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. 2021 Jul 28;11(8):386. doi: 10.1007/s13205-021-02926-1

Composition of nutrient media and temperature of cultivation imposes effect on the content of secondary metabolites of Nocardiopsis sp. isolated from a Siberian Cave

Denis V Axenov-Gribanov 1,✉, Maria M Morgunova 1, Ulyana A Vasilieva 1,2, Stanislav V Gamaiunov 1,3, Maria E Dmitrieva (Krasnova) 1, Ekaterina V Pereliaeva 1, Alexander Yu Belyshenko 1, Andriy N Luzhetskyy 4,5
PMCID: PMC8319253  PMID: 34350091

Abstract

Growth of human population leads to many global and medical problems. The problems include the crisis of health, antibiotic resistance, drug discovery, etc. Increasing antimicrobial resistance of microorganisms results in the need to screen natural products (incl. antibiotics and antimicrobial peptides) and their producers in different ecological niches. The purpose of this study was to estimate antibiotic activity and biotechnological potential of rare actinobacteria Nocardiopsis sp. The strain was isolated from Okhotnichya cave located in Siberia. Here, we cultivated the strain at 3 temperature modes (13 °C, 28 °C, 37 °C) in 11 liquid nutrient (rich and poor) media. Using modern assays of liquid chromatography and high-resolution mass spectrometry, we estimated the content and number of produced natural products, distribution of their masses, and potential rate of novel secondary metabolites. We demonstrated that minimal nutrient media with l-asparagine and SM25 media with malt extract were less productive at current experimental parameters. As it was shown, this strain was characterized by antibiotic properties against Bacillus subtilis when cultivated at 28 °C. Also, weak antibiotic activity of crude extracts was found in strain cultivation at 13 °C. Also, we detected a high number of novel amphiphilic and hydrophobic NPs produced by this strain. We demonstrated both the influence of the nutrient media composition and cultivation temperature on biosynthetic capabilities of rare strain Nocardiopsis sp. Finally, high level of natural products that were predicted as novel confirms high biotechnological value of rare genera of Actinobacteria that could be explained by the evolution of microorganisms in the isolated environment of cave ecosystem.

Keywords: Actinobacteria, Nocardiopsis sp., Cave, Natural products

Introduction

Growth of human population leads to many global and medical problems. The problems include the global crisis of health (Small et al. 2017), antibiotic resistance (Ventola 2015a, b; Haywood et al. 2021), drug discovery (Brown and Wright 2016; Negus 2018), etc. These result from a number of economical, technological and chemical reasons, low number of new classes of natural products, and increased antibiotic resistance of microorganisms (Roca et al. 2015). One of the potential findings for the screening of new natural products (NPs) and discovering new classes of NPs is studying the biosynthetic and genetic potential of rare microorganism species inhabiting specific ecosystems. Microorganisms are a highly studied and well-adapted group of live organisms. They synthesize secondary metabolites and are often used by humans in different medical, biotechnological, agricultural, and other industries (Margesin and Schinner 2001; Okafor 2016).

Among all microorganisms, actinobacteria are highly productive and effective systems of synthesis of different NPs with complex chemical structures. As is known, bacteria and fungi produce more than 70 000 NPs, incl. appr. 20 000 NPs and appr. 10 000 bioactive compounds produced by Actinobacteria. It includes molecules with antibiotic, neuroprotective, cytotoxic, and other activities (Subramani and Sipkema 2019). Around 76% (7 600) actinobacterial bioactive NPs were derived as secondary metabolites of Streptomyces genera. It is one of the most dominant and well-studied groups of actinobacteria. Other (rare) actinobacteria are less studied in the focus of diversity of produced NPs and their biological activity. There are many genera of actinobacteria related to the group of rare species and presented by species of Actinomadura sp., Microbacterium sp., Micromonospora sp., Pseudonocardia sp., Sinomonas sp., Nocardiopsis sp., and more than 340 other rare species (Azman et al. 2015).

One of the exciting representatives of rare actinobacteria is genus Nocardiopsis sp. (Bennur et al. 2015; Sun et al. 2010). This genus was first described by (Meyer 1976) when “Actinomadura dassonvillei (Brocq-Rousseu) Lechevalier and Lechevalier” and the former “Nocardia dassonvillei (Brocq- Rousseu) Liegard and Landrieu” were transferred from the genus “Actinomadura Lechevalier and Lechevalier” to a new genus named “Nocardiopsis”. Based on the distinct phylogenetic position, morphological features, and chemotaxonomic properties displayed by Nocardiopsis species, the authors of (Rainey et al. 1996) created a new family for the genus referred to as “Nocardiopsacea” (Bennur et al. 2015).

Characteristics of Nocardiopsis sp. species include the following features: the substrate mycelium may fragment into rods or cocci, production of aerial hyphae varies from sparse to abundant, and the cell wall contains 2,6-meso-diaminopimelic acid (meso-DAP) but no diagnostic sugar (Meyer 1976). Most of the species of the genus Nocardiopsis were isolated from saline or alkaline environments and can adapt well to extremely natural conditions (Bérdy 2012; Ding et al. 2010; Horikoshi 1999; Zhang et al. 2016). Also, representatives of this genus were found in close symbiotic interaction with marine sponges Dendrilla nigra (Selvin et al. 2009), Fasciospongia cavernosa (Gandhimathi et al. 2009), and molluscs Conus rolani (Lin et al. 2013). The last-mentioned study described γ-pyrones as secondary metabolites of symbiotic Nocardiopsis sp. with biological activity.

Thus, representative of Nocardiopsis sp. is a good model to study biosynthetic potential of rare strains. This study is intended to evaluate some aspects of biosynthetic potential of Nocardiopsis sp. isolated from a Siberian cave.

Materials and methods

Microbial strain, fermentation, and experimental design

To estimate biosynthetic potential, we chose a strain related to genus Nocardiopsis sp. (Nocardiopsis sp. IB2014I79-5, NCBI sequence ID MG971355). Earlier, the strain has been isolated from water of the underground lake in Okhotnichya cave in Siberia and previously mentioned in (Voytsekhovskaya et al. 2018).

The strain was cultivated in 30 mL of eleven original and modified production media in 250 mL shake flasks with baffles for 7 days at 28 °C at 180 rpm shaking rate (Kieser et al. 2000). The same nutrient media was used for the strain cultivation during 14 days at 13 °C and 5 days at 37 °C. The difference in time of cultivation was described by late stationary phase of microbial growth. Orbital shakers Multitron-standard (Infors HT, Switzerland) with 5 cm diameter of orbital movements were used for this study.

As it was mentioned before, eleven different liquid media were chosen to estimate primary antibiotic activity. These media were as follows: SM1 (soy flour, glucose, Na2SO4, pH 7.0), SM17 (soy flour, glucose, glycerol, soluble starch, peptone, yeast extract, NaCl, CaCO3, pH 6.4), SM12 (soy flour, glucose, peptone, meat extract, yeast extract, NaCl, CaCO3 pH 7.6), SM20 (maltose, peptone, meat extract, yeast extract, MgSO4 × 7 H2O, NaCl, pH 7.2), SM24 (yeast extract, peptone, glucose, KH2PO4, MgSO4 × 7 H2O, pH 6.2), SM25 (peptone, malt extract, glycerol, pH 6.5), SM27Ac (soy flour, glucose, peptone, meat extract, yeast extract, NaCl, CaCO3, pH 4.5), SM27N (pH 6.8–7.0) and SM27Al (SM27Ac at pH 4.5 and pH 8.7, respectively), R2 (malt extract, yeast extract, glucose, artificial sea water, pH 7.8), minimal medium (l-asparagine, K2HPO4, MgSO4 × 7 H2O, FeSO4 × 7 H2O, glucose, pH 7.2).

The grown liquid cultures were sonicated for 20 min. Then, secondary metabolites were extracted with the equal volume of ethyl acetate (Merck, Germany). The extraction was performed during 1 h on a rotator at 100 rpm at room temperature. The obtained crude extracts were evaporated in vacuo using IKA RV-10 rotatory evaporator (IKA, Germany) at 40 °C and dissolved in 500 uL of methanol–DMSO mixture (ratio 1:1, Merck, Germany) (Sarker et al. 2006). The obtained extracts were used for estimation of antimicrobial activity and dereplication analysis.

Assay of antimicrobial activity

Antimicrobial activity was estimated using the disk diffusion test. For this, 6 mm paper disks were loaded by 40 uL of crude extract, dried, and applied on the test cultures plated on solid nutrient media (Balouiri et al. 2016). Several nonpathogenic and model bacterial and yeast cultures were used, incl. Bacillus subtilis ATCC 66,337, Escherichia coli ATCC25922, Pseudomonas putida KT 2440 and Saccharomyces cerevisiae BY4742. For analysis, we used 24 h test cultures. Test cultures were obtained from the Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and the Helmholtz Institute for Pharmaceutical Research Saarland (Saarbrucken, Germany). The activity was estimated after 18–24 h.

LC–MS and dereplication analysis

Crude extracts were analyzed using ultra-high-performance liquid chromatography approaches with a mass spectrometry detector. We used the Ultimate 3000 (Dionex) UHPLC chromatography system with LTQ Orbitrap high-resolution mass spectrometer (Thermo Fisher Scientific, USA). Here, we used the general model for primary screening of NP and dereplication applied in the Helmholtz Institute for Pharmaceutical Research Saarland (Saarbrucken, Germany) and Saarland University (Saarbrucken, Germany). To separate crude extracts, we applied a linear gradient of acetonitrile (Merck, Germany) from 5 to 95% against 0.1% ammonium formate solution in water over time of 18 min with 0.5 mL/min flow rate. We used the ACQUITY UPLC BEH C18 Column, 130A, 1.7 µm, 2.1 mm X 100 mm. Mass detection in the range of m/z 200—2 000 was carried out under positive ionization. Data were collected and analyzed using Xcalibur software, version 3.0 (Thermo Fischer Scientific, USA). Screening for known NPs (dereplication) was performed using the Dictionary of Natural Products database (CRC Press 2019). To identify compounds in crude extracts, we used such parameters as a biological source of NPs (Nocardiopsis sp.) and the accurate molecular mass (Whittle et al. 2003). Compounds were considered to be identified when the difference between accurate mass was less than m/z 0.001, 10 ppm and biological source were identical with library data. The masses of NP were calculated using standard adducts protocol ([M + H], [M + Na], [M + NH4, etc.). Analysis of each sample was carried out for three times.

Results and discussion

Strain identification and estimation of biological activity of strain Nocardiopsis sp.

The strain Nocardiopsis sp. IB2014I79-5, (NCBI sequence ID MG971355) has been previously isolated from the water of underground lake in Okhotnichya cave in Siberia and mentioned in (Voytsekhovskaya et al. 2018). Based on phylogenetic analysis, we know that this strain forms a strict clade with representatives of other Nocardiopsis sp. The strain does not form a strict clade with strains isolated from other soils- or water-derived sources (Voytsekhovskaya et al. 2018). Based on data from Genbank and EZ BioCloud databases, the strain is characterized by high similarity with Nocardiopsis dassonvillei subsp. albirubida NBRC 13,392 (Similarity 99.49%). According to BacDive database, this strain is a mesophilic halophile, it is characterized by the ability to consume fructose, mannitol, and sucrose. Nocardiopsis dassonvillei subsp. albirubida NBRC 13,392 is related to Biosafety level 2, and risk group 2 (German classification).

According to performed experiments aimed at the strain cultivation in 11 different liquid nutrient media at 3 different temperatures we found, that this strain was endowed with activity against Gram-positive bacterium B. subtilis. First, it is worthy of note that the strain demonstrated relatively strong activity in the case of cultivation at 13 °C and 28 °C. We observed antibacterial activity for both temperatures in the case of cultivation in SM1 and SM12 media. The maximal antibiotic activity was observed when the strain was cultivated in SM27N and SM12 media at 28 °C. The absence of antibacterial activity was found while the strain was cultivated in SM24, SM25 medium, and in minimal nutrient media with l-asparagine. Second, it is worthy of note that the strain cultivation at 37 °C did not lead to onset of antimicrobial activity against chosen test cultures. We detected weak activity of the strain while it was growing in acidified SM27 (SM27Ac) nutrient media. Materials describing activity of crude extracts are presented in Table 1.

Table 1.

Antimicrobial activity of Nocardiopsis sp. IB2014I79-5 cultivated at different temperatures and nutrient media

Nutrient media Sm1 Sm17 Sm12 Sm20 Sm24 Sm25 Sm27Ac Sm27N Sm27Al R2 MM
Temperature of cultivation 13 °C
Antibiotic activity against test cultures
B. subtilis ** – ** – – – – – – – –
P. putida – – – – – – – – – – –
E. coli – – – – – – – – – – –
S. cerevisiae – – – – – – – – – – –
Temperature of cultivation 28 °C
Antibiotic activity against test cultures
B. subtilis ** ** *** ** – – *** *** *** ** –
P. putida – – – – – – – – – – –
E. coli – – – – – – – – – – –
S. cerevisiae – – – – – – – – – – –
Temperature of cultivation 37 °C
Antibiotic activity against test cultures
B. subtilis – – – – – – * – – – –
P. putida – – – – – – – – – – –
E. coli – – – – – – – – – – –
S. cerevisiae – – – – – – – – – – –
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*6–8 mm

**9–11 mm

***12–15 mm

Screening on natural products produced by strain Nocardiopsis sp.

There exists many screening approaches for definition of NPs in crude extracts and estimation of biosynthetic potential of microbial strains (Harvey 2008; Wolfender et al. 2010). One of the most popular and available techniques for dereplication of NPs in crude extracts is LC–MS (liquid chromatography–high-resolution mass spectrometry). Based on this approach, we found that the analyzed strain can be a producer for many NPs previously detected in Nocardiopsis sp. We established that the studied strain produces antimicrobial NPs related to Nacapyrone and Nocardiopyrone family (Fu et al. 2011; Ng et al. 2015; Schneemann et al. 2010). Thus, among the members of Nacapyrone, we detected Nocapyrone I ([M + H] 269.17471 Da; 2.4 ppm; syn. 2-(6-Hydroxyheptyl)-6-methoxy-3,5-dimethyl-4H-pyran-4-one), Nocapyrone C 6'-Deoxy ([M + H] 267.1954 Da; 2.6 ppm; syn. Nocapyrone L), Nocapyrone R ([M + H] 253.17975 Da; 2.8 ppm; syn. Nocapyrone H), Nocapyrone A ([M + H] 269.17471 Da; 2.4 ppm; syn. Nocapyrone N), and Nocapyrone G ([M + H] 209.117 Da; 4.1 ppm; syn. Nocardiatone B).

Nocardiopyrones were presented by Nocardiopyrone B Ac ([M + H] 253.14334 Da; 2.8 ppm; syn. Nocardiopyrone A), Nocardiopyrone A ([M + H] 253.17973 Da; 0.3 ppm; syn. 4-Hydroxy-3-methyl-6-(3-methylbutyl)-5-(2-methylpropyl)-2H-pyran-2-one).

Also, we detected Griseusin F 4'-Ketone ([M + H] 459.12738 Da; 3.97 ppm; syn. Griseusin G), and Griseusin D ([M + H] 433.11224 Da; 3.03 ppm). As is known, Nocapyrone G, Griseusin G and Griseusin D are characterized by cytotoxic and antimicrobial (against Gram-positive bacteria) activities (Ding et al. 2012; He et al. 2007; Li et al. 2007). Thus, the observed activity against B. subtilis shown in Table 1 can be explained by the presence of the above NPs.

Griseusin D was found in liquid culture when the strain was cultivated in four nutrient media (SM1, SM20, SM27N, Sm27Al) at 13 °C. In the case of cultivation at 28 °C, this NP was detected in six nutrient media (SM1, AM17, SM12, SM20Ac, SM27N, and Sm27Al). When the strain was cultivated at 37 °C, Griseusin D was found only in two nutrient media (SM1, SM27Ac). Thus, these two media were preferred by the strain to produce Griseusin D (Fig. 1).

Fig. 1.

Fig. 1

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The structural formulas of detected natural products produced by Nocardiopsis sp. at different temperatures and compositions of liquid nutrient media

In the case of the strain cultivation at 28 °C, we observed several compounds that could be highly responsible for antibiotic activity. This selection has been done using multiple sorting of data, definition of common and different peaks in mass chromatograms, and comparison of mass spectrometry data with antibiotic activity presented in Table 1. These NPs were characterized by the following retention time and masses: RT 6.86 ([M + H] 592.3695 Da), RT 8.37 ([M + H] 546.33279 Da), RT 12.65 ([M + H] 363.0861 Da), and RT 8.51 ([M + H] 560.3436 Da). The above masses were found in the crude extracts obtained from liquid culture of strain cultivated at 28 °C in SM1, SM17, SM12, SM20, SM27(Ac), SM27(N), SM27(Al), and R2. The above masses were absent in crude extracts of the strain cultivated at 28 °C in nutrient media SM24, SM25, and MM. This observation is in complete accord with the extracts' antibiotic activity against B. subtilis detected after strain cultivation at 28 °C.

The list of NPs, that could be responsible for antimicrobial activity of extracts obtained after cultivation at 37 °C is presented by compounds with RT 8.15 ([M + H] 399.14359 Da), RT 15.17 ([M + H] 407.3360 Da), and RT 15.36 ([M + H] 313.1634 Da). During cultivation in acidified medium SM27, these NPs were absent in liquid culture of the strain cultivated at 13 °C and detected in liquid culture of the strain cultivated at 28 °C, and 37 °C.

The list of NPs that could be responsible for antimicrobial activity of extract obtained after cultivation at 12 °C is presented by NPs with RT 10.01 ([M + H] 465.1174 Da), RT 10.1 ([M + H] 467.1331 Da), RT 11.17 ([M + H] 493.1487 Da), RT 14.46 ([M + H] 401.2895 Da), RT 18.56 ([M + H] 774.5643 Da), RT 19.36 ([M + H] 772.5488 Da). Possible NPs responsible for activity at 12 °C and 28 °C, but absent at 37 °C are presented by NP with the mass [M + H] 774.5643 Da and RT 18.56.

Paying attention to distribution of NPs, it should be noted that the following 10 NPs were found under almost all tested conditions of experiments: RT 2.64([M + H] 261.12335 Da), RT 3.26 ([M + H] 227.13882 Da), RT 3.74 ([M + H] 261.12314 Da), RT 4.95 ([M + H] 284.13922 Da), RT 10.19 ([M + H] 450.32092 Da), RT 10.87 ([M + H] 415.21109 Da), RT 11.59 ([M + H] 785.59161 Da), RT 17.53 ([M + H] 293.28375 Da), RT 18.7 ([M + H] 338.34134 Da), RT 20.8 ([M + H] 663.45276 Da). However, all mentioned NPs could not be preliminarily identified using the Dictionary of Natural Products database on accurate mass and biological source. Also, we found many NPs synthesized by the strain in a narrow range of tested parameters (nutrient media or temperature).

Analysis of obtained chromatograms includes only qualitative data obtained from one often used protocol of LC–MS. The data analysis does not include quantitative analysis of the above NPs. However, the obtained materials and approaches could help to organize preparative studies, purification of NPs, and selection of NPs responsible for biological activity. Also, this method of cultivation and analysis could be useful for determining the list of possible NPs with biological activity without long-term and high-scale fermentation.

Estimation of biosynthetic properties of strain Nocardiopsis sp.

Impact of temperature and content of nutrient media on biosynthesis of NPs

In our study, we applied the OSMAC (one strain–many compounds) strategy to estimate biosynthetic properties of the representative of rare strain Nocardiopsis sp. According to (Romano et al. 2018), the principles behind the cultivation-based approaches have been conceptualized in the OSMAC framework, which underlines how a single strain can produce different molecules when grown under different environmental conditions. Parameters such as the nutrient content, temperature, and rate of aeration can be easily changed, altering the global physiology of a microbial strain and, in turn, significantly affecting its secondary metabolism (Hewage et al. 2014; Romano et al. 2018).

Here, we estimated the number of NPs (peaks) that could be produced by the strain in different experimental conditions. Materials describing the number of NPs produced by strain when cultivated in different nutrient media at different temperatures are presented in Fig. 2. Visualized differences in mass chromatograms of the analyzed crude extracts are shown in Fig. 3.

Fig. 2.

Fig. 2

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Total number of natural products produced by Nocardiopsis sp. at different temperatures and compositions of liquid nutrient media

Fig. 3.

Fig. 3

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Visualized differences in mass chromatograms of analyzed crude extracts

Using the current LC–MS parameters, we have shown that the chosen strain could produce from 42 to 89 NPs under different experimental conditions. The minimal number of NPs was detected in crude extract of alkalified SM27 medium, while the strain was cultivated at 28 °C. Also, a small number of NPs was produced by the strain when cultivated in SM1 medium at 37 °C, and in SM25, and MM medium at 13 °C. The strain produced the highest number of NPs when grown in SM24 and R2 media at 28 °C.

Minimal nutrient media was characterized by the small difference between the number of NPs the strain produced when grown at different temperatures. Thus, we demonstrated that the content of NPs produced by the strain depends on the composition of nutrient media, and temperature of cultivation.

Impact of temperature and content of nutrient media on distribution of median masses of NPs

Based on the materials presented in Fig. 4, we estimated distribution of the median mass of NPs depending on their hydrophilic and hydrophobic properties and produced by Nocardiopsis sp. in liquid culture under cultivation at different temperatures and compositions of nutrient media. The common trend that we observed was the increase in the median mass of NPs with the rise of the hydrophobicity level. It could be explained by elution of long-chain fatty acids and/ or ionophoric antibiotics produced by the studied strain.

Fig. 4.

Fig. 4

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Distribution of median mass of NPs depends on their hydrophilic and hydrophobic properties and produced by Nocardiopsis sp. in liquid culture when cultivated at different temperatures and compositions of nutrient media

In the case of strain cultivation in SM1 nutrient medium, we found that the median masses of NPs eluted from the HPLC column in the hydrophilic range of linear gradient was appr. 296.6 Da–299.1 Da (± SD 77.2–84.9) instead of the median masses of NPs eluted from HPLC column in the hydrophobic range (598.4 Da–784.0 Da (± SD 175.5–193.2)). The cultivation of strain Nocardiopsis sp. in minimal media led to the synthesis of polar NPs with median molecular masses 235.1 Da–260.1 Da (± SD 32.1–34.7). The masses of nonpolar NPs eluted from the HPLC column in the hydrophobic range of linear gradient was appr. 488.3 Da–662.4 Da (± SD 128.1–223.1).

As we noted before, not only media composition influences the mass of NPs. In our study, we have shown the temperature-dependent changes in the masses of NPs. In the case of strain cultivation in SM20 medium, we found that NPs with the median mass 757.5 Da (± SD 79.13) were eluted in the interval from 14 to 16th min (relatively nonpolar NPs) when the strain was grown at 12 °C. At the same time, in the case of cultivation at 28 °C and 37 °C, median masses were lower and presented by 498.2 Da (± SD 105.4) and 459.8 Da (± SD 121.4), respectively. Another example of temperature-dependent changes was observed in strain cultivation in SM12 nutrient medium. The changes were observed in the average mass of NPs eluted after 17 min of the standard linear protocol. Among the hydrophobic NPs, the minimal average mass was shown for NPs synthesized at 37 °C (424.2 Da (± SD 119.8)) instead of NPs synthesized at 12 °C (757.5 Da (± SD 157.9)) or 28 °C (757.5 Da (± SD 177.5)).

Impact of temperature and content of nutrient media on the number of NPs and screening of novel NPs

As in the previous chapter, we also estimated distribution of the amount (number) of NPs depending on their hydrophilic and hydrophobic properties and produced by the studied Nocardiopsis sp. in liquid culture when cultivated at different temperatures and compositions of nutrient media (Fig. 5). We found the absence of any specific regularity of NPs distribution. However, we observed several preliminary findings in a number of predicted novel NPs: (1) the rate of predicted novel NPs (here, the NPs that do not match with NPs described in Dictionary of Natural Products database) studied for representatives of other Nocardiopsis sp. is higher in the hydrophobic part of analyzed chromatograms. Among polar NPs eluted from column (at the beginning of analysis), the number of known NPs was higher (up to 50%) than at the end of the analysis, when nonpolar NPs were eluted. (2) The rate of predicted novel NPs found in saline or alkalified media is lower than in the rich nutrient media. The last observation is well demonstrated in the case with alkalified SM27 and R2 nutrient media when the strain was cultivated at 28 °C and 37 °C, respectively. As we mentioned before, representatives of Nocardiopsis sp. are often related to halophile and/ or extremophile microorganisms and produce biologically active NPs (Chen et al. 2009; Subramani and Sipkema 2019; Tian et al. 2014).

Fig. 5.

Fig. 5

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Distribution of the number of NPs peaks depending on their hydrophilic and hydrophobic properties and produced by Nocardiopsis sp. in liquid culture when cultivated at different temperatures and compositions of nutrient media

Conclusion

Thus, our study was performed with the representative of a rare strain related to genus Nocardiopsis, previously isolated from a Siberian cave. Representatives of genera Nocardiopsis sp. are valuable for biopharmaceutical and biological study. Nowadays, several NPs produced by Nocardiopsis sp. were successfully commercialized by pharmaceutical giant Merck KGaA, (Darmstadt, Germany). Thus, alkaloid K-252a is used to inhibit protein kinases involved in cell signaling processes (Lazarovici et al. 1989; Matsuda and Fukuda 1988). It is used to inhibit nerve growth factors on PC1 2 cells and inhibit smooth muscle myosin light chain kinase. Nocardiopsis sp. produces another NP—apoptolidin A. The target of this molecule is mitochondrial F0F1-ATPase. Nowadays, due to commercial reasons, this molecule is produced by other strain of actinobacteria–Amicolatopsis sp. Apoptolidin A is a 20-membered macrolide shown to be selectively cytotoxic against several cancer cell lines and noncytotoxic against healthy cells (Salomon et al. 2001).

Based on the analysis of materials describing the strain antimicrobial activity and the experiment conditions (cultivation in different nutrient media and at different contrast temperatures), we have shown the possibility to concentrate helpful information from multiple chromatography/and mass spectrometry data necessary for screening and purification of NPs. In this study, we tested 33 experimental conditions of cultivation (3 temperatures of cultivation and 11 liquid nutrient media). Here we included the analysis of 413 peaks found in MS chromatograms. We predicted only one NP that can be responsible for activity against a model strain of B. subtilis in the case of growing the strain at 12 °C and 28 °C in liquid medium SM1.

Also, we detected a high number of novel NPs produced by this strain and demonstrated both the influence of nutrient media composition and temperature of cultivation on biosynthetic capabilities of rare strain Nocardiopsis sp. Here, we have demonstrated the advantages of rich nutrient media for cultivation of actinobacteria for primary screening of bioactive natural products.

Thus, as we mentioned before, rare (or extremophilic) strains of specific ecosystems, such as caves, are the unique sources for novel antibiotics and NPs. Rare strains are essential and valuable for biomedical and biotechnological development and for discovery of new NPs, and the agents for novel drugs.

Funding

This study was supported by Dr. Denis Axenov-Gribanov RFBR (18-29-05051) and Minobrnauki RF (МК-1245.2021.1.4)

Contributor Information

Denis V. Axenov-Gribanov, Email: denis.axengri@gmail.com

Maria M. Morgunova, Email: marymikhmorg@gmail.com

Ulyana A. Vasilieva, Email: uliana.andreevna.vasilyeva@gmail.com, https://www.scopus.com/authid/detail.uri?authorId=57218601877

Stanislav V. Gamaiunov, Email: utkaparazit@rambler.ru, https://www.scopus.com/authid/detail.uri?authorId=57164793400

Maria E. Dmitrieva (Krasnova), Email: marrieekrasnova@gmail.com, https://www.scopus.com/authid/detail.uri?authorId=57218599210

Ekaterina V. Pereliaeva, Email: cat.perelyaeva@gmail.com, https://www.scopus.com/authid/detail.uri?authorId=57218601455

Alexander Yu. Belyshenko, Email: al.belyshenko@gmail.com, https://www.scopus.com/authid/detail.uri?authorId=57189378141

Andriy N. Luzhetskyy, Email: alu11@helmholtz-hzi.de, https://www.scopus.com/authid/detail.uri?authorId=57204143369

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