Secondary Metabolites from Marine-Derived Bacillus: A Comprehensive Review of Origins, Structures, and Bioactivities

The marine is a highly complex ecosystem including various microorganisms. Bacillus species is a predominant microbialflora widely distributed in marine ecosystems. This review aims to provide a systematic summary of the newly reported metabolites produced by marine-derived Bacillus species over recent years covering the literature from 2014 to 2021. It describes the structural diversity and biological activities of the reported compounds. Herein, a total of 87 newly reported metabolites are included in this article, among which 49 compounds originated from marine sediments, indicating that marine sediments are majority sources of productive strains of Bacillus species Therefore, marine-derived Bacillus species are a potentially promising source for the discovery of new metabolites.


Introduction
The ocean is a highly complex ecosystem, a rich and underdeveloped treasure house containing a wide variety of biological resources including aquatic species and various microorganisms [1][2][3]. Natural products, especially small molecules isolated from biological sources, have long been regarded for their huge potential in human medicine and are still gaining traction [4]. Marine microorganisms produce many undiscovered molecules with unprecedented structures and pharmacological activities in an extreme living environment [2]. Therefore, it is commonly recognized that marine microbes constitute a promising source of novel metabolites with considerable therapeutically potential for new drug screening and development [3,[5][6][7][8].
Bacillus species is a predominant microbialflora widely distributed in marine ecosystems [9,10]. Bacillus species can grow rapidly and tolerate extremely adverse environmental conditions such as extreme ambient temperature, salinity and pH, high pressure and nutrient deficiency [11]. B. subtilis can adopt several responses when faced with the depletion of essential nutrients, including motility, secretion of extracellular enzymes, genetic transformation, antibiotic production, and finally sporulation [12]. The genus Bacillus is a prolific producer of bioactive metabolites, including more than 350 kinds of rod-shaped and Gram-positive bacteria [13]. Thereinto, B. subtilis, B. licheniformis and B. amyloliquefaciens possess potential value as therapeutic agent candidates on account of their ability to produce bioactive secondary metabolites [14][15][16][17].

Cyclic Lipopeptides
Cyclic lipopeptides (CLPs) are common secondary metabolites isolated from marinederived Bacillus. The CLPs are a class of metabolites with structural diversity produced by multifarious bacterial genera [42]. There are three families of CLPs being of particular importance, namely surfactins, iturins and plipastatins, all consisting of a short cyclic oligopeptide linked to the tail of a fatty acid [14]. Surfactin sequences comprise of seven amino acids and a β-hydroxy fatty acid chain containing 12-16 carbons [43]. The iturin family sequences are composed of heptapeptides and a β-amino fatty acid chain of 14-17 carbon atoms, which consists of bacillomycin D, F, L, Lc, iturin A, A L , C and mycosubtilin ( Figure 2) [44]. The plipastatin family comprise of ten amino acids and a β-hydroxy fatty acid containing 14-18 carbon atoms [45]. Figure 3 lists the structures of cyclic lipopeptides produced by marine-derived Bacillus species.   Compounds 1-5 belong to surfactin family. A new CLP surfactin named anteiso-C15 Ile2,7 surfactin (1) was isolated from B. velezensis SH-B74 in the China Center for Type Culture Collection (CCTCC), which collected from the marine sediments, comprising of an anteiso-C15 type saturated fatty acid chain, and a peptidic backbone of L-Glu1, L-Ile2, D-Leu3, L-Val4, L-Asp5, D-Leu6, L-Ile7 [46]. Compounds 1-5 belong to surfactin family. A new CLP surfactin named anteiso-C 15 Ile 2,7 surfactin (1) was isolated from B. velezensis SH-B74 in the China Center for Type Culture Collection (CCTCC), which collected from the marine sediments, comprising of an anteiso-C 15 type saturated fatty acid chain, and a peptidic backbone of L-Glu 1 , L-Ile 2 , D-Leu 3 , L-Val 4 , L-Asp 5 , D-Leu 6 , L-Ile 7 [46]. Rn-Glu 1 -Leu/Ile 2 -Leu 3 -Val 4 -Asp 5 -Leu 6 -Leu/Ile 7 (2)(3)(4)(5) belonging to surfactin homolog were isolated from B. licheniformis MB01 collected from sediments in the Bohai Sea, China [47]. Compounds 6-10 belong to the iturin family. A novel lipopeptide antibiotic bacillopeptin named bacillopeptin B 1 (6) and a known compound, bacillopeptin B (7) were detected in the fermentation broth of a marine sediment-derived B. amyloliquefaciens SH-B74 collected from sediments in the South China Sea. More precisely, compound 6 as a member of bacillopeptin family has the same amino-acid sequence and the same molecular weight as compound 7, but has a different fatty-acid residue [22]. Compounds 8-10 were characterized as cyclic lipopeptides with saturated β-amino fatty acid chain residues, iso-C14 mojavensin, iso-C16 mojavensin, and anteiso-C17 mojavensin, all of which were produced by a marine-derived B. mojavensis B0621A obtained from the mantle of a pearl oyster Pinctada martensii in the South China Sea [48]. In addition, plipastatin A1 (11), belonging to the plipastatin family, was obtained by solidphase extraction and reversed-phase high-performance liquid chromatograph (RP-HPLC) from the fermentation broth of a marine sediment-derived B. amyloliquefaciens SH-B74 in the CCTCC [49]. A new cyclic hexapeptide with three piperazic acids (N-OH-Thr, N-OH-Gly, β-OH-Leu) named dentigerumycin E (12) and two reported derivatives, 2-N, 16-N-deoxydenteigerumycin E (13) and dentigerumycin E methyl ester (14), were isolated from coculture of marine Streptomyces and Bacillus strains collected together from the intertidal mudflat in Wando, Republic of Korea. It is worth mentioning that only compound 12 showed antiproliferative and antimetastatic activities against human cancer cells, suggesting that 2-N-OH, 16-N-OH, and 37-OH (carboxylic acid) are essential for the activities [25]. Two novel cyclic lipopeptides, bacilotetrin A (15) and bacilotetrin B (16), possessing three leucines and one glutamic residue cyclized with a lipophilic 3-hydroxyl fatty acid, were isolated from B. subtilis 109GGC020 in the sediments from the Gageocho of southern reef, Republic of Korea [12]. Additionally, gageopeptins A (17) and B (18), two novel cyclic lipopeptides, were isolated from the same strain in the same sediments as above [50]. A new cyclic hexapeptide named bacicyclin (19) was purified from Bacillus sp. BC028 associated with the blue mussel Mytilus edulis collected from the western shore of the Baltic Sea in Germany [51].

Diketopiperazines
Cyclicpeptide diketopiperazines consist of residues of two amino acids and mevalonic acid [52]. Figure 4 lists the structures of diketopiperazines that were produced by marinederived Bacillus species.
Mar. Drugs 2022, 20, x FOR PEER REVIEW 7 of 32 Rn-Glu 1 -Leu/Ile 2 -Leu 3 -Val 4 -Asp 5 -Leu 6 -Leu/Ile 7 (2)(3)(4)(5) belonging to surfactin homolog were isolated from B. licheniformis MB01 collected from sediments in the Bohai Sea, China [47]. Compounds 6-10 belong to the iturin family. A novel lipopeptide antibiotic bacillopeptin named bacillopeptin B1 (6) and a known compound, bacillopeptin B (7) were detected in the fermentation broth of a marine sediment-derived B. amyloliquefaciens SH-B74 collected from sediments in the South China Sea. More precisely, compound 6 as a member of bacillopeptin family has the same amino-acid sequence and the same molecular weight as compound 7, but has a different fatty-acid residue [22]. Compounds 8-10 were characterized as cyclic lipopeptides with saturated β-amino fatty acid chain residues, iso-C14 mojavensin, iso-C16 mojavensin, and anteiso-C17 mojavensin, all of which were produced by a marine-derived B. mojavensis B0621A obtained from the mantle of a pearl oyster Pinctada martensii in the South China Sea [48]. In addition, plipastatin A1 (11), belonging to the plipastatin family, was obtained by solidphase extraction and reversed-phase high-performance liquid chromatograph (RP-HPLC) from the fermentation broth of a marine sediment-derived B. amyloliquefaciens SH-B74 in the CCTCC [49]. A new cyclic hexapeptide with three piperazic acids (N-OH-Thr, N-OH-Gly, β-OH-Leu) named dentigerumycin E (12) and two reported derivatives, 2-N, 16-Ndeoxydenteigerumycin E (13) and dentigerumycin E methyl ester (14), were isolated from coculture of marine Streptomyces and Bacillus strains collected together from the intertidal mudflat in Wando, Republic of Korea. It is worth mentioning that only compound 12 showed antiproliferative and antimetastatic activities against human cancer cells, suggesting that 2-N-OH, 16-N-OH, and 37-OH (carboxylic acid) are essential for the activities [25]. Two novel cyclic lipopeptides, bacilotetrin A (15) and bacilotetrin B (16), possessing three leucines and one glutamic residue cyclized with a lipophilic 3-hydroxyl fatty acid, were isolated from B. subtilis 109GGC020 in the sediments from the Gageocho of southern reef, Republic of Korea [12]. Additionally, gageopeptins A (17) and B (18), two novel cyclic lipopeptides, were isolated from the same strain in the same sediments as above [50]. A new cyclic hexapeptide named bacicyclin (19) was purified from Bacillus sp. BC028 associated with the blue mussel Mytilus edulis collected from the western shore of the Baltic Sea in Germany [51].

Linear Lipopeptides
Linear lipopeptide is a kind of lipopeptide, in which amino acids are connected in turn into linear, unconnected head and tail and no cyclic structure. Fatty acids are connected to α-amino groups or other hydroxyl groups at the N-terminal of the peptide chain [56]. Figure 5 lists the structures of linear lipopeptides that were produced by marine-derived Bacillus species.

Linear Lipopeptides
Linear lipopeptide is a kind of lipopeptide, in which amino acids are connected in turn into linear, unconnected head and tail and no cyclic structure. Fatty acids are connected to α-amino groups or other hydroxyl groups at the N-terminal of the peptide chain [56]. Figure 5 lists the structures of linear lipopeptides that were produced by marine-derived Bacillus species. Three newfound linear lipopeptides named gageostatins A (28), B (29) and C (30), comprising of hepta-peptides and new 3-β-hydroxy fatty acids yielded by a marine-derived bacterium B. subtilis from the culture broth [35]. Furthermore, three novel linear lipopeptides possessing di-and tetrapeptides and a new fatty acid, gageotetrins A (31), B (32) and C (33), were isolated from a marine B. subtilis [57]. Four unreported lipopeptides, gageopeptides A (34), B (35), C (36), and D (37) were isolated and identified from a marine-derived bacterium B. subtilis, which consisted of tetrapeptides and 3-β-hydroxy fatty acids [58]. The fatty acid of 28, 31, 32 and 34 was identical and determined as a 3-β-hydroxy-11-methyltridecanoic acid. Likewise, compounds 29 and 37 both possessed the same fatty acid, 3-β-hydroxy-9,11-dimethyltridecanoic acid. Moreover, the fatty acid unit of 33 and 36 was 3-β-hydroxy-8,10-dimethyldodecanoic acid. In particular, the absolute stereochemistry at C-3 of the fatty acids of linear lipopeptides 28-37 is R configuration except 30. Additionally, the configuration of the amino acid residues in 28-37 was found to be L-form, while Val in 28-30 was D-form. Besides, bacilysin (38), another identified dipeptide, was isolated from seaweed-associated B. amyloliquefaciens MTCC 10456 in Microbial Type Culture Collection and Gene Bank (MTCC) of Chandigarh in India. Notably, this is the first report on the co-production and isolation of anti-Malassezia spp. chemicals from marine Bacillus species [44]. In conclusion, all linear Lipopeptide mentioned above were obtained from the Gageocho in the southern reef (Republic of Korea) except 38.

Nonribosomal Peptides
Nonribosomal peptides (NRPs) are large enzyme complexes with a modular structure responsible for binding a particular amino acid. NRPSs of Bacillus are synthesized by large multimodular nonribosomal peptide-synthetase (NRPS) through Three newfound linear lipopeptides named gageostatins A (28), B (29) and C (30), comprising of hepta-peptides and new 3-β-hydroxy fatty acids yielded by a marine-derived bacterium B. subtilis from the culture broth [35]. Furthermore, three novel linear lipopeptides possessing di-and tetrapeptides and a new fatty acid, gageotetrins A (31), B (32) and C (33), were isolated from a marine B. subtilis [57]. Four unreported lipopeptides, gageopeptides A (34), B (35), C (36), and D (37) were isolated and identified from a marine-derived bacterium B. subtilis, which consisted of tetrapeptides and 3-β-hydroxy fatty acids [58]. The fatty acid of 28, 31, 32 and 34 was identical and determined as a 3-β-hydroxy-11methyltridecanoic acid. Likewise, compounds 29 and 37 both possessed the same fatty acid, 3-β-hydroxy-9,11-dimethyltridecanoic acid. Moreover, the fatty acid unit of 33 and 36 was 3-β-hydroxy-8,10-dimethyldodecanoic acid. In particular, the absolute stereochemistry at C-3 of the fatty acids of linear lipopeptides 28-37 is R configuration except 30. Additionally, the configuration of the amino acid residues in 28-37 was found to be L-form, while Val in 28-30 was D-form. Besides, bacilysin (38), another identified dipeptide, was isolated from seaweed-associated B. amyloliquefaciens MTCC 10456 in Microbial Type Culture Collection and Gene Bank (MTCC) of Chandigarh in India. Notably, this is the first report on the co-production and isolation of anti-Malassezia spp. chemicals from marine Bacillus species [44]. In conclusion, all linear Lipopeptide mentioned above were obtained from the Gageocho in the southern reef (Republic of Korea) except 38.

Nonribosomal Peptides
Nonribosomal peptides (NRPs) are large enzyme complexes with a modular structure responsible for binding a particular amino acid. NRPSs of Bacillus are synthesized by large multimodular nonribosomal peptide-synthetase (NRPS) through prolonging the active monomers of amino acid building blocks [59]. Figure 6 lists the structures of nonribosomal peptides produced by marine-derived Bacillus species.
Mar. Drugs 2022, 20, x FOR PEER REVIEW 10 of 32 prolonging the active monomers of amino acid building blocks [59]. Figure 6 lists the structures of nonribosomal peptides produced by marine-derived Bacillus species.  Two unreported compounds, bacillibactin B (39) and bacillibactin C (40), along with the known compounds Bacillibactin (41) and SVK21 (42), were discovered from Bacillus sp. named PKU-MA00093 from sponges, corals and sediments in the South China Sea. Additionally, compounds 43-48 were characterized as bacillomycin D, iso-C15 bacillomycin D, C15 bacillomycin D, iso-C16 bacillomycin D, C16 bacillomycin D, and anteiso-C17 bacillomycin D, respectively. They were isolated from Bacillus sp. PKU-MA00092 collected from sponges, corals and sediments in the South China Sea. Notably, this was the first time to report the structures of 45 and 47 with fully specified 1 H NMR and 13 C NMR data; their structures are highly similar except for the fatty acid moieties [60]. Compounds 45 and 47 in company with C14 bacillomycin D (49), were obtained from seaweedassociated B. amyloliquefaciens MTCC 10456 collected from seaweed in the MTCC, of Chandigarh, India [44]. Moreover, compounds 45 and 49 were also isolated from the methanol extract harvested from marine-derived B. megaterium CGMCC7086 obtained from the intestines of marine fish in the Yellow Sea of East China by two-step ultrafiltration and liquid chromatography-electronic spray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using a highly-efficient separation technique and identification method, more than 40 lipopeptides variants were identified from a Bacillus strain [23]. Besides, compound 47 was isolated from B. subtilis B38 strain [61]. Two unique bacillibactins, bacillibactins E (50) and F (51) were the first bacterial siderophores containing nicotinic and benzoic acid moieties isolated from a marine sponge Cinachyrella apion associated Bacillus sp. WMMC1349 collected from the west shore of Ramrod Key in Florida [24]. Two unreported compounds, bacillibactin B (39) and bacillibactin C (40), along with the known compounds Bacillibactin (41) and S VK21 (42), were discovered from Bacillus sp. named PKU-MA00093 from sponges, corals and sediments in the South China Sea. Additionally, compounds 43-48 were characterized as bacillomycin D, iso-C15 bacillomycin D, C15 bacillomycin D, iso-C16 bacillomycin D, C16 bacillomycin D, and anteiso-C17 bacillomycin D, respectively. They were isolated from Bacillus sp. PKU-MA00092 collected from sponges, corals and sediments in the South China Sea. Notably, this was the first time to report the structures of 45 and 47 with fully specified 1 H NMR and 13 C NMR data; their structures are highly similar except for the fatty acid moieties [60]. Compounds 45 and 47 in company with C14 bacillomycin D (49), were obtained from seaweed-associated B. amyloliquefaciens MTCC 10456 collected from seaweed in the MTCC, of Chandigarh, India [44]. Moreover, compounds 45 and 49 were also isolated from the methanol extract harvested from marine-derived B. megaterium CGMCC7086 obtained from the intestines of marine fish in the Yellow Sea of East China by two-step ultrafiltration and liquid chromatographyelectronic spray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using a highlyefficient separation technique and identification method, more than 40 lipopeptides variants were identified from a Bacillus strain [23]. Besides, compound 47 was isolated from B. subtilis B38 strain [61]. Two unique bacillibactins, bacillibactins E (50) and F (51) were the first bacterial siderophores containing nicotinic and benzoic acid moieties isolated from a marine sponge Cinachyrella apion associated Bacillus sp. WMMC1349 collected from the west shore of Ramrod Key in Florida [24].

Polyketides
Polyketides are a class of extremely large secondary metabolites assembled from simple acyl-coA compounds [62]. Bacillus species of marine origin was a potential source of bioactive compounds of polyketides and bacteriocins with significant antimicrobial activity against human pathogens [63]. Figure 7 lists the structures of polyketides that were produced by marine-derived Bacillus species.

Other Compounds
The secondary metabolites of marine-derived Bacillus species could be isolated from marine sediments, marine invertebrates (sponges, molluscs, and corals), and vertebrates (mainly fishes), as well as marine plants (mainly seaweed). Currently, there are 9 reported sources of Bacillus secondary metabolites. As shown in Figure 10C  The genus Bacillus comprises more than 350 species, some of which are used as antifungal agents, while others are promising producers of green pesticide [14]. As discussed above, a total of 10 identified species, including B. subtilis, B. amyloliquefaciens, B. megaterium CGMCC7086, B. mojavensis B0621A, B. licheniformis, B. siamensis, B. stratosphericus, B. pumilus RJA1515, B. polymyxa L 1 -9, and B. velezensis SH-B74 were reported as the producing strains of these described secondary metabolites. Among them, B. subtilis and B. amyloliquefaciens were the most prolific strains, with 24 (20.00%) and 17 (17.71%) metabolites identified, respectively ( Figure 10B). CLPs are ubiquitous in several Bacillus strains. However, linear lipopeptides are found predominantly in B. subtilis species, while the macrolactins are more preponderant in B. amyloliquefaciens species, suggesting the species-specific metabolites.
The secondary metabolites of marine-derived Bacillus species could be isolated from marine sediments, marine invertebrates (sponges, molluscs, and corals), and vertebrates (mainly fishes), as well as marine plants (mainly seaweed). Currently, there are 9 reported sources of Bacillus secondary metabolites. As shown in Figure 10C were collected from seaweed. In addition to these producing strains, only one Bacillus strain (B. megaterium CGMCC7086), which produced 45 and 49, was obtained from the intestines of marine fish and accounted for 1.74% of the reported total amount. Unfortunately, the environmental sources of the producing strain of 47 (B. subtilis B38), 73 and 74 (B. stratosphericus) were not described. From the above analysis, it can be concluded that marine sediments and sponges are more abundant sources of productive strains of marine-derived Bacillus, and which deserved much more attention in subsequent chemical studies.

Biological Activities
The producing strains, environmental sources, and biological activities of bioactive compounds from marine-derived Bacillus are listed. Most compounds possess a range of moderate to potent biological activities, including antimicrobial, antifungal, anticancer and other activities. Detailed descriptions of compounds with promising bioactivities are described as follows. Table 1 lists the potential antimicrobial Bacillus secondary metabolites. Compounds 2-5 exhibited antibacterial activity against Vibrio parahaemolyticus with a minimum inhibitory concentration (MIC) of 50 µg/mL. At the same time, they also displayed significant antibacterial activity against Escherichia coli, Vibrio cholerae, Vibrio harveyi, Pseudomonas aeruginosa, Staphylococcus aureus, and Proteus species, fully illustrating their broad-spectrum bacteriostatic characters [47]. Anti-staphylococcal activity of compounds 15 and 16 was evaluated against clinically methicillin resistant S. aureus (MRSA) strains (ATCC25923, XU212, SA1199B, RN4220) with MIC values from 8 to 32 µg/mL by the broth dilution assay, indicating better potential than that of the positive control norfloxacin (MIC, 2-64 µg/mL) [12]. Compounds 17 and 18 exhibited moderate antibacterial activity against Gram-positive bacteria (S. aureus, B. subtilis), and Gram-negative bacteria (Salmonella typhi and P. aeruginosa) with MIC values ranging from 16 to 32 µg/mL, compared to azithromycin (MIC, 2 µg/mL) [50]. Similarly, compounds 28-37 were active with MICs of 3-64 µg/mL when tested for the same bacteria as above, which proved they could be good biological probes for antimicrobial agents [35,57,58]. Among them, mixed compounds 28 + 29 were more active than individual ones [35]. Surprisingly, compound 19 displayed inhibitory activity against the clinically relevant strains S. aureus and Enterococcus faecalis with MIC values of 12 and 8 µM, respectively, compared to the positive control streptomycin (MIC, 2.09 µM and 5.24 µM), demonstrating the potential of marine microorganism as a hopeful source for the development of new antibiotics [51]. Moreover, compound 20 displayed strong antimicrobial activities against E. coli and S. aureus with MIC values of 16 µg/mL and 22 µg/mL, respectively, compared with ampicillin as positive control. Notably, this was the first report of antibacterial activity of diketopiperazine [53]. Compound 25 exhibited significant antimicrobial activity against fish pathogens, where the MIC was 31.25 mg/mL determined by the broth dilution assay method [55]. Compounds 52 and 53 were reported to display significant antibacterial activity against V. parahaemolyticus ATCC ® 17802™, Vibrio vulnificus, and Aeromonas hydrophila [64]. Compounds 54-57 exhibited antibacterial activities against human important clinical pathogens V. vulnificus and V. parahaemolyticus (inhibitory zone diameter greater than 15 mm, 100 mcg on disk) [63]. A new macrolactin 68 showed a moderate effect on A. hydrophila, V. parahemolyticus ATCC 17802 and V. vulnificus with inhibitory zone diameters of 18, 16, and 14 mm at concentration of 100 µg on disk compared with that of the control commercial antibiotics [68]. Difficidin analogues 69-72 displayed significant antibacterial activities against multidrug-resistant (MDR) bacteria containing methicillin-resistant S. aureus, vancomycin-resistant E. faecalis, and other drug-resistant strains, such as P. aeruginosa and Klebsiella pneumonia with the MICs of 0.002-0.009 µM, compared to positive controls (chloramphenicol and ampicillin with MIC of about 0.017-0.049 µM). Thereinto, a drug-likeness score of 70 was greater than those of other difficidin analogues, demonstrating its potential for pharmaceutical uses against the bottleneck of drug-resistant pathogens [13]. Compounds 73 and 74 exhibited higher activities against E. faecalis with MIC values of 0.05-0.8 µg/mL than the positive control (vancomycin and linezolid). In contrast, the antibacterial activities of 73 and 74 against V. parahaemolyticus, Photobacterium damselae, Shewanella algae, Bacillus amyloliquefaciens sspplantarum, and Pseudomonas stutzeri (0.5-8 µg/mL) were lower than those of the positive controls. Both compounds did not show cytotoxicity up to a concentration of 10 µM in the MTT assay [69]. Compounds 75-78 displayed antibacterial activity against P. aeruginosa PA-01 and Acinetobacter baumannii ATCC19606 with MIC values 58-64 µg/mL, while the positive control ofloxacin showed MIC values of 1-16 µg/mL [70]. Compounds 82 and 83 displayed good antimicrobial activities against P. aeruginosa, E. coli, Bacillus cereus, B. subtilis, S. typhi, and S. aureus with MICs values of 0.01-0.05 µM, while azithromycin exhibiting an MIC value of 0.003 µM [71]. Compound 86 displayed broad-spectrum antimicrobial activities against Salmonella typhimurium, MRSA, Listeria monocytogenes, A. hydrophila, S. aureus, Staphylococcus epidermidis, E. coli, and P. aeruginosa with MICs as follows: 16 µg/mL, 32 µg/mL, 0.25 µg/mL, 0.5 µg/mL, 8 µg/mL, 4 µg/mL, 4 µg/mL, and 8 µg/mL. Additionally, the minimum bactericidal concentration (MBC) values of 86 observed ranged from 1 to 64 µg/mL, while the lowest MBC value of 86 was 1 µg/mL for L. monocytogenes followed by A. hydrophila (2 µg/mL), E. coli (8 µg/mL), S. aureus (16 µg/mL) and S. epidermidis (16 µg/mL). On the other hand, the highest MBC values were obtained with MRSA (64 µg/mL), S. typhimurium (32 µg/mL) and P. aeruginosa (32 µg/mL) [73]. Compounds 86 and 87 exhibited inhibitory zone diameters of 1.3-9.7 mm against E. coli, B. subtilis, and S. aureus, wherein 87 showed higher inhibitory effect than 86, which proved they could be used as potential candidates for new antibacterial agents [74].      E. coli (9.7 ± 0.20 mm), B. subtilis (6.6 ± 0.16 mm), S. aureus (2.3 ± 0.12 mm) (inhibition zone diameter) [74] a Only the most competitive values are listed in this table due to limited space.

Antifungal Activities
The Bacillus secondary metabolites with potential antifungal activities are listed in Table 2. It was the first time reported that a surfactin type CLP 1 displayed an inhibitory effect on appressorium formation of rice blast causal pathogen Magnaporthe oryzae at concen-trations of 10 and 50 µM, indicating that 1 may be considered as potential green pesticide against M. oryzae as prospected [46]. Compound 6 had antagonistic activities against several plant pathogens (Valsa mali, Fusarium oxysporum f.sp. cucumerinum and Rhizoctonia solani) when the concentration was over 2 mg/mL by the paper disc-agar diffusion assay [22]. Compounds 8-10 displayed moderate and dose-dependent inhibition of growth with tested F. oxysporum (inhibitory zone diameter 5.31-7.33 mm) via paper disc-agar diffusion assay, when the concentrations ranged from 0.5 to 2.0 mm. The difference in antifungal activity between 8 and the other two compounds was statistically significant (p < 0.05). With respect to activity-structure relationships, a conclusion can be drawn that antifungal activity is closely related to the length of fatty acid chains, as well as C16 and C17 forms of mojavensins [48]. Research revealed that compound 11 has excellent activity in vitro on the suppression of the conidia germination of Botrytis cinerea (MIC, 50 µM) [49]. Compounds 17 (half maximal inhibitory concentration, IC 50 = 1 µg/mL) was an approximately 400-times-stronger inhibitor than compound 18 (IC 50 = 400 µg/mL) on the motility of zoospores of Phytophthora capsici with dose-dependent and time-dependent manners. It is also noteworthy that the zoospores blocked by 17 whereafter cleaved at a higher concentration (IC 50 = 50 µg/mL). It manifested that the methyl group at C-12 of the fatty acid in 17 is vital for activity. Therefore, compound 17 can be used to develop fungicides targeting P. capsici [50]. Moreover, compounds 17 and 18 possessed promising inhibitory activities against R. solani, Colletotrichum acutatum, B. cinerea with MIC values ranging from 4 to 8 µg/mL, which were comparable to that of amphotericin B (MIC, 1 µg/mL) [50]. Similarly, compounds 28-37 were active with MICs of 1-32 µg/mL when testing the same pathogenic fungi as above [35,57,58]. Among them, mixed compounds 28 + 29 were more active than individual ones. Additionally, the four novel non-cytotoxic lipopeptides 34-37 from marine-derived B. subtilis highlighted the research for novel safe antifungal agents [35,58]. Compounds 32, 34-37 could inhibit mycelial growth, conidiogenesis, conidia germination, morphological alterations in the germinated conidia, and wheat blast disease. Additionally, the MICs of the compounds 32, 34-37 were as follows: 1.5 µg/disk (32), 2.5 µg/disk (36), 2.5 µg/disk (37), 10.0 µg/disk (34), and 10.0 µg/disk (35), revealing 32 showed the highest mycelial growth inhibition of M. oryzae Triticum (MoT) among the tested compounds [75]. Compounds 45, 47 and 49 displayed antifungal activity against filamentous fungi and yeasts [45]. Additionally, compound 47 also exhibited antifungal activity against Candida albicans with an MIC value of 12.5 µg/mL [61]. Compounds 38, 60 and 62 showed significant antifungal activity against Malassezia spp. (Malassezia furfur ATCC 44344, Malassezia furfur ATCC 44344, and Malassezia globosa ATCC MYA 4612) with MIC values ranging from 38 to 330 µg/mL, while the positive control (ketoconazole) had MIC values ranging from 0.03 to 0.08 µg/mL. Therefore, these compounds reported against Malassezia spp. could be beneficial for application in the field of cosmetics and dermatology [44].

Cytotoxic Activities
The potential cytotoxic Bacillus secondary metabolites are listed in Table 3. Compound 12 displayed moderate cytotoxicity against the cancer cell lines including SK-HEP-1 (liver cancer), HCT116 (colorectal cancer), MDA-MB-231 (breast cancer), A549 (lung cancer), SNU638 (stomach cancer) and antimetastatic activity against breast cancer cells (MDA-MB-231) with IC 50 ranging from of 28 to 39 µM compared to positive control Etoposide (IC 50 , 0.42-6.21 µM). By contrast, compounds 13 and 14 did not exhibit antiproliferative and antimetastatic activities against human cancer cells, which indicated 2-N-OH, 16-N-OH, 37-OH (carboxylic acid) in 12 play an important role. More rarely, 12 was inactive towards normal epithelial cells (IC 50 > 50µM) [25]. Compounds 28-30 were cytotoxic towards breast cancer (MDA-MB-231), colon cancer (HCT-15), prostate cancer (PC-3), lung cancer (NCI-H23), stomach cancer (NUGC-3), and renal cancer (ACHN), with GI 50 values of 4.6 to 23.2 µg/mL compared with the positive control Adriamycin (GI 50 , 0.33 to 0.91 µg/mL). It is noteworthy that mixed compounds 28 + 29 demonstrated better activity than individuals, especially for lung cancer (NCI-H23) with the GI 50 value of 4.6 µg/mL [35]. Compounds 45-48 were observed with moderate cytotoxic activities towards human cancer cell lines MCF7 (breast cancer) and HepG2 (liver cancer) with IC 50 values ranging from 8.2 ± 0.2 to 2.9 ± 0.1 µM, whereas compounds 39-44 had no cytotoxic effects, suggesting that a C16-fatty acid of bacillomycin D analogues may be the key moiety with best cytotoxicities [60]. The MTS assay illustrated that compound 58 can promote cell proliferation at the concentration of 10 µM and 58-62 did not display obvious cytotoxic effect in the murine macrophage cell line RAW 264.7 at the employed concentrations (10-40 µM) [66]. Compound 84 possessed the inhibitory effects on human melanoma cell line MNT-1 and melanin synthesis in mouse melanoma cell line B16F10 [72]. Compound 85 displayed enzyme inhibitory activity against melanogenic enzymes, which could modulate melanogenesis by down-regulating melanogenic enzymes expression with minimal cytotoxicity in murine melanoma cells, such as MNT-1, B16F10 and human melanoma cell [72]. inhibitory activity (human melanoma cell line MNT-1 and melanin synthesis in mouse melanoma cell line B16F10), enzyme inhibitory activity, and anti-pigmentary activity [72] a Only the most competitive values are listed in this table due to limited space.

Other Activities
In the previous study, sponge epiphytic Bacillus was found to have an obvious inhibitory effect on the attachment of many kinds of diatoms. Compounds 21-27 could inhibit the attachment of diatoms to a certain extent in order to help the host sponges realize the chemical defense to Nitzschia closterium, so they can be used as marine natural products antifouling agent with high possibility and selectivity [54]. Compounds 50 and 51 exhibited siderophore activity at concentrations ranging from 1.28 mm to 1.25 µM, which were the first bacterial siderophores containing benzoic acid and nicotinic acid moieties [24]. Owing to the existence of the epoxy ring, compound 58 exhibited a significant inhibitory effect on the expression of various cytokines inducible and nitric oxide, compared with previously known macrolactins (59)(60)(61)(62). Additionally, 58 reduced the mRNA expression level of IL-1β with concentration-dependent manner [66]. Of note, in addition to cytotoxicity in melanoma cells, the anti-pigmentary effect of 85 was demonstrated in an artificial human skin model with comparable or superior effects to those of arbutin, bisabolol and kojic acid, indicating it could be potential as hypopigmenting agent [72].
In this view, most of the presented compounds display significant biological activities such as antimicrobial, antifungal, and anticancer. These compounds are also effective to a certain extent in inhibiting the attachment of diatoms, the expression of various cytokines inducible and nitric oxide exhibited siderophore activity, as well as enzyme inhibitory activity. Among them, antimicrobial (41.41%), antifungal (33.33%), and anticancer (10.10%) activities were dominant in evaluating the pharmacological potential of these metabolites ( Figure 11). Notably, a high proportion (54.84%) of the 49 marine sediment-derived metabolites exhibited moderate to potent bioactivity. Based on this, these impressive bioactivities indicate their potential to be new antibiotic candidates, biological control agents, and hypopigmenting agents.

Conclusions
This mini-review summarizes a total of 87 secondary metabolites produced by marine-derived Bacillus species reported in recent years. The described compounds were organized on the basis of their structural diversity, and biological activities. CLPs are ubiquitous in several Bacillus strains, meanwhile linear lipopeptides and macrolactins are found predominantly in B. subtilis and B. amyloliquefaciens species as species-specific metabolites. In addition, marine sediments and sponges are more abundant sources of productive strains of marine-derived Bacillus, and most marine sediment-derived metabolites exhibited moderate to potent bioactivity, which deserves much more attention in subsequent chemical studies. Therefore, marine-derived Bacillus species are a potential promising source for the discovery of new metabolites.

Conclusions
This mini-review summarizes a total of 87 secondary metabolites produced by marinederived Bacillus species reported in recent years. The described compounds were organized on the basis of their structural diversity, and biological activities. CLPs are ubiquitous in several Bacillus strains, meanwhile linear lipopeptides and macrolactins are found predominantly in B. subtilis and B. amyloliquefaciens species as species-specific metabolites. In addition, marine sediments and sponges are more abundant sources of productive strains of marine-derived Bacillus, and most marine sediment-derived metabolites exhibited moderate to potent bioactivity, which deserves much more attention in subsequent chemical studies. Therefore, marine-derived Bacillus species are a potential promising source for the discovery of new metabolites.