Exploring Structural Diversity of Microbe Secondary Metabolites Using OSMAC Strategy: A Literature Review

Microbial secondary metabolites (MSMs) have played and continue to play a highly significant role in the drug discovery and development process. Genetically, MSM chemical structures are biologically synthesized by microbial gene clusters. Recently, however, the speed of new bioactive MSM discovery has been slowing down due to consistent employment of conventional cultivation and isolation procedure. In order to alleviate this challenge, a number of new approaches have been developed. The strategy of one strain many compounds (OSMAC) has been shown as a simple and powerful tool that can activate many silent biogenetic gene clusters in microorganisms to make more natural products. This review highlights important and successful examples using OSMAC approaches, which covers changing medium composition and cultivation status, co-cultivation with other strain(s), adding enzyme inhibitor(s) and MSM biosynthetic precursor(s). Available evidences had shown that variation of cultivation condition is the most effective way to produce more MSMs and facilitate the discovery of new therapeutic agents.


INTRODUCTION
Microbial secondary metabolites (MSMs) have been recognized as the primary source of new compounds for drug discovery and development (Gunatilaka, 2006;Rateb et al., 2011b;Deng et al., 2013). Traditional chemical investigation of microorganism mainly focuses on extraction and isolation of structurally and highly active compounds from fermentation broth and mycelium. However, these processes are becoming inefficient due to high rate of the re-discovery of known MSMs. It is commonly believed that a large portion of microbial gene clusters are silenced under standard fermentation conditions (Scherlach and Hertweck, 2009;Wasil et al., 2013). By mining microbial genome and targeting biosynthetic gene clusters of MSM, researchers can exploit the potential of microbes in a more objective way, such as knocking down, introduction or heterologous expression of microbial genes, regulation of promoters, induction of mutations, or changing cultivation conditions to stimulate MSM genes expression (Schneider et al., 2008). Variation of cultivation condition has been deemed to be the simplest and most effective strategy, which is termed as "one strain many compounds (OSMAC)" by professor Zeeck and coworkers (Bode et al., 2002). On basis of extensive literature search, important and successful examples using OSMAC strategy are summarized in this review, which consists of variation of medium, changing cultivation condition, cocultivation with other strain(s), adding epigenetic modifier(s) or biosynthetic precursor(s).

VARIATION OF MEDIUM
Culture medium has a greater effect not only on microbe growth but also on metabolism. It has been reported that C/N ratio, salinity, and metal ion can regulate the degree and pattern of MSM gene expression and result in production of various secondary metabolites.

Medium Composition
Generally, carbon and nitrogen sources are major components in the culture medium. The carbon source not only provides the basis for building biomass and represents the source of energy for all heterotrophs but also delivers carbon units for secondary metabolites. The nitrogen source is required for the synthesis of essential proteins and nucleic acids, and likewise N-containing units for secondary metabolites. The type of used carbon and nitrogen sources is known to have a significant influence on microbial secondary metabolism (Ruiz et al., 2009;Singh et al., 2017). Furthermore, the C/N ratio is one of important factors that affect fermentation products (Karakoç and Aksöz, 2004;Brzonkalik et al., 2012;Dinarvand et al., 2013). Notably, the consumption of carbon and nitrogen-based medium components can greatly affects the pH of the cultivation broth, e.g., by formation of organic acids or the accumulation of basic ammonium. Thus, microorganisms cultured in medium containing different components may exhibit differently adapted metabolism and express specific sets of biosynthetic genes, which produced a differential biosynthesis of specialized metabolites (Ma et al., 2009).
One marine-derived strain Asteromyces cruciatus 763 was shown to produce a new pentapeptide lajollamide A (1), when cultivated in the Czapek-Dox broth contained arginine solely as nitrogen source rather than NaNO 3 , which was missed in the normal Czapek-Dox medium (Gulder et al., 2012). One sediment-derived Aspergillus niger BRF-074 produced a novel furan ester derivative (2), a compound has toxicity acidity against HCT-116 cancer cell line (Uchoa et al., 2017), when cultivated in MPDB (malt peptone dextrose broth) medium. But this compound failed to appear in PDB (potato dextrose broth) or PDYB (potato dextrose yeast broth) media. A fungus Aspergillus sp. from Waikiki Beach (Honolulu, HI), generated six isotopically labeled metabolites (3-8) when grown on the deuterium-enriched Czapek broth , whereas this strain was found to metabolite a novel prenylated indole alkaloid, waikialoid A (9) when cultivated in PDB medium. Bioassay results indicated that compound 9 possessed potent inhibitory effect on biofilm formation of Candida albicans with an IC 50 value of 1.4 µM (Wang Q.X. et al., 2012).
Five new polyketides (10)(11)(12)(13)(14) were detected in the crude extract of rice-based medium of a marine-derived Cladosporium sphaerospermum 2005-01-E3 . Another two new hybrid polyketides (15)(16) were accessed when the same strain was fermented on the soybean flour (Yu et al., 2015). The organic extract of Dothideomycete sp. CRI7 was elaborated by four comparative medium. The strain growing in PDB made with potato tubers led to the isolation of azaphilone derivatives (17-18) and a novel tricyclic polyketide (19). Only compound 19 exhibited a broad spectrum of cytotoxic activities (Senadeera et al., 2012). It is interesting that MSM production by strain CR17 was sensitive to sources of potato and malt extract used for the preparation of PDB and Czapek malt media, respectively. Three new polyketides (20-22) were produced when strain CR17 was grown in PDB broth prepared from a commercial potato powder instead of fresh tubers of potato, while this strain produced several other compounds (20-21 and 23-25) in Czapek malt medium. Compound 24 exhibited cytotoxic activity against cancer cell lines MOLT-3, HuCCA-1and A549 with IC 50 values of 17.4, 48.1, 46.5 µg/mL, respectively (Hewage et al., 2014). One fungus strain of Fusarium tricinctum isolated in Beni-Mellal, which can colonize the rhizomes of Aristolochia paucinervis, could afforded three new fusarielins (26-28). But these metabolites were not detected when cultivated in normal rice medium supplemented with fruit and vegetable juice. Bioassay results suggested that compound 26 possessed cytotoxic effect on human ovarian cancer cell line A2780 with an IC 50 value of 12.5 µM (Hemphill et al., 2017). A new diketopiperazine (29) was isolated from Eurotium rubrum MPUC136 cultured by wheat medium, which displayed more powerful bioactivity than the Czapek-Dox agar medium, and shown to have cytotoxicity against B 16 melanoma cell line with an IC 50 value of 60 µM (Kamauchi et al., 2016).
HPLC analysis of crude extracts of an actinomycete strain Lentzea violacea AS08 indicated different composition in three media including CYPS (casein yeast peptone), SCP-1 (starch casein peptone), and SC (starch casein) (Hussain et al., 2017). Only one new eudesmane sesquiterpenoid (30) and a new analog of virginiae butanolide E (31) were detected in SC medium, and compound 30 exhibited moderate cytotoxic effect on HCT-116 and A549 tumor cell lines with IC 50 values of 19.2 and 22.3 µM, respectively. One rhizosphere fungus Paraphaeosphaeria quadriseptata produced a known C18 polyketide monocillin I together with several analogs when incubated in PDA medium constituted with tap water (Wijeratne et al., 2004). However, the same fungal strain could make six new trihydroxybenzene lactones, cytosporones F-I (32-37), when the tap water was changed as distilled water (Paranagama et al., 2007). Similarly, one new naphthalopyran compound (38), which possesses an unusual oxygenated aromatic structure with a lactone bridge, could be metabolized by the fungus P. hordei grown on plant tissue agar such as macerated tulip and yellow onion, oatmeal and red onion, while it was not detected in CYA (caffeic acid agar), MEA (malt extract agar), and YES (yeast extract with supplements) media (Overy et al., 2005). When cultivated in rice medium, a hard coral-derived fungus Scopulariopsis sp. from the coastline of Red Sea was shown to afford six secondary metabolites including xanthone derivatives (39-40), phenolic bisabolane-type sesquiterpenes (41-42), one new alkaloid (43) and one new α-pyrone derivative (44) (Elnaggar et al., 2016). Interestingly, this strain could biosynthesize a new naphthoquinone derivative (45) and two new triterpenoids (46-47) in the protein-rich white bean medium (Elnaggar et al., 2017).

Salinity
Salinity is an important factor in determining many aspects of the chemistry of natural water and of biochemical process within cultivation system, and is a thermodynamic state variable that, along with temperature and pressure, governs physical characteristics like the osmotic pressure and enzymes involved in microbial growth and metabolism (Blunt et al., 2015). Suitable salinity is needed for normal microbial growth and high osmotic pressure makes cells dehydrated and affects microbial biochemical reactions (Poolman and Glaasker, 1998;Wang Y. et al., 2011).
Microorganisms exposed to different types of media supplemented with various halogens maybe trigger their synthesis pathway to restore osmotic imbalance, thus activating different hidden MSM biosynthetic gene clusters. Compare to that grown in seawater, one marine-derived fungus Aspergillus unguis CRI282-03 was shown to produce new brominated depsidones (93-95) and two new orcinol derivatives (96-97) in KBr medium and a new depsidone (98) in KI broth (Sureram et al., 2013). Bioassay results indicated that compounds 95 and 96 possesses aromatase inhibitory effects (Sureram et al., 2012). Nine new polyketides (99-107), which were absent in the broth contained KI or deionized water, were produced by the fungus Dothideomycete sp. CRI7 isolated from Tiliacora triandra when cultivated in the medium supplemented with KBr and seawater (Wijesekera et al., 2017).

Metal Ion
Metal ion affects physiological structure and function of microorganism. The interaction between metal ion and microbe is usually assumed in three pathways, including causing reactions in cells, conserving energy in the process of dissimilation, and assimilating reactions (Thorneley, 1990).
One marine-derived strain Ascotricha sp. ZJ-M-5 was shown to produce a new 3,4-split ring lanolin alkyl triterpene (119) and a new cyclonerols derivative (120), when cultivated in eutrophic medium made up with sea salt (Xie et al., 2013a,b). However, three new caryophyllene derivatives (121-123) were detected in modified Czapek Dox medium, while compound 122 was absent in the fermentation broth without Mg 2+ (Wang W.J. et al., 2014). Strain Aspergillus sclerotiorum C10WU derived from hydrothermal vent sediment in Taiwan (China) could produce three new alkaloids (124-126) under normal medium. However, this strain metabolized one unelucidated compound due to the low amount available together with aspochracin when grown in the stressed culture medium with Cu 2+ as a supplement. Likewise, two compounds, namely deoxytryptoquivaline and tryptoquivaline A (127-128), were purified from the normal extract of A. clavatus C2WU, while only metabolite 129 was found in normal medium containing Cu 2+ and Cd 3+ (Jiang et al., 2014). A novel antibacterial cyclodepsipeptide, named NC-1 (130), was produced by a red soil-derived strain Streptomyces sp. FXJ1.172 when cultured in GYM (glucoseyeast extract-malt extract) medium added with ferric ion (Liu M. et al., 2016).

CULTIVATION CONDITION
Suitable cultivation conditions, such as appropriate temperature, pH, oxygen concentration, and cultivation status, are essential for the growth and biochemical reactions of microorganisms. However, many biosynthetic genes of MSMs are not expressed under normal culture conditions, thus it is essential to change the cultivation condition to activate these silent gene clusters to diversify their MSMs.

Temperature
Chemical diversity of MSM is directly influenced by microbe enzyme activity, which is susceptible to cultivation temperature. The normal function of microbial enzyme is dependent on appropriate temperature. Generally, the higher the cultivation temperature is, the faster the enzyme deactivation rate will be (Feller et al., 1994). For example, when the temperature was lower than 30 • C, secondary metabolites of an uncoded strain Streptomyces sp. were composed of chlortetracycline, while only tetracycline was synthesized when cultivation temperature went up to 35 • C (Cui et al., 1996).
pH During microbe fermentation process, the decomposition and utilization of nutrients as well as the accumulation of secondary metabolites usually causes the variation of medium pH (Gibson et al., 1988;Tan et al., 1998). It affects not only the activity of each enzyme, but also the surface charge of the membrane. The nature and permeability of cell membrane could change the rate of utilization of substrate, thus affecting the growth of microorganisms and biosynthesis of final products. Chemical study of one desert-derived strain Nocardiopsis alkaliphila nov. YIM-80379 led to isolation of two new pyran-2-one derivatives (131-132) when cultivated on Gause's synthetic agar slants with pH = 10. However, the neutral medium was unsuitable for its growth (Hozzein et al., 2004;Wang et al., 2013c). Acidic medium (pH = 5) dramatically increased the production of bioactive compounds of a mangrovederived fungus Rhytidhysteron rufulum AS21B, including two new antitumor spirobisnaphthalenes (133-134). However, these compounds were not detected in neutral medium (Siridechakorn et al., 2017).

Oxygen Concentration
Changes in oxygen supply can affect the biochemical reactions and activate different set of functional gene clusters for different secondary metabolites production (Sato, 1990). For example, [ 13 C]-labeled acetates and a small amount of [ 18 O 2 ] were used to investigate the biosynthetic pathway of aspinonene (135) in the culture broth of Aspergillus ochraceus DSM-7428. It is interesting that aspyrone (136) was produced by increasing dissolved oxygen concentration during fermentation, accompanied by reduced amounts of compound 135 under an oxygen enriched atmosphere (Fuchser et al., 1995).

Cultivation Status
A growing body of evidence has indicated that cultivation status can directly affect microbe metabolic process, including solid or liquid, static or dynamic. Compared with solid and static cultivation, liquid and dynamic modes not only ensure the full contact of microorganisms and nutrients, but also affect their biochemical reactions by changing oxygen supply and activating functional gene clusters. Till now, MSMs from 12 genera had been investigated under different fermentation status, including Arthrinium, Aspergillus, Myxotrichum, Nodulisporium, Lentinus, Paraphaeosphaeria, Penicillium, Pestalotiopsis, Phomopsis, Spicaria, Streptomyces, Ulocladium.

Aspergillus
By comparison of solid and liquid fermentation products of an endophytic strain A. fumigates LN-4 from stem bark of Melia azedarach L., their HPLC profiles were obviously different . Strain A. versicolor ZLN-60 could produce two new cyclic pentapeptides (147-148) and four new prenylated diphenyl ethers (149-152) in static liquid condition (Zhou et al., 2011;Gao et al., 2013). Biological tests indicated that compound 151 displayed moderate cytotoxicity against Hela and K562 cancer cell lines with IC 50 values of 31.5, 48.9 µM, respectively. However, further purification of its crude extract of solid medium resulted in the detection of four other novel cyclic peptides (153-157) (Peng et al., 2014). Chemical study of one marine-derived fungus A. terreus cultivated in 11 different culture conditions indicated that static agar was ideal for the production of antifungal lovastatins (158-159) and 7-desmethylcitreoviridin (160), which were absent in the shaking fermentation (Adpressa and Loesgen, 2016).
Three novel penipanoids (230-232) were characterized from one marine-derived strain P. paneum SD-44 grown in rice medium . The exploration of changing fermentation conditions of P. paneum SD-44 to a seawater-based culture broth under dynamic fermentation condition gave five new anthranilic acid derivatives (233-237). Metabolites 233 and 237 exhibited inhibitory activity toward human colon cancer RKO cell lines with IC 50 values of 8.4, 9.7 µM, respectively . One deep sea-derived fungus Penicillium sp. F23-2 biosynthesized terpenoids, diketopiperazines, and meleagrin alkaloids when incubated in sea-water-based culture medium under static condition (Du et al., 2009(Du et al., , 2010, whereas five new nitrogen-containing sorbicillinoids (238-242) were metabolized by this stain when cultivated in PYG (peptone yeast glucose) medium under shaking status (Guo et al., 2013).

Phomopsis
An endophytic fungus Phomopsis sp. sh917 isolated from fresh stems of Isodon eriocalyx var. laxiflora collected in Kunming Botanical Garden of China, was shown to produce six new polyketides (268-273) on solid rice medium but metabolize a new polyketide (274) in shaking liquid FM4 medium .

Streptomyces
One marine-derived stain Streptomyces sp. CHQ-64 was found to produce six new antifungal polyene-polyols (294-299) and two new cytotoxic hybrid isoprenoid alkaloids (300-301) in liquid medium under shaking condition, while this strain made only one new hybrid isoprenoid alkaloid (302) under static condition (Che et al., 2012(Che et al., , 2013(Che et al., , 2015(Che et al., , 2016. When cultivated in liquid Gause's No. 1 medium, strain Streptomyces sp. DT-A37 could produce a new ring-opened lactam (303), while in rice medium one unknown holomycin (304) and two new cyclopropaneacetic acids (305-306) were detected (Ding et al., 2017). Strain Streptomyces sp. HZP-2216E cultured in 2216E solid medium, GYM solid medium and GMSS (Gause's medium with sea salt) liquid medium resulted in isolation of two new compounds of 23-O-butyrylbafilomycin D (307), streptoarylpyrazinone A (308) a unique indolizinium alkaloid streptopertusacin A (309). It was noted that compound 307 showed potent activity in suppressing the proliferation of the four tested glioma cell lines with IC 50 values in a range from 0.35 to 2.95 µM and antibacterial activity with MIC value of 7.4 µM for MRSA and IC 50 values of 0.44 to 0.98 µM for glioma cells (Zhang et al., 2017c,d).

CO-CULTIVATION WITH OTHER STRAIN(S)
In one culture medium, the relationship between one strain and other(s) may be competitive, antagonistic or friendly. Co-cultivation of two or more strains usually has positive effect of an enhanced production of known compounds or an accumulation of cryptic compounds that are not detected in axenic culture (Bohni et al., 2013;Marmann et al., 2014). This effect maybe results from the production of enzymes that activate metabolite precursors or that other strain(s) may induce epigenetic modifications of the producer strain.

Fungus and Other Fungal Strain
An endophytic strain Acremonium sp. Tbp-5 from the European yew (Taxus baccata L.) could produce new lipoaminopeptides (320-322) when co-cultivated with Mycogone rosea DSM 12973 (Degenkolb et al., 2002). Chemical investigation of the mixed fermentation broth of two epiphytic strains Aspergillus sp. FSY-01 and FSW-02 from marine mangrove Avicennia marina led to the isolation of a novel alkaloid (323), which had antibacterial activity against Bacillus dysenteriae, B. proteus, and E. coli . The production of 2-alkenyl-tetrahydropyran analogs (224-326) was provoked by Chaunopycnis sp. CMB-MF028 in the mixed culture with a partner strain Trichoderma hamatum CMB-MF030, which were isolated from the inner tissue of marine pulmonate false limpet (Shang et al., 2017). Co-cultivation of Monascus sp. J101, used as the producer of Monascus pigment, with Saccharomyces cerevisiae KCCM 11371 or A. oryzae KCCM 11773 on the solid sucrose medium could result in two folds of accelerated cell growth and 30-40 folds of increased pigment production (Shin et al., 1998). Strain J101 was shown to stimulate cell growth and reproduction by interacting with S. cerevisiae, which resulted in production of more hydrophobic pigments compared to the mono-culture (Suh and Shin, 2000a,b). When co-cultivated with Beauveria felina, one marine-derived P. citrinum could biosynthesize two new compounds (327-328) featuring in a unique tetracyclic framework, whereas neither strain could produce these compounds in axenic medium. Antimicrobial assay showed that compounds 327 and 328 had strong inhibitory effects on human pathogens S. aureus and E. coli (Meng et al., 2015a). Penicillium sp. IO1 derived from mediterranean sponge Ircinia oros could produce a new fusarielin analog (329). However, co-cultivation of Penicillium strains IO1 and IO2 resulted in the accumulation of two known compounds norlichexanthone and monocerin, which were not detected in axenic controls (Chen et al., 2015a). Four new polyketides (330-333) were detected in a dual culture of the deep-sea-derived fungus Talaromyces aculeatus and a mangrove-derived fungus P. variabile, while these compounds were not identified in single culture. Compounds 333 displayed strong cytotoxicity against A549, K562, HCT-116, HeLa, MCF-7 and HL-60 human cancer cell lines with IC 50 values ranging from 1.2 to 9.8 µM (Zhang et al., 2017b).
One novel 1-isoquinolone analog (334) and its methyl ester (335) were detected in mycelia and culture filtrate of mixed fermentation of two endophytic fungi Nos. 1924 # and 3893 # , whereas these compounds were not traced in axenic medium under the same conditions (Zhu and Lin, 2006). The formation of new antibiotics (336-337) was emerged during co-cultivation of a multi-antibiotic stable mutant strain of Rhodococcus fascians and a strain Streptomyces padanus, neither of which was capable of yielding an antibiotic (Kurosawa et al., 2008). An terrestrial bacterium Tsukamurella pulmonis TP-B0596 co-cultured with strain Streptomyces sp. NZ-6, coincided with stimulation of three new metabolites (338-340) of unprecedented di-andtricyclic macrolactams (Hoshino et al., 2015). The yield of red pigment was detected in the dual induction of T. pulmonis TP-B0596 and S. lividans TK23. Co-cultivation of T. pulmonis and S. endus S-522 resulted in the production of one new antibiotics called alchivemycin A (341) (Onaka et al., 2011). A soil-dwelling actinomycetes S. coelicolor was shown to significantly improve the yield of red compound undecylprodigiosin, when co-cultured with Corallococcus coralloides (Schäberle et al., 2014).

Bacterium and Other Bacterial Strain
Only two new macrolactams (342-343) were detected in the co-culture broth of a rare actinomycete Micromonospora wenchangensis HEK-797 and Tsukamurella pulmonis TPB0596, whereas the axenic medium of strain HEK-797 produced a polyene macrolactam (344), which was possibly the precursor of compounds 342 and 343 (Hoshino et al., 2017). Investigation of the interaction of the portable predator Myxococcus Xanthus and Streptomyces coelicolor showed that actinorhodin production of S. coelicolor was raised up to 20-fold and stimulated aerial mycelium production (Pérez et al., 2011). Co-cultivation of two sponge-derived actinomycetes, Nocardiopsis sp. RV163 and Actinokineospora sp. EG49, induced ten reported compounds, including diketopiperazine, angucycline, and β-carboline derivatives, while only three natural products were isolated in mono-culture (Dashti et al., 2014). Mixed culture of Pseudomonas maltophilia 1928 and S. griseorubiginosus 43708 resulted in the production of one peptide antibiotic, biphenomycin A (345) (Ezaki et al., 1992). However, the accumulation of biphenomycin A, which could be obtained from the transformation of biphenomycin C (346), was inhibited in single culture of strain 1928 (Uchida et al., 1985;Ezaki et al., 1993). Interspecies interactions between Streptomyces coelicolor M145 with other actinomycete stains (Amycolatopsis sp. AA4, Streptomyces sp. E14, Streptomyces sp., SPB74 and S. viridochromogenes DSM 40736) resulted in the production of at least 12 different versions of a molecule called desferrioxamine (Traxler et al., 2013).

Fungus and Bacterium
Co-cultivation of one fungal strain A. terreus with B. cereus and B. subtilis resulted in the yield of two novel butyrolactones (347-348), which were absent in single culture medium (Chen et al., 2015b). An endophyte Chaetomium sp. from the Cameroonian plant Sapium Ellipticum (Euphorbiaceae) was shown to produce two novel shikimic acid analogs (349-350) and four new butenolide derivatives (351-354) when co-cultivated with Pseudomonas aeruginosa, while none of these chemicals was traced in axenic medium (Ancheeva et al., 2017). Strain Bacillus subtilis 168 trpC2 was shown to greatly activate the biosynthesis of three novel chemicals (355-357) of fungal endophyte Fusarium tricinctum during co-culture process. And these compounds were not duplicated in axenic fungal culture (Ola et al., 2013).
Co-cultivation of one marine fungus Libertella sp. CNL-523 symbiotic on an ascidian collected from the Bahamas and a fellow strain Thalassospira sp. CNJ-328 resulted in the production of four new diterpenoids (358-361). Compound 360 exhibited remarkable cytotoxicity against HCT-116 human adenocarcinoma cell line with an IC 50 value of 0.76 µM (Oh et al., 2005). A new pyridone alkaloid (362) was isolated from the mixed culture extract of Paecilomyces lilacinus and Salmonella typhimurium, which had 57.5 ± 5.50% of AChE inhibition (Teles and Takahashi, 2013). Co-culture of an endophyte Pestalotiopsis sp. from Drepanocarpus lunatus with B. subtilis was found to biosynthesize two novel sesquiterpenoids (363-364) while new compounds 365 and 366 emerged in axenic culture . The mixed cultivation of Trichoderma sp. 307 colonizing in Clerodendrum inerme and one bacterium Acinetobacter johnsonii B2 led to the production of two new sesquiterpenes (367-368) and three novel de-O-methyl lasiodiplodins (369-371). Compounds 369 and 370 displayed potent α-glucosidase inhibitory effect with IC 50 values of 25.8 and 54.6 µM, respectively (Zhang et al., 2017a).
Chemical study of an endophytic stain Aspergillus austroafricanus from Eichhornia crassipes led to the isolation of a highly oxygenated heterodimeric xanthone (372) and a new sesquiterpene (373) in axenic culture. Mixed fermentation of A. austroafricanus with B. subtilis or S. lividans afforded several diphenyl ethers, including one new austramide (374) . Two novel N-formyl alkaloids (375-376) were characterized from a mixed fermentation of A. fumigatus and S. peucetius. Compound 376 displayed in vitro cytotoxic effect on cancer cell line NCI-60 with an IC 50 value of 1.12 µM (Zuck et al., 2011). Seven known diketopiperazine alkaloids associated with ergosterol and 11-O-methylpseurotin A were traced in response to the supplement of A. fumigatus MBC-F1-10 to an established culture of S. bullii, whereas neither strain metabolized these compounds when cultivated alone (Rateb et al., 2013). Co-cultivation of A. fumigatus MR2012 with S. leeuwenhoekii C34 in ISP2 medium resulted in the yield of a new luteoride derivative (377) and a new pseurotin derivative (378). None of these compounds could be detected in axenic culture. When strain MR2012 was co-cultivated with strain C58, a lasso peptide chaxapeptin (379) was made, which displayed significant inhibitory effect on human lung cancer cell line A549 (Elsayed et al., 2015;Wakefield et al., 2017).
Physical interaction of A. nidulans RMS011 with S. hygroscopicus was found to trigger biosynthesis of four new aromatic polyketides (380-383), which were absent in the axenic medium (Schroeckh et al., 2009). A new polyketide glycoside (384) was formed in the dual induction of two Gram-positive bacteria, S. tendae KMC006 and Gordonia sp. KMC005, which were obtained from an acidic mine drainage sample (Park et al., 2017). In response to S. coelicolor A3(2) M145, strain A. niger N402 was shown to be apt to produce 2-hydroxyphenylacetic acid and cyclic dipeptide cyclo(Phe-Phe). Biotransformation of a new hexadienedioxic acid (385) and a new phenol derivative (386) was achieved by co-culture of these strains . More interestingly, co-cultivation of one marinederived fungus Emericella sp. CNL-878 with Salinispora arenicola CNH-665 resulted in the higher yields of two novel antimicrobial cyclic depsipeptides (387-388) than axenic culture (Oh et al., 2007).

EPIGENETIC MODIFIER
Epigenetic modifiers are those chemicals that are able to change microbial characteristics in correspondence to alteration of their epigenetic status, such as DNA methyltransferase (DNMT) inhibitor and histone deacetylase (HDAC) inhibitor. The addition of these modifiers usually suppresses the activity of related enzymes in the biosynthetic pathway and promotes the progress of other metabolic pathways (Seyedsayamdost, 2014).

DNA Methyltransferase Inhibitor
DNA methylation is a process by which methyl groups are added to DNA. When located in a gene promoter, DNA methylation typically acts to repress gene transcription and causes chromatin structure changes in the corresponding regions, preventing the binding of specific transcription factors and suppressing gene expression (Araujo et al., 2001). 5-Azacytidine (5-AC) is the most common DNMT inhibitor used to modify the function of microbe DNA followed by repressing gene transcription. Chemical investigation of a marine-derived fungus Aspergillus sydowii afforded three novel bisabolane-type sesquiterpenoids (389-391) when its culture medium was supplemented with 5-AC (Chung et al., 2013b). An entomopathogenic fungus Cordyceps indigotica yielded a novel aromatic polyketide glycoside (392) when cultivated in PDB media, while the strain produced another unusual glycoside (393) when supplement with 5-AC (Asai et al., 2012e). Several other examples that adding 5-AC as epigenetic modifier in culture medium could lead to the production of new metabolites were also reported, such as novel diethylene glycol phthalate esters (394-400) from a marine-derived strain Cochliobolus lunatus TA26-46 (Chen et al., 2016), a new benzoic acid (401) from Pestalotiopsis microspora (Yang et al., 2017), one new coumarin (402) from P. crassiuscula NBRC 31055 associated with Fragaria chiloensis , and novel meroterpenes (403-404) from P. citreonigrum (Wang et al., 2010).

Histone Deacetylase Inhibitor
The acetylation or deacetylation of histone affects its binding to DNA in microbe. There are many chemical modifications in the tail of histone that regulate the gene expression. The introduction of hydrophobic acetyl group into the N-terminal lysine residues of histone could increase the electrostatic attraction and steric hindrance between histone and DNA, which is conducive to facilitate the depolymerization of DNA and the binding of transcription factors (Fukuda et al., 2006;Cole, 2008). Suberoyl bishydroxamic acid (SBHA), suberoylanilide hydroxamic acid (SAHA), and nicotinamide are the most common HDAC chemicals used to inhibit the deacetylation and facilitate gene transcription and expression in microbes (Moore et al., 2012).

Multiple Chemical Epigenetic Modifiers
Interactions between epigenetic features play an important role in regulation of gene expressing or silencing in microorganisms, such as DNA methylation and histone modification. Many references that looked into the combined effect of epigenetic processes suggested that these chemicals could regulate the activity of genomic regions of varying sizes, from single genes to entire domains and chromosomes. Epigenetic markers could also interact with other nuclear proteins to work together to form chromatin structures and to create genomic functional discrete regions that induce the production of new secondary metabolites (Tammen et al., 2013).
One symbiotic strain Alternaria sp. from medicinal plant Datura stramonium Linn. was shown to produce four new aromatic polyketides (454-457) and a new tenuazonic acid (458) when incubated in medium containing 5-AC and/or SBHA. While these compounds were absent in normal culture medium. Interestingly, the yield of these secondary metabolites was higher in the medium of adding HDAC and DNMT inhibitors than that of addition of any other inhibitors (Sun et al., 2012). Chemical investigation of one marine-derived fungus Aspergillus sp. SCSIOW2 or SCSIOW3, exposed with an integration of SHBA and 5-AC, led to production of three new eremophilane-type sesquiterpenes (459-461) together with a new diphenylether-Oglycoside (462) . Bioactivity tests indicated that the glycosylated compound 462 exhibited a protective activity toward free radicals with an EC 50 value of 20.8 µM. One strain Cladosporium cladosporioides from a tidal pool was found to display different responses to the treatment with 5-AC and SHBA. Exposure of C. cladosporioides to 5-AC resulted in substantially increased biosynthesis of three oxylipins (463-465), whereas SHBA induced the yield of two new perylenequinones (466-467) (Williams et al., 2008).
Concomitant supplement of SHBA and N-phthalyl-Ltryptophan (DNMT inhibitor) to the fermentation medium of an entomopathogenic fungus Gibellula formosana induced the formation of two new highly oxidized ergosterols (468-469) and five new isariotin analogs (470-474) (Asai et al., 2012a). The same method was applied to expand MSM profile of Isaria tenuipes, which resulted in the yield of one new polyketide (475) (Asai et al., 2012b). An endophytic strain Leucostoma persoonii from red mangrove was subject to large-scale cultivation with sodium butyrate (HDAC inhibitor) and 5-AC, which resulted in the increased yield of known cytosporones and the production of one new cytosporone (476) (Beau et al., 2012). Three novel aromatics (477-479) were produced by Pestalotiopsis acacia from Taxus brevifolia when its culture medium was supplemented with SHBA and 5-AC (Yang and Li, 2013). Application of this approach also led to production of a new glycolipid ustilagic acid C (480) by Ustilago maydis .

OTHER FACTORS Enzyme Inhibitor
Beside DNMT and HDAC, other microbial enzymes also played important role in the regulating the biosynthesis of secondary metabolites, such as monooxygenase and hydroxylase. Some chemicals can selectively inhibit the activity of these enzymes in the biosynthetic pathway and promote the progress of other metabolic pathways, such as metyrapone, tricyclazole, jasplakinolide, and DMSO.

Biosynthetic Precursor
Biosynthetic precursor refers to one chemical that is apt to be directly incorporated into the final product. Adding various biosynthetic precursors in the fermentation medium may change biosynthesis pathways of secondary metabolites and result in the production of novel compounds (Ramm et al., 2017).

CONCLUSION
Microorganisms are susceptible to culture conditions, such as medium composition, temperature, pH, salinity, culture status, axenic or mixed culture, epigenetic modifier, biosynthetic precursor, and so on. Variation of these factors may result in changing chemical diversity of secondary metabolites. Traditional culture method of microbe is limited to the expression of a large number of metabolic pathways that many MSMs could not be biologically synthesized. A growing body of evidence has suggested that OSMAC strategy can provide a simple, quick and effective approach for enhancing chemodiversity of MSM to obtain new drug leads through activating silent gene clusters. Moreover, employment of this strategy could avoids the waste of time and resources caused by multiple inoculation, screening, culturing and separation in comparison with mutation strategy (Fang et al., 2014) and ribosome engineering (Ochi et al., 2004). Nowadays, the rate of discovery of new MSM is getting lower and the possibility of the rediscovery of known compounds is higher than before. Therefore, OSMAC strategy would be an important alternative way to alleviate this challenge. There is a great need for new method to assist in isolating and identifying novel bioactive MSMs, such as bioassay-guided isolation, microbe genomes mining (Hug et al., 2018) and LC-MS/MS based molecular networking analysis .

AUTHOR CONTRIBUTIONS
RP made a draft of the review. XB and JC searched and collected the references. HZ and HW conceived and revised this review.