Effect of adsorbing resins on the rapamycin biosynthesis by Streptomyces hygroscopicus VKM Ac-2737D

Rapamycin, a metabolite of Streptomyces hygroscopicus, has a wide range of therapeutic applications in medicine. However, the possibility of its use is significantly limited, since the existing natural strains of this microorganism have rather low productivity that negatively influences the cost of the substance and the possible volumes of its industrial production. At the same time, the commercial use of overproducing strains is also very problematic, since they secrete a large number of metabolites suppressing both the growth of microbial culture and the biosynthesis of a target product. A possible way to solve the problem includes the addition of adsorbents to a fermentation medium for the binding of excess metabolites. In this study, five different selective adsorbing resins, DIAIONTM HP20, DIAIONTM HP21, DIAIONTM HP 20SS, Chromolite, and LPS-500 were tested to provide optimal conditions for the rapamycin biosynthesis by the earlier developed highly-active S. hygroscopicus strain VKM Ac-2737D. The experiments were performed in a 15-L bioreactor using the optimized fermentation medium developed earlier and supplemented with the tested resins. The highest rapamycin yield (1420±5 mg/L) was obtained using a Chromolite 15AD2 adsorbing resin; this result exceeds the earlier obtained yield by 11.4%. The sorptive capacity of this resin was 92% that may significantly facilitate the processes of rapamycin isolation and purification under industrial conditions. The results of the study may be of certain interest for the further scaling-up of the industrial rapamycin production.


Introduction
Rapamycin, which is also known as sirolimus, is a secondary metabolite of the actinomycete bacterium Streptomyces hygroscopicus . The structure of this nitrogen-containing compound belonging to macrolide antibiotics (Dutta et al., 2014) is shown in Fig. 1. Rapamycin is characterized by multiple effects on the organism, the main of which are the capability for cell division suppression used in the therapy of oncogenic diseases (Murugan, 2019) and immunosuppressive properties used to prevent graft rejection or any other immune diseases (Waldner et al., 2016;Yoo et al., 2017). Besides, some researchers consider rapamycin as a remedy for delaying aging and also for prevention and therapy of age-associated diseases, such as atherosclerosis, osteoarthritis, neurodegenerative ad heart diseases (Blagosklonny, 2017;Bagherpour et al., 2018).

231
Effect of adsorbing resins on the rapamycin Ukrainian Journal of Ecology, 10(6), 2020 Everolimus, a chemical derivative of rapamycin, is also widely used in the therapy of oncologic diseases. Everolimus actively inhibits the growth and proliferation of fibroblasts, as well as the tumor, endothelial, and unstriated muscle tissues of blood vessels and is considered a promising drug for the therapy of a progressive hormone-dependent HER2-negative breast cancer in women in a post-menopause period and the therapy of adult patients suffering from the renal angiomyolipoma, nonfunctional neuroendocrinal tumors of the gastrointestinal or pulmonary origin, and progressive neuroendocrinal tumors of the pancreas (Chan et al., 2010;Hasskarl, 2018).
In the first studies of the rapamycin biosynthesis by S. hygroscopicus, the productivity of strains cultured in a 250-L bioreactor for 96 h did not exceed 87 mg/L (Sehgal et al., 1975); in the case of 6-day cultivation in 500-mL flasks, it did not exceed 100 mg/L . The analysis of existing publications and patents shows no significant changes in the situation: the productivity of existing industrial strains remains within 700-1000 mg/L (Zhao & Zhao, 2012;Cao et al., 2019) that increases the cost price of the substance and reduces possible volumes of its industrial production. Thus, the use of rapamycin in medicine is still limited, even despite its wide therapeutic potential. In this connection, the development of a strain with high biosynthetic activity concerning rapamycin and the development of technology suitable for the industrial production of this antibiotic represent very relevant tasks.
Earlier a highly active strain S. hygroscopicus R 33-41 was obtained at the Laboratory of Biotechnology of Physiologically Active Substances of the Federal Research Center "Fundamentals of Biotechnology" of the Russian Academy of Sciences (Moscow) by an undirected multi-step induced UV mutagenesis. The productivity of this strain reached 655±5 mg/L of rapamycin, more than 15-fold exceeding that of the initial strain S. hygroscopicus АТСС 29253 (Savelyeva et al., 2017a). The obtained strain was deposited in the All-Russian Collection of Microorganisms at the Skryabin Institute of Biochemistry and Physiology of Microorganisms with the accession number VKM Ac-2737D. The further optimization of the nutrient medium composition combined with the full experimental factorial planning, steepest ascent method, and the movement along the concentration gradient method provided an additional increase in the productivity of this strain under submerged cultivation in flasks or a bioreactor up to 1215 ± 5 (Savelyeva et al., 2017b) or 1275 ± 5 (Dzhavakhiya et al., 2019) mg/L of rapamycin, respectively, providing a 30-fold increase compared to the productivity of the initial strain S. hygroscopicus АТСС 29253.
Strains obtained by the non-directed induced mutagenesis and characterized by improved productivity to the target biologically active compounds are known to have impaired metabolic pathways (Egorov, 1989). Due to this fact, the cultivation of such strains is accompanied by the secretion of a large number of metabolites suppressing both the culture growth and the biosynthesis of a target compound (Phillips, 2013). One of the ways to reduce the cytotoxic activity of the secreted metabolites is the use of adsorbing resins during fermentation (Frykman et al., 2006;Phillips et al., 2013). Adsorbing resins are widely used in the biotechnological production of biologically active compounds (Vojshvillo et al., 2007;Pathapat et al., 2018). Nevertheless, this method is not universal and requires some studies to choose the appropriate selective absorbent for a target metabolite, since the same adsorbent may provide different effects for different processes (Phillips et al., 2013). The correctly chosen adsorbent may significantly increase the yield of a target product (Jia et al., 2006;Zhang et al., 2012). For example, our earlier studies on the effect of adsorbing resins on the virginiamycin biosynthesis in S. virginiae VKM Ac-2738D showed the use of a Diaion HP21 resin increases the productivity of this strain . Taking into account a high need in the efficient rapamycin production technology suitable for a large-scale realization, the purpose of this study was the selection of a selective adsorbing resin providing optimal conditions for the rapamycin biosynthesis by S. hygroscopicus ВКМ Ac-2737D.

Strain cultivation and storage
In this study the Streptomyces hygroscopicus strain VKM Ас-2737D obtained from the initial S. hygroscopicus strain ATCC 29253 by a multi-step selection combined with the use of mutagenic factors and the directed selection and deposited in the All-Russian Collection of Microorganisms was used. The strain was maintained and stored using R1 agarized medium of the following composition (g/L): agar, 22.0; soybean flour, 1.0; soluble starch, 10.0; MgSO4, 1.0, K2HPO4, 0.5 (pH 6.8 ± 0.1). For maintenance, the strain was cultured at 29 ºC for 10-12 days with periodical re-inoculations onto a fresh medium. For long-term storage, the culture was freeze-fried and stored in sealed ampoules in a refrigerator.

Vegetation and fermentation media
To obtain seed material, 25-mL tubes containing agar medium with a work culture were washed with 5 mL of sterile physiological solution. The resulted spore suspension was transferred to 500-mL Erlenmeyer flasks containing 100 mL of nutrient medium consisting of the following components (g/L): soybean flour, 21.0; L-lysine, 6.0; yeast extract, 6.0; glucose, 20.0 (pre-sterilization pH 6.8 ± 0.1). The flasks with the seed culture were incubated on a shaker for 46-48 h at 220-240 rpm and 29 ºC. The quality of a seed material was controlled by the microscopic inoculation of diagnostic media (meat-extract agar and Sabouraud glucose agar) and examination of the basic cultivation parameters, such as a pH value at the end of fermentation (should be 6.2-6.8) and the biomass content (no less than 8%). Ready seed culture (10 vol.%) was aseptically transferred into 500-mL mL Erlenmeyer flasks containing 100 mL of the same nutrient medium and 20g/L of an adsorbing resin added before sterilization. The fermentation was carried out for 214-216 h at 29 ºС and 250 rpm.
Prior inoculation, the medium was sampled for the microbiological and biochemical control. Then the medium cooled to 28 ± 1°С was inoculated with 1 L of seed material grown in vegetation medium as described above. During fermentation, a constant control and regulation of pH, рО2, and temperature was performed. The рО2 level was regulated by changes in the volume of air uptake per a volume of culture broth (manually) and in the rotation speed of a stirring device (automated mode). Culture broth pH was automatically regulated by sterile 20% NaOH solution fed by a peristaltic pump. During the first 48 h, the pH value was maintained within 6.6-6.8. Starting from 72 h of fermentation, a continuous supply of the bioreactor with 50% sterile glucose solution was carried out in an automated mode with the simultaneous maintenance of the pH and pO2 values at the earlier determined optimum levels (6.6-6.8 and 30%, respectively). Determination of a crude biomass content in culture broth Aliquots of culture broth (10-15 mL) were passed through a stainless steel mesh (hole size <0.1 mm) to separate resin particles. Then the samples were transferred to centrifuge tubes with the known weight and centrifuged at 4500 rpm for 10 min. After a supernatant removal, the tube with mycelial sediment was weighted. The biomass of each sample was determined in three parallel repetitions and calculated using the following formula: where a is the weight of a sample-containing tube prior centrifuging (g); b is the weight of a sample-containing tube after centrifuging (g); and c is the tube weight (g).

Quantification of a rapamycin content in fermentation broth
Rapamycin was extracted from 10-mL samples of fermentation broth (including those containing the sorbent) by their mixing with a double volume of ethyl acetate followed by a 2-h incubation under constant stirring at a room temperature. After a completion of the extraction process, 2 mL of the ethyl acetate extract (or emulsion) was transferred to a new tube and centrifuged at 13400 rpm for 5 min, then 0.5 mL of the upper liquid layer was sampled and evaporated. The resulted solid residue was thoroughly mixed with 0.5 mL of methanol and analyzed by HPLC (see below). To determine the content of rapamycin absorbed by tested resins, resin-containing fermentation broth was filtered through a stainless steel mesh (hole size < 0.1 mm), and the collected resin was washed with distilled water. The further rapamycin extraction and HPLC sample preparation was carried out separately for the resin and filtered fermentation broth as described above. The HPLC analysis was performed using an Agilent 1200 chromatographic system (Agilent Technologies, USA) with a Zorbax C18-SB column (250 × 4.6 mm, Agilent Technologies, USA) filled with octadecyl silica gel (5 μm). The mobile phase was acetonitrile/distilled water (75/25), the flow rate was 1.0 mL/min at 60°C. The sample volume was 10 μL. The absorbance was measured at 278 nm. A commercial rapamycin preparation (Sigma-Aldrich, USA) dissolved in the methanol was used as the standard. The retention time was 9 min. Statistical treatment All fermentations were carried out in three replications. The obtained data were statistically treated using the MS Excel program package.

Results
Effect of adsorbing resins on the rapamycin biosynthesis in flasks The results of the assessment of the efficiency of adsorbing resin addition in relation to the rapamycin yield are shown in Table  2. According to these data, only DIAION HP20SS and Chromolite 15AD2 demonstrated a positive influence on the strain activity resulting in an increased rapamycin yield comparing to the control. The maximum total rapamycin yield (1330 mg/L) was obtained using the Chromolite 15AD2 resin; it exceeded the yield in the control variant by 10%. Cultivation of S. hygroscopicus VKM Ас-2737D in the presence of other resin types resulted in a slight inhibition of the rapamycin production. The minimum yield of the antibiotic (1065 mg/L), which was 12% less than in the control, was observed for the LPS-500 variant. In spite of the obtained results, one can note that all tested resins demonstrated a high sorption capacity (85-92%) representing a significant advantage of their use in biotechnological processes.
Assessment of the rapamycin production by S. hygroscopicus VKM Ас-2737D in a 15-L bioreactor in the presence of the Chromolite

15AD2 adsorbing resin
To scale the rapamycin production by the S. hygroscopicus strain in the presence of the Chromolite 15AD2 adsorbing resin, a 15-L bioreactor with the working volume of 10 L was used. The results of the experiment are shown on Fig. 2. According to them, the active rapamycin biosynthesis started in the beginning of the stationary growth phase (96 h). The maximum antibiotic yield (1420 ± 5 mg/L) was observed after 216 h of fermentation; this result exceeds the earlier obtained level by 11.4% (Dzhavakhiya et al., 2019). Therefore, along with the fermentation medium optimization, the rapamycin yield can be additionally increased by the use of adsorbents, such as the Chromolite 15AD2 resin, during fermentation.

Discussion
Biotechnological methods of production of various compounds including antibiotics using metabolic processes in microorganisms represent an efficient tool for the production of valuable biologically active compounds without a need for expensive chemical synthesis. However, there are some limitations to the use of biological methods on an industrial scale (Stark, 2003). One of such factors is the self-inhibition of the biosynthetic activity of microorganisms by the generated products. To overcome the cytotoxic action of the produced compound, one can in situ remove it from the reaction medium (Bechtold, 2009;Phillips, 2013). To maximally use the advantages of this approach, highly selective removal of a target compound on an adsorbing phase is required (Bechtold, 2009). However, as it has been already mentioned earlier, the use of synthetic adsorbing resins is not a universal method, and the suitable resin type should be determined for each biosynthetic process, since even resins with similar physical characteristics may provide either increase or reduction of the yield of a target compound in culture broth (Lam, 1995).
In our case, we tested five polystyrene/divinylbenzene adsorbing resins with similar characteristics (Table 1). DIAION HP20 and DIAION HP21С resins are highly cross-linked polymers with spherical particles characterized by a porous structure and the surface area within 500-1200 m²/g of dry matter. They are used for adsorption refining of peptides, proteins, polyphenol compounds, cephalosporin C, and for the chromatographic separation and purification of fermentation products (Kamiyama, 1994). DIAION HP20SS developed based on DIAION HP20 is characterized by small-size particles and controlled pore size distribution and is used for separation and purification of a wide range of molecules, from small peptides and oligonucleotides to large proteins (Rani, 2020). Chromolite 15AD2 is a macroporous adsorbent used for the reverse-phase chromatography of organic compounds and can successfully replace hydrophobic silica gel sorbents of the C8 and C18 type. It works at any pH values of eluents and regenerating solutions and is characterized by a rigid polymeric matrix and a uniform particle size that provides a high separation efficiency. LPS-500 is a highly cross-linked polystyrene sorbent characterized by a rigid matrix of the optimal porosity and used for the sorption refining of flavonoids, peptides, proteins, drug substances, and radioactive isotopes. There are some examples of a successful application of some of the above-mentioned adsorbing resins in the processes of antibiotic biosynthesis. The use of the DIAION HP20 resin made it possible to remove the toxic action of the teicoplanin antibiotic on the growth of its producer, Аctinoplanes teicomyceticus, which resulted in a 4.2-fold increase of its final titer and reduced the number of required extraction stages (Lee, 2003). A similar positive effect was reported for the use of DIAION HP20 in the biosynthesis of thailandepsin A, a promising antitumor agent produced by Burkholderia thailandensis E264 (Liu, 2012). The use of the DIAION HP21 resin in the process of fermentation of Streptomyces achromogenes v. rubradiris provided a fourfold increase in the rubradirin yield (Marshall, 1990). A comparative analysis of the effect of the chosen adsorbents on the final rapamycin yield showed that only two adsorbing resins, НР20SS and Chromolite 15AD2, provided a 5% (1271 mg/L) and 10% (1330 mg/L) increase of this parameter, respectively, comparing to the control. These sorbents added to the fermentation medium before fermentation can a highly selective (90 and 92%, respectively) sorption of the target antibiotic from the culture broth that provides a significant simplification of the rapamycin extraction scheme. Moreover, the sorbents provide almost complete removal of related compounds (minor components, pigments, protein products, salts, etc.), which remain in the culture broth. Other testing resins (DIAION HP20, DIAION HP21, and LPS-500) showed slightly reduced rapamycin yield. These results can be explained base on the features of sorbent particles (Table 1). For example, the pore size in LPS-500 resin, which showed the worst result, is rather heterogeneous and reaches 500Å that exceeds the corresponding parameter of other adsorbents. Since all resin types were added to the fermentation medium before fermentation, this resin could absorb any medium components or metabolites important for the rapamycin biosynthesis; their deficiency, in turn, could result in the reduction of the final antibiotic concentration. To clarify the reasons for the observed phenomenon, further investigations are needed. The main advantages of the Chromolite 15AD2 resin are the relatively low cost, excellent physical and chemical stability, thermal stability, and high exchange capacity. The resistance of this sorbent to organic solvents makes it reusable for the rapamycin production process. In this study, this resin was successfully used for the scaling of the rapamycin biosynthesis for a 15-L bioreactor providing an 11.4% increase in the final yield of this antibiotic comparing to the earlier obtained results (Dzhavakhiya et al., 2019). Thus, the result of this study may be of certain interest for the further scaling up of the process of industrial rapamycin production by S. hygroscopicus.