Streptomyces : Still the Biggest Producer of New Natural Secondary Metabolites, a Current Perspective

: There is a real consensus that new antibiotics are urgently needed and are the best chance for combating antibiotic resistance. The phylum Actinobacteria is one of the main producers of new antibiotics, with a recent paradigm shift whereby rare actinomycetes have been increasingly targeted as a source of new secondary metabolites for the discovery of new antibiotics. However, this review shows that the genus Streptomyces is still the largest current producer of new and innovative secondary metabolites. Between January 2015 and December 2020, a signiﬁcantly high number of novel Streptomyces spp. have been isolated from different environments, including extreme environments, symbionts, terrestrial soils, sediments and also from marine environments, mainly from marine invertebrates and marine sediments. This review highlights 135 new species of Streptomyces during this 6-year period with 108 new species of Streptomyces from the terrestrial environment and 27 new species from marine sources. A brief summary of the different pre-treatment methods used for the successful isolation of some of the new species of Streptomyces is also discussed, as well as the biological activities of the isolated secondary metabolites. A total of 279 new secondary metabolites have been recorded from 121 species of Streptomyces which exhibit diverse biological activity. The greatest number of new secondary metabolites originated from the terrestrial-sourced Streptomyces spp.


Introduction
Indiscriminate use of antibiotics has led to a rise in antimicrobial resistance [1]. This dramatically increases the demands for research and discovery of new drugs and antibiotics. Natural products isolated from microorganisms as well as their semi-synthetic derivatives and synthetic analogues have historically been one of the most important sources of antibiotics [2]. Nature includes a large number of microbial species including at least 1.5 million fungi and as many as 5 × 10 12 distinct microbial species [1]. However, only a small fraction of about 250,000 to 300,000 living species, mainly in oceans and rainforests, have been identified and documented [1,3]. Worryingly, over the past few decades, there has been a significant decrease in the discovery of new natural product-derived medicines from 20 to 30 approved drugs per decade to only 3 to 4 newly marketed drugs, which has triggered many uncertainties in the medical industries [1]. This decline in translation from

Isolation Methods
Streptomycetes are ubiquitous in nature and have colonized a wide range of ecologically important terrestrial habitats. To isolate Streptomycete species from environmental samples, sophisticated research techniques and correlated studies are needed to mimic the native environmental conditions. A diverse suite of isolation methods has been used to successfully isolate new strains of Streptomycetes [36]. These include different methods of pre-treatment, the use of specific selective media under specific laboratory conditions, the use of supplements, and modification of the incubation time and temperature [37][38][39][40]. In particular, it is important to understand the physiological and biochemical conditions of the sampled environment. However, some bacterial cells cannot be cultured using culture-dependent methods or modern laboratory techniques and are referred to as "viable but not culturable" (VBNC) [41]. For this, high throughput sequencing metagenomic studies have shown that a large number of microbial communities remain unculturable from environmental samples [42].

Extreme Environments
Extreme environments are characterized by high salinity, high or low pH, arid conditions, low nutrient and oxygen content, high or low temperatures, and high exposure to UV rays, and which would be detrimental to "normal" conditions as required for human survival [43]. In recent years, researchers have focused more on the extreme environment as a potent source of new species of Streptomycetes with biological activity [44]. This group of bacteria has the ability to survive under multiple such conditions (polyextremophilic) because they possess distinctive adaptive characteristics such as the production of specific enzymes, switching between different metabolic modes (i.e., heterotrophy and autotrophy) and antibiosis [39].
Thirty-six new strains of Streptomyces were reported from various extreme environments between 2015 and 2020 ( Table 1). In order to isolate new strains of Streptomycetes from these samples collected from these environments, it is essential to consider various factors such as pH, temperature, nutrients required, as well as the use of pre-treatments. Culturing Streptomyces from samples collected from these environments does not necessarily require extreme conditions to obtain new Streptomyces spp. [45]. The pretreatments applied activate the endospores, which grow on the isolation media [46,47].
Selective pretreatment is carried out to eliminate Gram-negative, fastidious bacteria and unwanted microorganisms [12]. There are different pre-treatment methods used for environmental samples, including chemical pretreatment, physical pretreatment, and heating. Samples from extreme environments are subjected to various chemical pretreatments, including the addition of chemicals to the samples and dilution with deionized water. Physical treatments have also been applied to samples, which involves shaking the sample on a rotary shaker or a tumble shaker and heat treatment of the sample involving wet heat treatment or dry heat treatment. Streptomyces spores are very resistant to exogenous chemicals and temperature extremes due to the complex chemical compositions of their cell wall [70]. This is advantageous when performing pretreatments to selectively isolate Streptomyces spp. Some of the commonly used pretreatments for the isolation of Streptomyces from an extreme terrestrial environment include distilled water with added NaCl [38,52,71,72], air-drying [53,64,68] pretreatment by ultrasound [56,69] and agitation on a rotary stirrer [37,40,54,57,66,69]. Dry heat treatment and wet heat treatment are the most frequently used pretreatments. Notably, a combination of physical and chemical treatment [66] or two different physical treatment methods [69] have been shown to be significant and effectively isolate strains of Streptomyces. On the other hand, there are samples from extreme environments, which are not subjected to any pretreatment [51,54,60,61,65,73]. Turkey [38] Streptomyces taklimakanensis sp. nov. Desert Gauze's No. 1 medium 1 supplemented with Nystatin (100 mg mL −1 ) and nalidixic acid (50 mg mL −1 ) North-West China [40] Streptomyces alkaliterrae sp. nov.

Soil from peat swamp forest
Humic acid vitamin (HV) agar supplemented with nalidixic acid (25 µg mL −1 ) and nystatin (50 µg mL −1 ) Thailand [51] Streptomyces Medium for the isolation of alkalophilic actinomycetes at pH 10.0 or 11.0 (after autoclaving) . Na 2 CO 3 , or NaOH were separately sterilized and used for adjusting the pH India [8] Refer to Supplementary Table S1 for the composition of each media. The superscript ( 1,2 ) on some media indicates slight changes in the amount of ingredients used.

Symbionts
Microorganisms are the most common symbiotic partners of eukaryotes. They live either in mutualism with the host organism or may be parasitic to the host organism [77]. Streptomycetes are not only free species, but have also evolved to live in symbiosis with other animals, fungi and plants [78]. Similar to extreme conditions, Streptomyces have developed specific adaptive strategies and it is therefore very important to have knowledge of the sample environment to successfully isolate them [78]. In addition, it is important to know the different environmental factors such as pH, temperature, specific nutrients necessary for the preparation of isolation media [12].
Different parts of plants are sampled for the isolation of Streptomycetes, including tree bark [79], leaf litter [80], bulbil [81], roots [82][83][84], stem [85][86][87], fruits [88], seeds [89] and phylloplane [90] (Table 2). Mosses have also been recorded as a source of novel Streptomyces spp. [91]. Studies have proven that Streptomyces are very important for the growth and development of plants as they play an important role in nutrient uptake, have high absorption of tropospheric di-hydrogen and they also play an important role in forests by actively participating in biodegradation of biopolymers, which increases the fertility of forests soil [92].
Humic acid vitamin agar is typically used to isolate new Streptomyces spp. from plant samples [70,[77][78][79][80][81][82][83]. There are also other isolation media that have been used for isolation, including potato dextrose agar [93] and vitamin arginine agar [88] (Table 2). Different carbon sources have been used in these isolation media, including humic acid, glucose, dextrose, methanol and starch. The main sources of carbon-nitrogen in isolation media are beef or yeast extracts. Additionally, supplements such as nalidixic acid and cycloheximide may be added to the medium to reduce fungal and fastidious bacterial growth as shown in (Table 2).
Plant samples are often pretreated using a range of chemical, physical and thermal methods. Chemical pretreatment includes different concentrations of NaCl [82,88], sodium hypochlorite [89], Lodewyckx pretreatment [81] and hydrogen peroxide [81]. The only heat treatment applied is air-drying, which involves spreading the sample evenly on clean sheets and leaving it at room temperature to remove moisture from the sample [79,83]. This pretreatment is effective because desiccation selectively kills other common bacteria and fungi and activates Streptomyces spores [94]. In addition, sonic oscillation can also be applied [80].
Additionally, fungi and lichens are also a source of new Streptomycetes. For example, symbiotic Streptomycetes that reside on fungus farming ants are beneficial because they protect the fungal garden and ants against pathogenic fungi [78]. Streptomycetes have also been shown to suppress phytopathogenic fungi [95]. Streptomycetes are mutually important for fungi because they promote mycorrhizal symbiosis [96], which indirectly benefits plants. For successful isolation of Streptomycetes from fungal samples, vitamin arginine agar [97] and potato dextrose agar [98] (Table 2) were used. No pretreatment was applied to isolate the new Streptomycetes from fungi from the data reviewed. Streptomyces capparidis sp. nov.

Culture of mushroom Tremella fuciformis
Potato dextrose agar (PDA) medium (200 gpotato tissue, 20 g glucose, 20 g agar and 1000 mL deionized water, pH 5.6); cycloheximide (100 µg mL −1 ) China [98]  Furthermore, several studies have shown that insects are also an important host of Streptomyces. They are beneficial for the insect microbiome because they influence the production of metabolites and the biosynthetic potential to inhibit and resist pathogens [99]. The main areas of insects that studies generally focus on are the intestinal region [100,101], the cuticle [102], and the head region [93,[103][104][105][106] (Table 3). Studies have shown that Streptomycetes live in symbiosis with insects and strengthen their defensive mechanism by producing chemicals for ecological adaptation [99]. For example, endosymbiotic Streptomyces live in the antennal glands of female solitary wasps, where they are secreted as a white matter that the larvae absorb and wrap around their cocoon as a defense mechanism [107].
As with other samples, to isolate Streptomycetes from insects, different carbon sources (humic acid, starch, methylcellulose and oats) can be added to the isolation media [93,100,102,106] as well as cycloheximide and nalidixic acid have been the main supplements in isolation settings [102][103][104][105][106]. Unlike samples from plants and extreme environments, only two different pretreatment procedures have been applied to insect-derived samples from the data collected, which include physical pretreatment where the sample was shaken in a rotary shaker at 180 r.p.m. at 28 • C for 30 min [102][103][104][105][106], or a chemical pre-treatment where samples were surface sterilized in 70% ethanol [100].

Soil and Sediments
Actinomycetes represent up to 50% of the total population of Actinobacteria found in the soil (varies on different soil) [111]. They play a major role in the soil by biodegrading biopolymers such as lignocellulose, cellulose and hemicellulose [112][113][114]. Streptomyces also play an important role in biogeochemical cycles due to their high ability to produce the enzyme hydrogenase, which actively participates in the hydrogen cycle [92]. In addition, Streptomyces also influence the structure of soil microbial communities [115]; they are involved in the decomposition of plant litter and the formation of organic matter in the soil [116], as well as weathering of rocks [117]. New strains of Streptomycetes have been isolated from different soil samples, including rhizospheric soil [118][119][120], free soil [121][122][123], forest soil [124][125][126], wetland soil [127], and the sediments and the soil of the savannah. There were 16 new Streptomyces spp. isolated and reported from the rhizosphere (Table 4), which underlines the importance of Streptomyces spp. to plants. Streptomyces in the rhizosphere are essential for plant growth and development as they enhance root and shoot growth, biological nitrogen fixation, mineral solubilization, and they also serve as biological control agents against insects, pests and pathogens [10].
For the preferential isolation of Streptomycetes from soil, a number of isolation methods have been reported to enhance the growth of Streptomyces. Different isolation agars that could selectively isolate Streptomyces spp. have been used. Humic acid vitamin agar is prolific in the isolation of novel Streptomyces spp. isolates (Table 4). Other commonly used isolation media include starch casein agar and Gauze synthetic agar (Table 4).  China [137] Streptomyces tritici sp. nov.

Tropical forest soil
Starch-casein-nitrate agar within the pH range of 7.0-7.2 and supplemented with cycloheximide (50 µg mL −1 ), nystatin (50 µg mL −1 ) and nalidixic acid (20 µg mL −1 ) China [126]   Different pretreatment techniques have also been applied to samples, such as ultrasonic treatment [140], orbital shaking [166] and water bath sonication [163], and are crucial to activate Streptomycete spores and inhibit the growth of unwanted microbes [168] (Table 5). Subjecting the sample to heat treatment for 1 h at 120°C [146] is an important pre-treatment because actinomycete spores, including Streptomycetes, are more resistant to desiccation than other facultative and Gram-negative anaerobic bacteria [94]. In addition, the selective isolation of actinobacteria by suspending the sample in 1.5% phenol for 30 min is a chemical pretreatment used [138] that disrupts the cell wall of other common bacteria and fungi and improves the growth of Streptomyces [94]. Furthermore, treatment of samples by airdrying for a week [121,130,147] or two [129,136] is a commonly used pretreatment method (Table 5). Moreover, different carbon sources (cellulose, glucose, chitin, starch, dextrose, mannitol and proteose) and combined nitrogen-carbon sources (casein, yeast extract, malt extract, tryptone and peptone) have been added to the media for successful isolation of Streptomycetes [128,129,133,136] from soil sediments (see Table 4 and Supplementary Notes on Supplementary Table S1). Highlighted data shows that soil and sediment are the main source of new Streptomyces spp. in the terrestrial environment ( Figure 1), followed by the extreme environment and other symbionts.    Head of an ant sodium succinateasparagine agar at pH 7.2 and supplemented with cycloheximide (50 mg L −1 ) and nalidixic acid (20 mg L −1 ). [103] Head of an ant (Camponotus japonicus Mayr) sodium succinateasparagine agar at pH 7.2 and supplemented with cycloheximide (50 mg L −1 ) and nalidixic acid (20 mg L −1 ) Humic acid vitamin (HV) agar containing cycloheximide (50 mg L −1 ) and nalidixic acid (25 mg L −1 )

Pre-Treatment Terrestrial Source Isolation Medium Incubation Time/ Temperature References
Subjected to a seven-step surface sterilization procedure: a 60 s wash in sterile tap water containing cycloheximide (100 mg L −1 ) and nalidixic acid (20 mg L −1 ), followed by a wash in sterile water, a 5 min wash in 5% (v/v) NaCl, a 10 min wash in 2.5% (w/v) Na 2 S 2 O 3 , a 5 min wash in 75% (v/v) ethanol, a wash in sterile water and a final rinse in 10% (w/v) NaHCO 3 for 10 min, and then the rinsed root sample was Dried at 100 • C for 15 min. To summarize, the data collected have shown that soil and sediments are prominent sources of novel Streptomyces spp. from the terrestrial environment as shown in Figure 1 below, followed by extreme environments and other symbionts.

Novel Streptomycetes Species Isolated from Marine Environments
Despite the fact that marine Actinobacteria are less studied than terrestrial Actinobacteria, studies have revealed that marine sources of Actinomycetes harbor some of the most important bioactive metabolites for industrial and medical applications [12,169]. However, it is still not clear whether these organisms, in particular Streptomyces spp., are also present in terrestrial sources or exclusive to marine environments [170,171]. Several studies have found that actinobacterial spores are generally dormant and wash away from terrestrial ecosystems in runoff and rivers to the ocean floor and remain dormant [171]. There, at the bottom of the ocean, they will often be exposed to harsh conditions such as high pressure, high salinity and nutrient deficiency. As a result, they will evolve genetically over time and produce a secondary metabolite profile distinct from terrestrial actinobacteria [171]. However, to date, knowledge about the chemistry, distribution and biodiversity of marine Streptomyces and other genera of marine Actinobacteria is still limited [172]. In addition, marine ecosystems are extremely dynamic and it is very difficult to access varying ocean depths for sampling [173]. However, after the development of SCUBA, microbial ocean studies accelerated for the discovery of new drugs [173]. Marine Streptomyces are not only found in seawater and sediments, but also in a wide range of biological sources, including sponges, algae, corals, fish, jellyfish and mangroves [174]. In this review, only two marine sources were reported as sources of new Streptomyces spp. namely marine invertebrates and sediments.

Isolation Methods
Actinobacteria adapt well and successfully colonize different marine ecosystems where they exhibit a wide range of morphologically, physiologically and metabolic diver- To summarize, the data collected have shown that soil and sediments are prominent sources of novel Streptomyces spp. from the terrestrial environment as shown in Figure 1 below, followed by extreme environments and other symbionts.

Novel Streptomycetes Species Isolated from Marine Environments
Despite the fact that marine Actinobacteria are less studied than terrestrial Actinobacteria, studies have revealed that marine sources of Actinomycetes harbor some of the most important bioactive metabolites for industrial and medical applications [12,169]. However, it is still not clear whether these organisms, in particular Streptomyces spp., are also present in terrestrial sources or exclusive to marine environments [170,171]. Several studies have found that actinobacterial spores are generally dormant and wash away from terrestrial ecosystems in runoff and rivers to the ocean floor and remain dormant [171]. There, at the bottom of the ocean, they will often be exposed to harsh conditions such as high pressure, high salinity and nutrient deficiency. As a result, they will evolve genetically over time and produce a secondary metabolite profile distinct from terrestrial actinobacteria [171]. However, to date, knowledge about the chemistry, distribution and biodiversity of marine Streptomyces and other genera of marine Actinobacteria is still limited [172]. In addition, marine ecosystems are extremely dynamic and it is very difficult to access varying ocean depths for sampling [173]. However, after the development of SCUBA, microbial ocean studies accelerated for the discovery of new drugs [173]. Marine Streptomyces are not only found in seawater and sediments, but also in a wide range of biological sources, including sponges, algae, corals, fish, jellyfish and mangroves [174]. In this review, only two marine sources were reported as sources of new Streptomyces spp. namely marine invertebrates and sediments.

Isolation Methods
Actinobacteria adapt well and successfully colonize different marine ecosystems where they exhibit a wide range of morphologically, physiologically and metabolic diversity. Marine Streptomyces may require special growth conditions, which require knowledge and experience to prepare isolation media. To mimic such marine environments, researchers must have in-depth knowledge of the different abiotic factors in the sampled environment to successfully isolate new marine Streptomycetes. Since marine habitats are halophilic environments, salt supplements are important ingredients that are added to the isolation medium to provide osmotic values similar to seawater [175]. In addition, the NaCl added to the medium serves to protect the halophilic bacterial cells from changes in osmotic pressure between the external and internal environment of the bacteria [176]. Similar to the terrestrial environment, culture independent studies using high throughput sequencing are used to study marine microbial communities that are not culturable in the laboratory. At the same time, the knowledge gained over time from these culture independent studies on the morphological and physical characteristics of marine Streptomycetes has led to better strategies for growth and culture media to recover these previously uncultured Streptomyces [36,177].

Invertebrates
About 89% of organisms living in the marine environment are invertebrates [178]. This is clearly reflected by the high number of microbial symbionts associated with this group. These microbial symbionts have produced medically important natural products and studies have shown that Actinomycetes are the most prolific producers of marine novel antibiotics with about 80% of reported compounds from marines' microorganisms originating from actinomycetes [178]. This can be seen by the great diversity of actinobacteria colonizing marine habitats. Streptomyces are also known to be abundant in marine habitats [167,173]. For successful isolation of marine Streptomyces, different concentrations of sodium salts were added to the medium of different marine samples. Some of the isolation media supplemented, 50% (v/v) sea water [179,180], 3% NaCl [181,182] and even up to 70% seawater can be added to the growth media [183]. In addition, isolation media are made specifically to isolate Streptomycetes spp. from marine samples [182]. In addition to humic acid isolation agar [179,184], other isolation agars such as actinomycete isolation agar [181], inorganic salt-starch agar [183] and starch and casein [180] have also been used to isolate new Streptomycetes from the marine environment. In comparison, humic acid and starch are the only two sources of carbon supplemented reported for us for the isolation of marine invertebrate Streptomyces [179,181,182]. Among these supplements, casein is the main source of carbon-nitrogen [180][181][182]. Other isolation media have been summarized in Table 6. Notably, a sample underwent chemical pretreatment using 3% NaCl [184]. Three percent NaCl has proven to be the optimum concentration that supports actinobacterial growth compared to concentrations lower or higher than 3% [185]. Moreover, the addition of NaCl to the medium selectively inhibits other fastidious microbes by altering the ionic strength of the medium, thus generating an osmotic shock for the microbes resulting in dehydration and growth retardation resulting in cell death [186,187]. Furthermore, NaCl addition also disrupts the solubility of oxygen by disrupting enzymatic functions, thus reducing the growth rate of fastidious bacteria and fungi [188]. and nalidixic acid (20 mg mL −1 ).
China [180] Refer to Supplementary Table S1 for the composition of each media.

Sediments
Marine sediments represent 63.5% of the Earth's surface [190], constituting inorganic and organic products from erosion of landmasses, volcanic activities and biochemical activities in the ocean [191]. Apparently, this ecosystem is the most under-sampled marine habitat [192], presumably due to the inaccessibility of the deep-sea floor. Marine sediments have a remarkable diversity of microbial communities constituting approximately 0.18-3.6% of the Earth's total living biomass [193,194].
In this context, sediments refer to shallow- [195] to deep-water sediments [196], sandy beaches [197] and mangrove sediments [34,198,199] (Table 7). Reports have suggested that these habitats harbor a great number of microbial species, which are still underexplored [12,172]. This is clearly reflected from the data collected at the time of writing where deep-sea sediments are the least sampled for Streptomyces (Table 7) compared to sediments from mangrove forests. A total of 20 new Streptomyces species were isolated from marine sediments between 2015 and 2020 (Table 7) Microorganisms in seawater play an important role in the marine food chain by recycling and breaking down organic matter and other biochemical processes [200]. From this study, 11 out of 20 new Streptomycetes spp. from marine sediments were isolated from mangrove habitats ( Table 7).
Streptomycetes from marine sediment samples were isolated using several isolation media. One of the most widely used media is the International Streptomyces Project (ISP2) media [34,[201][202][203][204] (Table 7). As seen with marine invertebrate-derived samples, NaCl is also an important ingredient in the isolation medium of marine sediments. These sodium sources include both fresh and aged seawater [196,199] or NaCl solution [205,206] to give the medium an ionic strength similar to that of the sampled environment. In addition, different carbon sources (chitin, dextrose, glucose and soluble starch) combined with carbon-nitrogen sources (casein, peptone, malt extract, yeast extract and tryptone) were added to the medium to successfully isolate them. Furthermore, the media were also supplemented with nystatin and cycloheximide [196,197,199,204,[207][208][209][210]. China [199] Streptomyces qaidamensis sp. nov. Sand Gause's synthetic agar medium 2 at pH 7.2 supplemented with nalidixic acid (25 µg mL −1 ) China [197] Streptomyces monashensis sp. nov. Mangrove soil ISP2 agar Malaysia [34] Streptomyces euryhalinus sp. nov. Sediment in a mangrove forest Enrichment medium at pH 7.5 India [198] Streptomyces colonosanans sp. nov. Sediment in mangrove soil ISP 2 medium supplemented with cycloheximide (50 µ g mL −1 ) and nalidixic (20 µg mL −1 )
More than 70% of the new Streptomyces spp. were isolated from marine sediments and only 7 (29%) of the new Streptomyces spp. from the marine environment were isolated from marine invertebrates (Figure 2).
More than 70% of the new Streptomyces spp. were isolated from marine sediments and only 7 (29%) of the new Streptomyces spp. from the marine environment were isolated from marine invertebrates (Figure 2). The pre-treatment of samples is also an important procedure for marine sediments, with heat being the most commonly applied pre-treatment (Table 8). Of the 27 novel Streptomyces spp. isolated from marine samples, less than half of the samples, constituting only 11 new Streptomyces spp., have undergone some form of pre-treatment. The pre-treatments carried out were either treatment by the wet method [201,208,213] or by dry heat [199,207]. Chemical pre-treatment is used when a source of sodium is added such as 3% NaCl [205] or 3.3% seawater [210]. Sodium modifies the tonicity of the isolation medium and thus selectively inhibits the growth of unwanted microbes [186]. The pre-treatment of samples is also an important procedure for marine sediments, with heat being the most commonly applied pre-treatment (Table 8). Of the 27 novel Streptomyces spp. isolated from marine samples, less than half of the samples, constituting only 11 new Streptomyces spp., have undergone some form of pre-treatment. The pretreatments carried out were either treatment by the wet method [201,208,213] or by dry heat [199,207]. Chemical pre-treatment is used when a source of sodium is added such as 3% NaCl [205] or 3.3% seawater [210]. Sodium modifies the tonicity of the isolation medium and thus selectively inhibits the growth of unwanted microbes [186]. SM3 agar Gauze s medium 2 at pH 7.0 supplemented with filter sterilized solutions of cycloheximide (50 µ g mL −1 , nalidixic acid (10 µ g mL −1 , novobiocin (10 µ g mL −1 and nystatin (50 µ g mL −1 ) and supplemented with sterile seawater (3.3%, w/v)

Summary
In summary, the data collected have shown that terrestrial environment has been the source of a higher number of novel Streptomyces spp. (80%) compared to the marine environment (20%) as shown in Figure 3.

Summary
In summary, the data collected have shown that terrestrial environment has been the source of a higher number of novel Streptomyces spp. (80%) compared to the marine environment (20%) as shown in Figure 3.

Streptomyces as Source of Antibiotics
Streptomyces spp. have the genetic capacity to produce an average of 30 secondary metabolites [99], making them the most prolific producers of antibiotics. This genus produces about 80% of the total antibiotics sourced from the phylum Actinobacteria [11,16] and produces two thirds of the antibiotics from natural sources that are currently available for public use [7]. The production of secondary metabolites by Streptomycetes is abundant; when resources are limited, they produce aerial hyphae, which divide into spores that can withstand adverse conditions [214]. This is an important factor for successful colonization by Streptomycetes in normal and extreme environments. Strepomycete secondary metabolites protect the vegetative bacterial cell by sequestering heavy metals such as iron, protecting against UV rays, inhibiting other competitors and playing a major role in quorum sensing [15,99].
The discovery of Streptomycetes as a source of antibiotics began in 1940 when the antibiotic actinomycin, commonly used as chemotherapeutic agent for the treatment of a variety of cancers, was discovered, filling the void left by penicillin that was ineffective against tuberculosis and certain Gram-negative pathogens [215]. Two years later, streptothricin was isolated from Streptomyces lavendulae [216] followed by streptomycin from Streptomyces griseus [217]. Approximately 12,400 bioactive compounds used clinically and in agriculture were produced by the genus Streptomyces throughout the years, such as the immunosuppressive tacrolimus produced by S. tsukubaensis, the anti-tumor platenolides were obtained from S. platensis, and the insecticide avermectin, as a few examples [6,218]. However, there was a sharp decline in the discovery of such bioactive compounds from 1985 to 2006 [2]. In addition, in the past 30 years, only two Streptomyces-sourced antibiotics have been approved for clinical treatment of systemic infections [16]. This has led researchers to focus their attention on the production of bioactive compounds from other genera of actinobacteria, commonly referred to as "rare Actinomycetes" [219]. A recent study has shown that Streptomyces spp. are no longer considered a potential source of new antibiotics given that no compound isolated from Streptomycetes underwent clinical trials between 2007 and mid-2013 [12], while three compounds isolated from rare marine Actinomycetes are currently undergoing clinical trials [12]. However, data collected from 2015 to 2020 show that a high number of new secondary metabolites were isolated from 20% 80%

Marine Environment
Terrestrial Environment Figure 3. Distribution of novel Streptomyces spp. recorded in terrestrial and marine environment between 2015 and 2020, with 80% sourced from terrestrial environments and 20% reported from the marine environment.

Streptomyces as Source of Antibiotics
Streptomyces spp. have the genetic capacity to produce an average of 30 secondary metabolites [99], making them the most prolific producers of antibiotics. This genus produces about 80% of the total antibiotics sourced from the phylum Actinobacteria [11,16] and produces two thirds of the antibiotics from natural sources that are currently available for public use [7]. The production of secondary metabolites by Streptomycetes is abundant; when resources are limited, they produce aerial hyphae, which divide into spores that can withstand adverse conditions [214]. This is an important factor for successful colonization by Streptomycetes in normal and extreme environments. Strepomycete secondary metabolites protect the vegetative bacterial cell by sequestering heavy metals such as iron, protecting against UV rays, inhibiting other competitors and playing a major role in quorum sensing [15,99].
The discovery of Streptomycetes as a source of antibiotics began in 1940 when the antibiotic actinomycin, commonly used as chemotherapeutic agent for the treatment of a variety of cancers, was discovered, filling the void left by penicillin that was ineffective against tuberculosis and certain Gram-negative pathogens [215]. Two years later, streptothricin was isolated from Streptomyces lavendulae [216] followed by streptomycin from Streptomyces griseus [217]. Approximately 12,400 bioactive compounds used clinically and in agriculture were produced by the genus Streptomyces throughout the years, such as the immunosuppressive tacrolimus produced by S. tsukubaensis, the anti-tumor platenolides were obtained from S. platensis, and the insecticide avermectin, as a few examples [6,218]. However, there was a sharp decline in the discovery of such bioactive compounds from 1985 to 2006 [2]. In addition, in the past 30 years, only two Streptomyces-sourced antibiotics have been approved for clinical treatment of systemic infections [16]. This has led researchers to focus their attention on the production of bioactive compounds from other genera of actinobacteria, commonly referred to as "rare Actinomycetes" [219]. A recent study has shown that Streptomyces spp. are no longer considered a potential source of new antibiotics given that no compound isolated from Streptomycetes underwent clinical trials between 2007 and mid-2013 [12], while three compounds isolated from rare marine Actinomycetes are currently undergoing clinical trials [12]. However, data collected from 2015 to 2020 show that a high number of new secondary metabolites were isolated from Streptomycete species. A total of 279 new natural products with diverse bioactivities were discovered from 121 Streptomyces spp. between 2015 and 2020.

Terrestial Streptomyces as a Source of Antibiotics
Actinomycetes from terrestrial environments produce a large number of bioactive compounds. The data collected showed that despite the decrease in bioactive compounds isolated from Streptomycetes in terrestrial samples, a high number of new secondary metabolites are still isolated from this environment. Seventy-three terrestrial Streptomyces spp. were isolated between 2015 and 2020 as sources of 173 new bioactive compounds, the majority of which show significant antibiotic bioactivity.

Marine Streptomyces as a Source of Antibiotics
Marine natural products (MNPs) are also a prolific source of novel antibiotics [220]. Actinomycete sources alone account for approximately 80% of novel antibiotics derived from the marine environment [178]. Studies targeting specific and understudied marine microbial phyla can result in a greater likelihood of finding specific marine compounds, since most compounds isolated from marine microorganisms are closely related to compounds isolated from terrestrial microorganisms [221]. Unfortunately, the marine environment is one of the most under-explored environments, but still holds as a promising source of new and innovative natural products [220], which is clearly illustrated in Figure 4. Nair et al.
(2020), highlighting the urgent need to explore marine habitats for new microbial bioactive compounds [220]. A total of 106 new bioactive compounds have been discovered from 48 Streptomyces spp. sourced from the marine environment between 2015 and 2020.
Microbiol. Res. 2022, 13, FOR PEER REVIEW 24 Streptomycete species. A total of 279 new natural products with diverse bioactivities were discovered from 121 Streptomyces spp. between 2015 and 2020.

Terrestial Streptomyces as a Source of Antibiotics
Actinomycetes from terrestrial environments produce a large number of bioactive compounds. The data collected showed that despite the decrease in bioactive compounds isolated from Streptomycetes in terrestrial samples, a high number of new secondary metabolites are still isolated from this environment. Seventy-three terrestrial Streptomyces spp. were isolated between 2015 and 2020 as sources of 173 new bioactive compounds, the majority of which show significant antibiotic bioactivity.

Marine Streptomyces as a Source of Antibiotics
Marine natural products (MNPs) are also a prolific source of novel antibiotics [220]. Actinomycete sources alone account for approximately 80% of novel antibiotics derived from the marine environment [178]. Studies targeting specific and understudied marine microbial phyla can result in a greater likelihood of finding specific marine compounds, since most compounds isolated from marine microorganisms are closely related to compounds isolated from terrestrial microorganisms [221]. Unfortunately, the marine environment is one of the most under-explored environments, but still holds as a promising source of new and innovative natural products [220], which is clearly illustrated in Figure  4. Nair et al. (2020), highlighting the urgent need to explore marine habitats for new microbial bioactive compounds [220]. A total of 106 new bioactive compounds have been discovered from 48 Streptomyces spp. sourced from the marine environment between 2015 and 2020.

New Compounds from Streptomyces spp. with Bioactivity
All new compounds from Streptomyces spp. as reported between 2015 and 2020 are reviewed below. Despite the fact that a high number of new compounds were reported during the timeframe covered by this review, only a selection of the structures with significant biological activity as stated in their respective articles are presented.

New Compounds from Streptomyces spp. with Bioactivity
All new compounds from Streptomyces spp. as reported between 2015 and 2020 are reviewed below. Despite the fact that a high number of new compounds were reported during the timeframe covered by this review, only a selection of the structures with significant biological activity as stated in their respective articles are presented.

Antibacterial Activity
Infections by pathogenic bacteria are a leading cause of death worldwide. Unfortunately, the resistance to antibiotics acquired by pathogenic bacteria has led to an increasing number of untreatable bacterial diseases [220]. Thus, the need to scour natural habitats for new antibacterial compounds has increased. Between 2015 and 2020, 92 new compounds were reported from 39 Streptomyces spp. with antibacterial activity against a wide range of bacterial pathogens, including two of the multidrug-resistant pathogens (Table 9). This shows that Streptomyces spp. are undoubtedly still the current leading producer of antibacterial agents. Figure 5 showed some examples of new compounds isolated from Streptomyces spp. with significant antibacterial activity. Table 9. Novel/new antibacterial bioactive compounds isolated from Streptomyces spp. between 2015 and 2020.

Novel/New Antibacterial Compound
Chemical Class Antibacterial Activity Sample Environment Ref.

Anticancer Activity
Cancer is a major health crisis and is also a major cause of death globally [246]. Scientific fields devoted to treating cancer have developed rapidly and discoveries in therapeutic methods such as chemotherapy, immunotherapy, radiotherapy and surgery offer effective treatments for cancerous tumors [260]. Natural resources have a high potential in the production of anticancer compounds. A suitable source of anticancer compounds is from Streptomyces spp. [260]. During the study period of this review, a total of 82 new anticancer compounds were isolated from 38 Streptomyces spp. (Table 10). Figure 6 showed some examples of new compounds isolated from Streptomyces spp. with significant anticancer activity.

Anticancer Activity
Cancer is a major health crisis and is also a major cause of death globally [246]. Scientific fields devoted to treating cancer have developed rapidly and discoveries in therapeutic methods such as chemotherapy, immunotherapy, radiotherapy and surgery offer effective treatments for cancerous tumors [260]. Natural resources have a high potential in the production of anticancer compounds. A suitable source of anticancer compounds is from Streptomyces spp. [260]. During the study period of this review, a total of 82 new anticancer compounds were isolated from 38 Streptomyces spp. (Table 10). Figure 6

Enzyme Inhibitor/Inducer Activity
Streptomycetes also produce metabolites with enzyme modulatory activity. There were 27 new compounds derived from 11 Streptomyces spp. during the period of study and these compounds exhibit both enzymes inducing and/or inhibitory activity (Table 11).  Figure 7 showed some examples of new compounds isolated from Streptomyces spp. with significant enzyme modulatory activity.

Antifungal
New antifungal drugs are urgently needed to alleviate infectious diseases caused by pathogenic fungi. At present, drug resistant fungi are evolving continuously, so the need to find new antifungal drugs is increasing. For example, the multi-drug resistant fungi Candida albicans, Aspergillus fumigatus, and Candida glabrata have all been shown to be resistant to azole drugs after their drug binding sites mutated, thereby reducing binding affinity. In addition, other therapeutic antifungal drugs were also ineffective against these multi-drug resistant species. [293][294][295][296]. Interestingly, several compounds obtained from Streptomyces spp. have antifungal properties that could be utilized in fighting against drug-resistant and fungal pathogens. 33 new antifungal compounds were reported from nine Streptomyces spp. between 2015 and 2020 (Table 12). Figure 8 showed some examples of new compounds isolated from Streptomyces spp. with significant antifungal activity.

Other Biological Activity
There were also new natural products from Streptomycetes, which show other biological activities as described in Table 13 below. A total of 18 different bioactivities were recorded from 23 Streptomyces spp., which produced 45 bioactive compounds in total between 2015 and 2020 (Table 13). Figure 9 showed some examples of new compounds isolated from Streptomyces spp. with other biological activity.   Trienomycin J−L Macrolide Inhibited nitric oxide production Soil moss [301] Herbicidin L Adenosine-nucleoside Antiparasitic Soil [269] Simamycin Prenylated nucleoside Induces differentiation of preadipocytes into matured adipocytes Soil [307] Oxachelin CSpoxazomicin D Oxazoline carboxamide, peptide Potent neuroprotectives Soil [308] Aotaphenazine Phenazine Overcome tumor necrosisFactor-related apoptosis-inducing ligand (TRAIL).
Soil [309] Aotaphenazine Phenazine Enhances the levels of apoptosis inducing proteins Soil [309] Inubosin B Acridine alkaloid Ngn2 promoter activity and induces mRNA expression of genes related to neural stem cell differentiation.

Antifungal
New antifungal drugs are urgently needed to alleviate infectious diseases caused by pathogenic fungi. At present, drug resistant fungi are evolving continuously, so the need to find new antifungal drugs is increasing. For example, the multi-drug resistant fungi Candida albicans, Aspergillus fumigatus, and Candida glabrata have all been shown to be resistant to azole drugs after their drug binding sites mutated, thereby reducing binding affinity. In addition, other therapeutic antifungal drugs were also ineffective against these

Other Biological Activity
There were also new natural products from Streptomycetes, which show other biological activities as described in Table 13 below. A total of 18 different bioactivities were recorded from 23 Streptomyces spp., which produced 45 bioactive compounds in total between 2015 and 2020 (Table 13). Figure 9 showed some examples of new compounds isolated from Streptomyces spp. with other biological activity.  Streptomyces spp. are still a potential source of new and interesting secondary metabolites with diverse bioactivities. The significant biological activity of new secondary metabo-lites obtained from the genus Streptomycetes is dominated by antibacterial activity, followed by anticancer, antifungal and enzyme modulatory activities as shown in Figure 10.

Summary
Streptomyces spp. are still a potential source of new and interesting secondary metabolites with diverse bioactivities. The significant biological activity of new secondary metabolites obtained from the genus Streptomycetes is dominated by antibacterial activity, followed by anticancer, antifungal and enzyme modulatory activities as shown in Figure  10.

Conclusions and Future Perspectives
During the six years of study (January 2015 to December 2020), a high number of new Streptomyces spp. were isolated from terrestrial and marine environments using different isolation procedures. This includes different pre-treatment methods such as chemical, physical and thermal treatments that were used with various selective isolation media to promote the isolation of a total of 135 new Streptomycetes. From this total, 108 new Streptomyces spp. (80%) were sourced from terrestrial habitats and 27 (20%) from marine habitats. Additionally, a total of 279 new natural products have been isolated from 121 Streptomyces spp. with diverse biological activities. A high number (91) of the new natural products shows antibacterial activity followed by anticancer and antifungal effects.
Streptomyces species are undoubtedly a potential source of pharmaceutically important drugs. Despite the tireless efforts of Scientists to discover bioactive metabolites from other prokaryotic sources, including rare actinomycetes, and synthetic drug production, species of the genus Streptomyces are still recognized as a major producer of microbial metabolites. A thorough knowledge and understanding of microbial physiology and metabolism is essential for the successful isolation of novel Streptomyces spp. Culture independent studies have also shown that there are large numbers of Streptomycetes and new natural products that are remain undiscovered under typical laboratory conditions [14,157]. This should be a guide for the future selective isolation procedure to target these Streptomyces spp. and activate their silent biosynthetic gene clusters, which are not expressed under typical laboratory conditions, for new drug discovery. In addition, more effort should be invested in the marine environment for the discovery of new Streptomyces spp. and their associated bioactivities.

Conclusions and Future Perspectives
During the six years of study (January 2015 to December 2020), a high number of new Streptomyces spp. were isolated from terrestrial and marine environments using different isolation procedures. This includes different pre-treatment methods such as chemical, physical and thermal treatments that were used with various selective isolation media to promote the isolation of a total of 135 new Streptomycetes. From this total, 108 new Streptomyces spp. (80%) were sourced from terrestrial habitats and 27 (20%) from marine habitats. Additionally, a total of 279 new natural products have been isolated from 121 Streptomyces spp. with diverse biological activities. A high number (91) of the new natural products shows antibacterial activity followed by anticancer and antifungal effects.
Streptomyces species are undoubtedly a potential source of pharmaceutically important drugs. Despite the tireless efforts of Scientists to discover bioactive metabolites from other prokaryotic sources, including rare actinomycetes, and synthetic drug production, species of the genus Streptomyces are still recognized as a major producer of microbial metabolites. A thorough knowledge and understanding of microbial physiology and metabolism is essential for the successful isolation of novel Streptomyces spp. Culture independent studies have also shown that there are large numbers of Streptomycetes and new natural products that are remain undiscovered under typical laboratory conditions [14,157]. This should be a guide for the future selective isolation procedure to target these Streptomyces spp. and activate their silent biosynthetic gene clusters, which are not expressed under typical laboratory conditions, for new drug discovery. In addition, more effort should be invested in the marine environment for the discovery of new Streptomyces spp. and their associated bioactivities.