Culturable Microorganisms Associated with Sea Cucumbers and Microbial Natural Products

Sea cucumbers are a class of marine invertebrates and a source of food and drug. Numerous microorganisms are associated with sea cucumbers. Seventy-eight genera of bacteria belonging to 47 families in four phyla, and 29 genera of fungi belonging to 24 families in the phylum Ascomycota have been cultured from sea cucumbers. Sea-cucumber-associated microorganisms produce diverse secondary metabolites with various biological activities, including cytotoxic, antimicrobial, enzyme-inhibiting, and antiangiogenic activities. In this review, we present the current list of the 145 natural products from microorganisms associated with sea cucumbers, which include primarily polyketides, as well as alkaloids and terpenoids. These results indicate the potential of the microorganisms associated with sea cucumbers as sources of bioactive natural products.


Introduction
Sea cucumbers are marine invertebrates that belong to the class Holothuroidea of the phylum Echinodermata. Globally, there are about 1500 species of sea cucumbers [1], which are divided into three subclasses: Aspidochirotacea, Apodacea, and Dendrochirotacea, and can be further divided into six orders: Aspidochirotida, Elasipodida, Apodida, Molpadida, Dendrochirotida, and Dactylochirotida [2].
Sea cucumbers are found in benthic areas and the deep sea worldwide [3]. They play an important role in marine ecosystems and occupy a similar niche to earthworms in terrestrial ecosystems [4]. Sea cucumbers obtain food by ingesting marine sediments or filtering seawater [5] and provide a unique, fertile habitat for a variety of microorganisms, including bacteria and fungi [6]. However, since most microorganisms are unculturable under conventional laboratory conditions [7], this review primarily focuses on culturable sea-cucumber-associated microorganisms.
Sea cucumbers have been used in medicine in Asia for a long time [8]. For example, an ointment derived from the sea cucumber Stichopus sp. 1 is used to treat back and joint pain in Malaysia [9]. Compounds isolated from sea cucumbers have a variety of biological and pharmacological activities, such as anticancer, antiangiogenic, anticoagulant/antithrombotic, antioxidant, antiinflammatory, antimicrobial, antihypertension, and radioprotective properties [10,11]. A phase II clinical trial of a sea cucumber extract, called TBL-12, has been conducted in patients with untreated asymptomatic myeloma [12]. Many studies have shown that the microorganisms associated with marine animals, such as sponges and ascidians, are the true producers of marine natural products [13][14][15][16]. Therefore, investigating sea-cucumber-associated microorganisms is essential for discovering new compounds with potential as novel active drugs. For the past 20 years, there has been an increasing effort made by researchers on diversity and bioactive compounds of microorganisms associated with sea cucumber. However, previously, no comprehensive review article as such has ever been published about this field. an increasing effort made by researchers on diversity and bioactive compounds of microorganisms associated with sea cucumber. However, previously, no comprehensive review article as such has ever been published about this field. This review discusses the biodiversity of the culturable microorganisms associated with sea cucumbers and the chemical structure and bioactive properties of the secondary metabolites produced by these microorganisms.

Culturable Microorganisms Associated with Sea Cucumbers
The sea cucumbers used for the isolation of culturable microorganisms belong to five genera (Holothuria, Cucumaria, Stichopus, Apostichopus, and Eupentacta) in four families (Holothuriidae, Stichopodidae, Cucumariidae, and Sclerodactylidae) ( Table 1). The dominant species is Apostichopus japonicus, which accounts for about 41% of the total sea cucumber population. In second place, Holothuria leucospilota accounts for about 27% of the total sea cucumber population (Table S1).
Sea cucumbers harbor a rich and diverse assortment of microorganisms. A variety of microorganisms, including bacteria and fungi, have been isolated from sea cucumbers. Most of the isolation conditions (medium, temperature, and aeration) are common. There

Culturable Microorganisms Associated with Sea Cucumbers
The sea cucumbers used for the isolation of culturable microorganisms belong to five genera (Holothuria, Cucumaria, Stichopus, Apostichopus, and Eupentacta) in four families (Holothuriidae, Stichopodidae, Cucumariidae, and Sclerodactylidae) ( Table 1). The dominant species is Apostichopus japonicus, which accounts for about 41% of the total sea cucumber population. In second place, Holothuria leucospilota accounts for about 27% of the total sea cucumber population (Table S1).
Sea cucumbers harbor a rich and diverse assortment of microorganisms. A variety of microorganisms, including bacteria and fungi, have been isolated from sea cucumbers. Most of the isolation conditions (medium, temperature, and aeration) are common. There are some papers on the diversity of culturable bacteria associated with sea cucumbers, which plays a very important role in understanding the digestion and diseases of sea cucumbers [4,6,17,25,33]. Because marine-derived fungi had shown potential to synthesize 3 of 21 pharmaceutical compounds with bioactivities, researchers usually directly isolate fungi associated with sea cucumbers for the separation of active natural products [21,28,29], except one paper about the diversity and bioactivity of fungi associated with sea cucumbers [22].
The anthraquinone compounds coniothyrinone A (10) and lentisone (11) were isolated from the fungus Trichoderma sp. associated with a sea cucumber that was collected from Chengshantou Island in the Yellow Sea in Weihai City, China [44]. Compounds 10 and 11 were isolated for the first time from fungi of the genus Trichoderma, and they had weak antiangiogenicc activity. Compound 10 showed pronounced antibacterial activity against three common marine pathogens, Vibrio parahaemolyticus, Vibrio anguillarum, and Pseudomonas putida, and the MIC values were 6.25, 1.56, and 3.13 µM, respectively. Compound 11 showed inhibitory effect against V. parahaemolyticus, V. anguillarum, and P. putida, with MIC values of 12.5, 1.56, and 6.25 µM, respectively [44].

Alkaloids
Alkaloids have been identified as a class of nitrogenous organic compounds derived from plants [55,56]; although they are most commonly found in plants, alkaloids can also be isolated from marine organisms and marine microorganisms [57,58].

Terpenoids
Terpenoids, which are widely found in nature and in numerous species, have various structures and are divided into monoterpenes (C 10 ), sesquiterpenes (C 15 ), diterpenes (C 20 ), and sesterterpenes (C 25 ) [61]. Although most known terpenoids have been isolated from plants [62], they are also produced by marine microorganisms [63].
The fungus Aspergillus sp. H30, derived from the sea cucumber Cucumaria japonica, which was collected from the South China Sea, produced a meroterpenoid called chevalone B (75) that exhibited weak antibacterial activity [36].
Three compounds, (+)-butyrolactone IV (129), butyrolactone I (130), and terrelactone A (131), were isolated from the fungus Aspergillus terreus, associated with the sea cucumber A. japonicus, collected from the Yellow Sea in China [47]. Compounds 129 and 130 showed moderate antiangiogenic activity when evaluated using a zebrafish assay. The inhibition ratio of compound 129, at a concentration of 100 µg/mL, was 43.4% and that of compound 130, at a concentration of 10 µg/mL, was 28.7% [47].
Cerebroside (141) was isolated from the fungus Alternaria sp., associated with sea cucumber from the sea near Zhifu Island in Yantai, China [28].

Summary of the Natural Products Isolated from Microorganisms Associated with Sea Cucumbers
From 2000 to 2021, 145 natural products were isolated from microorganisms associated with sea cucumbers. The numbers of compounds isolated in 2008, 2014, and 2020 were significantly higher than the numbers isolated in other years ( Figure 3). The compounds isolated from sea-cucumber-associated microorganisms are mainly polyketides, alkaloids, and terpenoids (Figures 4 and 5), which account for 28%, 18%, and 32% of the total isolated compounds, respectively ( Figure 4). Most of these compounds were isolated from sea-cucumber-associated fungi (Figure 4), and many of them have demonstrated bioactivities, including cytotoxicity, antimicrobial, enzyme-inhibiting, antiviral, and antiangiogenic activities, and the downregulation of ROS and NO production ( Figure 6).

Summary of the Natural Products Isolated from Microorganisms Associated with Sea Cucumbers
From 2000 to 2021, 145 natural products were isolated from microorganisms associated with sea cucumbers. The numbers of compounds isolated in 2008, 2014, and 2020 were significantly higher than the numbers isolated in other years ( Figure 3). The compounds isolated from sea-cucumber-associated microorganisms are mainly polyketides, alkaloids, and terpenoids (Figures 4 and 5), which account for 28%, 18%, and 32% of the total isolated compounds, respectively ( Figure 4). Most of these compounds were isolated from seacucumber-associated fungi (Figure 4), and many of them have demonstrated bioactivities, including cytotoxicity, antimicrobial, enzyme-inhibiting, antiviral, and antiangiogenic activities, and the downregulation of ROS and NO production ( Figure 6).

Conclusions
Sea cucumbers have been extensively utilized in medicine in Asia for a long time, and a variety of compounds with pharmacological activities have been isolated from sea cucumbers [10]. The actual producers of these marine natural products may be sea-cu-

Conclusions
Sea cucumbers have been extensively utilized in medicine in Asia for a long time, and a variety of compounds with pharmacological activities have been isolated from sea cucumbers [10]. The actual producers of these marine natural products may be sea-cu-

Conclusions
Sea cucumbers have been extensively utilized in medicine in Asia for a long time, and a variety of compounds with pharmacological activities have been isolated from sea cucumbers [10]. The actual producers of these marine natural products may be seacucumber-associated microorganisms. Sea cucumbers harbor a rich and diverse assortment of microorganisms. Over the past 20 years, seventy-eight genera of bacteria belonging to 47 families in four phyla, and 29 genera of fungi belonging to 24 families in the phylum Ascomycota have been cultured from sea cucumbers. A total of 145 natural products have been isolated from sea-cucumber-associated microorganisms. These compounds are polyketides, terpenoids, alkaloids, and others, and many have been shown to have various biological activities. Sea-cucumber-associated microorganisms have great potential for the production and isolation of high-value bioactive compounds.