadiazin derivative sclerotiorumin C (**18**), a pyrrole derivative 1-(4-benzyl-1*<sup>H</sup>*-pyrrol-3-yl) ethanone (**19**), aluminumneohydroxyaspergillin (**20**) and ferrineohydroxyaspergillin (**21**), together with one known compound ferrineoaspergillin (**22**) (Figure 6) [23]. Compounds **18**–**21** were only produced in the co-culture process.

**Figure 6.** Chemical structures of **18**–**22**.

Compound **20** exhibited potent toxicity towards brine shrimp with medium lethal concentration (LC50) value of 6.1 μM and high selective cytotoxicity towards histiocytic lymphoma U937 cell line with an IC50 value of 4.2 μM. **19**, **21**, and **22** showed moderate toxicity against brine shrimp with LC50 values of 46.2, 11.5 and 27.8 μM, respectively. **21** and **22** possessed mild cytotoxicity against U937 with IC50 values of 42.0 and 48.0 μM, respectively. These results suggested that the aluminum complex skeletons of compounds showed more potent toxicity and cytotoxicity than ferricomplex structures of compounds [23–26]. Moreover, aspergillic acid and **16** also showed more potent inhibitory activities than neohydroxyaspergillic acid and hydroxyaspergillic acid against *B. subtilis*, *E. coli*, *S. aureus* and *Candida albicans* [27].

The co-culture of mangrove fungi *Phomopsis* sp. K38 and *Alternaria* sp. E33 led to the identification of one new diimide derivative, (-)-byssochlamic acid bisdiimide (**23**) and a novel nonadride derivative, (-)-byssochlamic acid imide (**24**) (Figure 7) [28,29]. Ebada et al. (2014) investigated the mycelial extract of a co-cultivation of marine fungal strains *Aspergillus.* BM-05 and BM-05ML, and identified two alkaloids, protuboxepin A (**25**) and oxepinamide E (**26**) (Figure 7) [30]. **23**–**24** were only found in the co-culture process.

**Figure 7.** Chemical structures of **23**–**26**.

Compound **23** exhibited moderate inhibitory activity against HepG2 and Hep-2 with IC50 values of 51 μg/mL and 45 μg/mL, respectively. **24** had moderate antifungal activities against *Fusarium oxysporum* and *Fusarium graminearum* with MIC values of 60 μg/mL and 50 μg/mL, respectively [28,29,31]. **25** possessed anti-proliferative activity against human breast cancer adenocarcinoma MDA-MB-231, human acute promyelocytic leukemia HL-60, hepatocellular carcinoma Hep3B, chronic myelogenous leukemia K562 and rat fibroblast 3Y1 cell lines with IC50 values of 130, 75, 150, 250 and 180 μM, respectively [32,33]. **26** showed transcriptional activation on liver X receptor α (LXRα) with a half maximal effective concentration (EC50) value of 12.8 μM. It was known that LXR was an important target in drug discovery; LXR agonists had been proven to exhibit remarkable therapeutic effects on diabetes, atherosclerosis, Alzheimer's disease and anti-inflammation. Therefore, **26** was worthy of consideration as a potential lead compound for drug discovery [34].

#### 2.1.2. Alkaloids Derived from the Co-Cultures of Marine Fungi and Bacteria

The alkaloids derived from the co-culture of marine fungi and bacteria were tallied to be 41 isolates (Figures 2D and 3) and can be described as follows; prenylated 2,5-diketopiperazines (2,5-DKPs) were isolated from the co-culture of marine *Penicillium* sp. DT-F29 isolated from marine sediments of Dongtou country, China, and *Bacillus* sp. B31 collected from marine sediments of Changzhi Island, China [35], including ten novel metabolites, 12-β-hydroxy-13-butoxyethoxyfumitremorgin B (**27**), diprostatin A (**28**), 12-hydroxy-13α-ethoxyverruculogen TR-2 (**29**), hydrocycloprostatin A (**30**), 12-β-hydroxy-13<sup>α</sup>butoxyethoxyverruculogen TR-2 (**31**), hydrocycloprostatin B (**32**), 26-α-hydroxyfumitremorgin A (**33**), 25-hydroxyfumitremorgin B (**34**), 12-β-hydroxy-13α-methoxyverruculogen (**35**), 25-hydroxyfumitremorgin A (**36**) and thirteen known isolates, verruculogen TR-2 (**37**), 12-α-hydroxy-13-α-prenylverruculogen TR-2 (**38**), 12-hydroxyverruculogen TR-2 (**39**), 13-prenyl fumitremorgin B (**40**), 12-β-hydroxy-13-<sup>α</sup>methoxyverruculogen TR-2 (**41**), cycloprostatin C (**42**), cyclotryprostatin B (**43**), spirotryprostatin C (**44**), 12,13-dihydroxyfumitremorgin C (**45**), neofipiperzine C (**46**), prenylcycloprostatin B (**47**), fumitremorgin B (**48**) and fumitremorgin A (**49**) (Figure 8)

**Figure 8.** Chemical structures of **27**–**49**.

The secondary metabolites profile of the co-culture of *Streptomyces* sp. and *Aspergillus flavipes*, obtained from marine sediments of the Nanji Islands of the same habitat, showed an induced biosynthesis of a series of known cytochalasans, including rosellichalasin (**50**), and five aspochalasins (aspochalasin E **51**, aspochalasin P **52**, aspochalasin H **53**, aspochalasin M **54** and 19,20-dihydro-aspochalasin D **55**) (Figure 9) [36]. The chromatographic purification of the combination culture extract from marine-derived *Aspergillus fumigatus* MR2012 and *Streptomyces leeuwenhoekii* C34 led to the isolation of two novel compounds, luteoride D (**56**) and pseurotin G (**57**), along with the known isolates, nocardamine (**58**), terezine D (**59**), 11-*O*-methylpseurotin A (**60**) and lasso peptide chaxapeptin (**61**) [37]. In addition, seven known compounds, notoamide D (**9**), speramide B (**62**), notoamide E (**63**), stephacidin A (**64**), notoamide R (**65**), protuboxepin B (**66**) and 3,10-dehydrocyclopeptine (**67**) (Figure 9) were identified from the mixed-fermentation of the marine-derived fungus *Aspergillus versicolor* isolated from sponge *Agelas oroides* and *B. subtilis* [38].

**Figure 9.** Chemical structures of **50**–**67**.

Compounds **27**, **28**, **38**–**40**, **44** and **46**–**49** displayed strong inhibitory effects on bromodomaincontaining protein 4 (BRD4) at 20 μM. Notably, **39** and **48** exhibited the most inhibitory activity with 72.7% and 80.4%, compared with the positive control, BRD4 inhibitor (+)-JQ1 (85.7%) [35]. As reported in the previous study, BRD4 protein was a member of the bromodomain and extra-terminal domain (BET) family that carried two bromodomains and was associated with mitotic chromosomes. Bromodomains targeted genetic and epigenetic alterations and regulated chromatin remodeling, which were important therapeutic targets for major diseases, such as neurological disorders, obesity, cancer and inflammation [39,40]. Thus, these compounds further deserved development and research for the treatment of major diseases. Li et al. (2012) reported that **41** had potent inhibitory activities against *Fusarium oxysporum* f. sp. *Niveum*, *Alternaria alternate*, *Fusarium oxysporum* f. sp. *vasinfectum* and *Fusarium solani* with MIC values of 6.25–25 μg/mL and moderate brine shrimp toxicity (LC50 60.7 μg/mL) [41]. The occurrence of **41** could be involved in protecting microbes against invasion by other competing microbes. Therefore, **41** could be considered as a promising lead compound for developing new fungicides. Cui et al. reported **43** could completely inhibit the G2/M phase of tsFT210 cells at concentrations >29.4 μM [42]. Furthermore, Wang et al. (in 2008) showed that **44** had selective cytotoxicity against four cancer cell lines, MOLT-4, HL-60, A-549 and BEL-7402 [43].

Cytochalasans were fungal metabolites that were structurally identified by the presence of a reduced isoindone nucleus connected with a macrocyclic ring [44]. Six cytochalasans (**50**–**55**) showed strong toxicity against *Streptomyces* sp. with 50–80% inhibition at 2–16 μg/mL, and most of them even

exhibited 60% inhibition at 2 μg/mL, but they had no any effect on the fungus *A. flavipes* at the same concentration. This indicated that cytochalasans could help *A. flavipes* to compete with *Streptomyces* sp., which was an important support for their potential ecological role. All cytochalasans also exhibited obvious toxicity against human cell lines, as cytochalasans had the ability to inhibit, specifically, the actin filament elongation by blocking the polymerization sites [45–47]. Thus, all six compounds (**50**–**55**) exhibited powerful toxicity against *Streptomyces* sp. at 2–16 μg/mL with inhibition rate of 50–80%. Notably, most of these compounds displayed strong inhibitory activity with inhibition rate of 60% even at 2 μg/mL, whereas none of them had antimicrobial activity against the marine-derived producer *A. flavipes* at the same concentration. These findings implied that the co-culture through microbial physical contact could stimulate the expression of silent gene cluster that was responsible for the production of cytochalasans.

The cyclic siderophore, nocardamine (**58**), had inhibitory effects on the proliferation of human tumor cell lines: SK-Mel-5 with an IC50 value of 18 μM, T-47D with an IC50 value of 6 μM, PRMI-7951 with an IC50 value of 14 μM and SK-Mel-28 with an IC50 value of 12 μM [48]. Compared with the pure cultures, some novel metabolites were observed in the mixed culture. Two fungal prenylated indole metabolites, **56** and **59**, which were not traced before in *A. fumigatus*, were induced. Both of them had an oxazino [6,5-*b*]indole nucleus which was not previously found in nature. Additionally, the yield of compound **61** was obviously higher than that of the monoculture of *Streptomyces leeuwenhoekii* C58. It was the first time that a bi-lateral cross talk was proved, which resulted in dual induction of both fungal and bacterial metabolites in the same culture conditions. **64** displayed cytotoxic activities toward mouse lymphoma cell line L5178Y with an IC50 value of 16.7 μM and in vitro toward testosterone-dependent prostate LNCaP cells with an IC50 value of 2.1 μM [49].

#### 2.1.3. Alkaloids Derived from the Co-Cultures of Different Marine Bacteria

Thirteen alkaloids were isolated from the co-culture of different marine bacteria (Figures 2E and 3); the structures of these isolates were listed in Figure 10. The average yields of five known tryptamine derivatives, N-acetyltryptamine (**68**), N-propanoyltryptamine (**69**), bacillamide C (**70**), bacillamide B (**71**) and bacillamide A (**72**) using the co-fermentation of marine strain *Streptomyces* sp. CGMCC4.7185 and *Bacillus mycoides* isolated from marine sediments of the Nanji Island (China, 27◦42 N, 121◦08 E), were 14.9, 2.8, 9.6, 13.7 and 3.0 mg/L, respectively, which were all undetectable under simple culture conditions [50]. This was the first report of applying a microorganism co-culture system to enhance the yields of known compounds [50].

In 2018, El-Hawary et al. identified four indole alkaloids—a novel brominated oxindole alkaloid saccharomonosporine A (**73**), a novel convolutamydine F (**74**) and two known compounds, (*S*) 6-bromo-3-hydroxy-3-(1H-indol-3-yl) indolin-2-one (**75**) and vibrindole (**76**)—from the mixed fermentation culture of two sponge-associated actinomycetes, *Saccharomonospora* sp. UR22 and *Dietzia* sp. UR66 collected from the Red Sea sponge *Callyspongia siphonella* [51].

Two sponge-associated actinomycetes, *Actinokineospora* sp. EG49 isolated from the Red Sea sponge, *Spheciospongia vagabunda*, and *Nocardiopsis* sp. RV163 derived from the Mediterranean sponge, *Dysidea avara*, were co-cultivated together and yielded a novel 5a,6,11a,12-tetrahydro-5a,11a-dimethyl-1,4-benzoxazino[3,2-*b*][1,4]benzoxazine (**77**) and three known metabolites, N-(2-hydroxyphenyl)- acetamide (**78**), 1,6-dihydroxyphenazine (**79**) and 2,2,3,3-tetrahydro-2,2-dimethyl-2,2-bibenzoxazole (**80**) [52].

Pim-1 kinase is a well-established oncoprotein in several tumor entities, such as prostate cancer, pancreatic cancer, colorectal cancer and myeloid leukemia. Inhibition of Pim-1 kinase would prevent the growth of tumor cells. Compounds **73** and **75** exhibited potent Pim-1 kinase inhibitors with IC50 values of 0.3 μM and 0.946 μM, respectively. Docking studies showed the binding model of **73** and **75** in the ATP pocket of Pim-1 kinase. They also exhibited obvious antiproliferative activity against human promyelocytic leukemia HL-60 (IC50 2.8 and 4.9 μM) and human colon adenocarcinoma HT-29

(IC50 3.6 and 3.7 μM). This indicated that **73** and **75** could act as potential Pim-1 kinase inhibitors that mediated the inhibitory effects on the growth of tumor cells [51].

**Figure 10.** Chemical structures of **68**–**80**.

In addition, only compound **79** was documented against *Trypanosoma brucei*(IC50 19 μM), *Bacillus* sp. (11 mm inhibition zone diameter) and *Actinokineospora* sp. EG49 (15 mm inhibition zone diameter) [52]. The yield of **79** was very high in the co-culture process. However, it was not detected in the single microbial culture. Co-culture strategy not only enhanced the chemical diversity of the metabolites but also increased the production of metabolites undetected in the single microbial culture.
