Deep-Sea Natural Products from Extreme Environments: Cold Seeps and Hydrothermal Vents

The deep sea has been proven to be a great treasure for structurally unique and biologically active natural products in the last two decades. Cold seeps and hydrothermal vents, as typical representatives of deep-sea extreme environments, have attracted more and more attention. This review mainly summarizes the natural products of marine animals, marine fungi, and marine bacteria derived from deep-sea cold seeps and hydrothermal vents as well as their biological activities. In general, there were 182 compounds reported, citing 132 references and covering the literature from the first report in 1984 up to March 2022. The sources of the compounds are represented by the genera Aspergillus sp., Penicillium sp., Streptomyces sp., and so on. It is worth mentioning that 90 of the 182 compounds are new and that almost 60% of the reported structures exhibited diverse bioactivities, which became attractive targets for relevant organic synthetic and biosynthetic studies.


Introduction
Extreme environments refer to areas close to the limits of life, such as cold seeps, hydrothermal vents, polar and hot regions, or marine areas with high salinity [1]. Because of the extreme conditions of pressure, temperature, or high concentrations of toxic elements, unique organisms are more likely to appear. Compared with other ecosystems, extreme environments have not been fully developed and utilized, due to the limited conditions and difficult sampling. In recent years, with the progress of technology and the further exploration of the deep sea, scientists have gradually realized the uniqueness of natural products from extreme environments [2].
Cold seeps are typical deep-sea, chemosynthetically driven ecosystems, characterized by methane-rich fluid emissions and distinctive sulfur oxidation-reduction reactions, which lead to a high abundance of specialized cold-seep microorganisms [3]. The temperature of cold seeps is 2-4 • C, basically the same as the temperature around the seafloor. Microorganisms and animals from deep-sea cold seeps, which could be a new source of biomedically important compounds, due to their unique habitat, are only beginning to be investigated. The great potential for natural product discovery in deep-sea cold seep organisms will undoubtedly accelerate the investigation of new drugs [4].
Hydrothermal vents are formed when water heated in the Earth's crust and magma are forced explosively to the surface through rock fissures in volcanic regions. Since ocean hydrothermal vents are among the most dynamic environments on Earth, secondary metabolite diversity of this extreme environment is considerably high [5]. With advances in sample collecting techniques, deep-sea hydrothermal vents might be potential hot spots for natural product discovery [6].
Therefore, this review covers papers on metabolites isolated from deep-sea extreme environments, including cold seeps and hydrothermal vents, using databases such as SciFinder, Web of Science, and so on. The structures of these compounds and details of the source organisms and depth of collection are presented along with relevant biological activities of the metabolites and synthetic studies. A total number of 182 compounds are presented in this review, with 132 cited references.

Marine Animals
Marine animals generally contain high proportions of n-3 polyunsaturated fatty acids (PUFAs) [7], in particular, long-chain PUFAs, such as DHA and EPA. There is increasing evidence that specific dietary patterns including, for example, n-3 PUFAs may be beneficial in reducing breast cancer risk [8,9]. However, some bivalve symbiotic bacteria were found to contain a novel n-4 or n-7 family, which appears to be an adaptation to the extremely high pressure and low temperature of seawater [10].
Novel fatty acids (1-10) ( Figure 1) were purified from the two cold-seep-derived mussels Bathymodiolus japonicus and B. platifrons, collected at a depth of 1209 m at latitude 35 • 18 N and longitude 139 • 13 E in the Northern Pacific Ocean and a depth of 978 m at latitude 27 • 47 N and longitude 126 • 54 E in the East China Sea. The major PUFAs in the two mussels belong to unusual n-4 and n-7 methylene-interrupted PUFAs. B. japonicus and B. platifrons could maintain fluidity in plasma membrane lipids by accumulation of n-4 family methylene-interrupted PUFAs [11]. The cold-seep clam Calyptogena phaseoliformis, collected in the Japan Trench at a depth of 6354-6367 m, yielded eight novel fatty acids (11)(12)(13)(14)(15)(16)(17)(18) (Figure 1). They were determined by gas chromatography-mass spectrometry analysis of 4,4-dimethyloxazoline derivatives. The major fatty acids present in C. phaseoliformis lipids belong to the n-4 family non-methyleneinterrupted PUFAs [12].

Penicillium sp.
Penicillium fungi have received remarkable interest as an important source of novel natural products encompassing diverse chemical structures and bioactive properties [55,56].
A cold-seep-derived actinomycete belonging to the Streptomyces olivaceus OUCLQ19-3 genus was found to contain two new (72 and 73) and six known (74-79) (Figure 9) dixiamycins. In the antibacterial test, compounds 72-79 exhibited significant growth inhibition against several multi-drug-resistant (MDR) strains, with MIC values ranging from 0.78 to 6.25 µg/mL; among these, 72, 73, and 76-79 were more potent than the positive control tetracycline [4]. Dixiamycins A (77) and B (76) are the first examples of atropisomerism naturally occurring in N-N-coupled atropo-diastereomers [68]. A unique method of electrochemical dimerization of carbazoles and carbolines enabled the first total synthesis of dixiamycin B (76) [69]. Sulfadixiamycin A (79) was found to have selective yet moderate antimycobacterial properties, with a MIC value of 25 mg/mL [70].

Halomonas sp.
Halomonas is a kind of Gram-negative bacterium which has strong adaptability and a wide range of adaptability to temperature, salinity, and oxygen. It may have important application values in sewage treatment and bioremediation [71].
An immune-enhancing exopolysaccharide, EPS2E1 (80) Bacteria belonging to the Vibrio family are short in shape and named for their curvelike arcs. They are usually found in freshwater or seawater and also in the intestines of humans or fish. Some species are pathogenic to fish or humans [73]. Vibrio species can produce compounds with attractive biological activities, including antibacterial, anticancer, and antivirulence activities [74].

Bacillus sp.
Marine Bacillus species produce versatile secondary metabolites, including lipopeptides, polypeptides, fatty acids, polyketides, and coumarins. These structurally diverse compounds exhibit a wide range of biological activities [76].
A bacterial strain isolated from the cold-seep-derived fungus Bacillus sp. CS30 which was collected in the South China Sea in October 2017 (119 • 17 09.655" E, 22 • 06 5.169" N), exhibited strong growth inhibition against M. grisea. Two purified antifungal agents were isolated which belong to the surfactin family and were named surfactin CS30-1 and surfactin CS30-2 (82 and 83). Both of them showed antifungal activity, since they could induce the generation of reactive oxygen species (ROS) and caused serious damage to the cell wall and cytoplasm [77].

Others
Three novel series of non-isoprenoidal dialkyl glycerol diethers were tentatively identified in carbonate crusts precipitated from methane-rich bottom-waters and pore-waters associated with Mediterranean mud volcanoes (84-86) ( Figure 10). All of the reported sedimentary compounds represent the first detailed report on the occurrence of alkyl diethers in a non-thermophilic setting, and the cyclopropyl and cyclohexyl moieties as observed in the series I and II components are unique for ether lipids [78].

Geobacillus sp.
Geobacillus is a Gram-positive bacterium, rod-shaped, and either paired or chained, and its optimum growth temperature is 65-70 degrees [123].
In 2017, the bacterium Geobacillus sp. E263, collected from a deep-sea hydrothermal vent in the East Pacific, was reported to contain a novel quinoid compound (166) (Figure 18). The research indicated that 2-amino-6-hydroxy- [1,4]-benzoquinone (166) could trigger the apoptosis of gastric cancer cells and breast cancer cells by inducing the accumulation of intracellular reactive oxygen species [124].

Halomonas sp.
Six new amphiphilic siderophores, loihichelins A-F (167-172) (Figure 19), were obtained from cultures of the deep-sea hydrothermal vent and sulfide rock bacterium Halomonas sp. LOB-5, which was collected from Marker 17 (depth of 1714 m) at Loihi Seamount. These siderophores showed a potential role in the promotion of Mn(II) and Fe(II) oxidation [125]. In addition, the reports on loihichelins A-F were the first publications on new natural products from ocean hydrothermal vent environments.

Comprehensive Overview and Outlook
We provide a comprehensive overview of the sources and bioactivities of the 182 natural products from the deep-sea extreme environments described up to March 2022. It was observed that cold-seep-derived compounds could be divided into four parts, namely, marine animals (24%), fungi (56%), bacteria (17%), and others (3%). In general, they mainly come from Aspergillus, Bathymodiolus, and Curvularia, according to the number of compounds (Figure 21), suggesting that these genera would be subjected to the focus of future research. The secondary metabolites isolated from hydrothermal vents are found in three parts. At the domain level, 78% of the natural products were derived from fungi, while 19% originated from bacteria, among which Aspergillus and Penicillium were the main source of natural products ( Figure 22).  By comparing and analyzing the activities of secondary metabolites derived from cold seeps and hydrothermal vents, it was found that almost 60% of the 182 compounds had biological activities, and their activities were diverse ( Figure 23). Among them, antibacterial and antitumor activities are reported most frequently. Some cold-seep-derived compounds also have antifungal and anti-epileptic activities, while hydrothermal vent-derived natural products also include plant growth regulation and oxidant activities. In general, that secondary metabolites derived from cold seeps and hydrothermal vents have novel and diverse biological activities may be due to their extreme and special environments.

Conclusions
There were 86 natural products isolated from cold seeps, while 96 secondary metabolites were isolated from hydrothermal vents. The sources of the compounds are represented by the genera Aspergillus sp., Penicillium sp., and so on. There are 90 new compounds among the 182 compounds. Around 60% of the deep-sea natural products were reported to possess bioactivity. For example, an exopolysaccharide, EPS364 (81), from cold-seep Vibrio alginolyticus 364, was investigated for its mechanism of inhibiting the growth and adhesion of liver cancer cells, which has proved to be the basis for a promising anticancer drug [75]. A hydrothermal vent-derived compound, deoxytryptoquivaline (142), showed strong binding to three important targets of SARS-CoV-2 and so has promise for being further investigated as a possible multitarget drug against COVID-19 [104]. These novel and diverse activities indicate that deep-sea extreme environments might facilitate the production of functional natural products. Moreover, the total synthesis or biosynthesis of some compounds was described. For example, the total synthesis pathway of (−)-6-epi-ophiobolin N (28), which was isolated from cold-seep sediments, was reported [27]. Dixiamycins A (77) and B (76), which were separated from a cold-seep environment sample, were reported in an unusual oxidative cyclization strategy for tailoring indolosesquiterpene biosynthesis [132] and in a possible route for total synthesis [69], respectively. These synthesized compounds either have a wide range of sources, diverse activities, or unique molecular skeletons rarely discovered in nature. This further indicates that the natural products derived from extreme environments, such as cold seeps and hydrothermal vents, have great potential and are a treasure to be further developed.

Conflicts of Interest:
The authors declare no conflict of interest.