1. INTRODUCTION:

Rapamycin (Rap), also known as sirolimus, is a 31-membered macrocyclic natural product that was isolated for the first time about a half-century ago^[1]. Rap is a multifunctional, extraordinary drug with a wide range of biological activities started as an antifungal^[1,2] exerting strong antimicrobial activity against Candida albicans, then extended to include immunosuppression^[3,4], anticancer activity^[5], neuroregenerative and neuroprotective function^[6-8].

Also its role as an anti-aging and lifespan extension agent is still under speculation^[9-11]. In addition, its ability to prevent coronary arteries restenosis after angioplasty has been well established^[12]. Raphas gained FDA approval as a drug used to prevent organs-rejection in kidney transplants operations^[13] and as an eluting stent drug preventing restenosis of coronary arteries following angioplasty ^[14,15].

As an immunosuppressant, Rapacts via a mechanism that is completely different to that of cyclosporine A, and it has the fabulous advantage to be 150 times as active as cyclosporine A with remarked lower toxicity ^[16]. The action mechanism of Rap has been well studied^[17]. It acts through inhibiting mTOR (the mechanistic target of Rap, which is regulating different mammalian proteins and is essential for many growth-related, motility, and surviving cellular activities) by binding and forming a complex with its intracellular receptor, FK506-binding protein 12 (FKBP12). That formed complex interacts directly with the FKBP12-Rapamycin-binding (FRB) domain of mTOR^[18], followed by inhibiting mTOR^[19]. Rap also affects the second phase of T-cell activation through the inhibition of growth promoting cytokine signaling transduction pathway with mTOR^[20,21].

The insufficient yield of Rap microbial production resulted in increasing its cost. The price for sirolimus oral tablet (concentration of 0.5 mg) is about $226 /30 tablets, which is very expensive in comparison with other generic drugs^[22]. Many studies have been conducted to enhance the yield of Rapmicrobial production in order to decrease its cost^[12]. Screening for potent producer strains, changing medium composition and growth conditions are the key points to focus on to increase Rap production. For example, using L-lysine as an additive to the production medium increased the yield of produced Rap^[23]. On the other hand, studies using Plackett-Burman experimental design and Response Surface Methodology (RSM) were conducted to verify the most significant variables affecting Rap production yield from Streptomyces hygroscopicus^[24,25]. Yield optimization strategies including genetic algorithms and artificial neural networks were also applied^[23]. Recent studies had reported different strain improving techniques. However, random mutagenesis technique showed advantageous results than those of molecular approaches^[19,24,26].

Indeed, the importance of Rap as a unique surprising drug having endless list of clinical bioactivities and a special efficient potency with a very high price justified the efforts aiming at employing novel methodologies and strategies for increasing its production. In an attempt to find out new effectors stimulating the production of Rap, the current investigation intended to screen many non-conventional factors, which were thought to have a physical or nutritional effect, to realize their role in the production of Rap.

2. MATERIALS AND METHODS:

2.1 Producing strain:

The organism used in this study, Streptomyces hygroscopicus ATCC 29253, was purchased from Microbiological Resources Centre in Cairo (Cairo MIRCEN), Egypt. It was grown on slants of oat meal medium (contained oat meal, 20 g/l; agar, 20 g/l; pH 7) for 10 days at 28ºC. After that, spores were collected by adding 4 ml of 10% (v/v) glycerol to each slant. Spore suspensions of 25.8 x 10⁶ CFU/ml were then prepared and distributed in cryopreservation vials 1 ml/each, and stored at -20ºC till needed^[27].

2.2 Inoculum preparation:

One milliliter of thawed spore suspension (25.8 x 10⁶ CFU/ml) was inoculated into 50 ml starch casein broth (contained in g/l: starch, 10; casein, 0.3; KNO₃, 2; NaCl, 2; K₂HPO₄ 2; MgSO₄.7H₂O, 0.05; CaCO₃, 0.02; FeSO₄.7H₂O, 0.01; pH 7) in 250-ml Erlenmeyer flask. The flask was then incubated at 28±2ºC for 5 days at 150 rpm. Three milliliters of the grown culture were used to inoculate 50 ml of fermentation medium.

2.3 Production medium and fermentation process:

The medium used in this study for production of Rap composed of (g/l): soybean meal, 20; D (+) mannose, 20; KH₂PO₄, 5. Components have been dissolved in tap water and pH was adjusted to 6^[27]. Fermentation process was initiated by inoculating each 50-mlproduction medium contained in 250-ml Erlenmeyer flask with 3 ml of inoculum culture that was prepared as described previously. Then, the flasks were incubated at 25 ºC ±2 for 5 days at 150 rpm^[27]. All experiments were performed in triplicates.

2.4 Estimation of the microbial growth:

Packed cell volume percentage (PCV%) was employed in the current study as rough estimate of growth^[28]. It was determined by placing a 3-ml sample of whole fermentation medium into 10-ml tube and centrifuging at 3500 rpm for 5 minutes. By transferring the supernatant to graduated cylinder, its volume was determined and thus the volume of packed cell precipitate was calculated. The percentage of packed cell volume to the total volume of whole fermentation medium sample is the desired estimate of growth. All data were expressed as the arithmetic mean and the standard errors from triplicate-flask preparations.

2.5 Rap Extraction:

At the end of fermentation, aliquots of 3 ml were taken where microbial growth was separated by centrifugation at 3500rev/min for 5 minutes and extracted twice by shaking with 3 ml methanol for 30 minutes. Then the obtained extracts were pooled to be assayed for Rap concentration.

2.6 Estimation of Rap:

Determination of Rap was achieved by paper-disc agar diffusion method as described by Kojima et al. ^[16]. The assay was conducted in agar plates of assay medium seeded with Candida albicans ATCC 10231 as the indicator strain. Assay medium composed of (g/l): peptone, 2; glucose, 5; agar, 11; pH 6. Five μl of the methanolic extract of cells were loaded onto paper discs (Whatman no. 3) of 6 mm diameter. The discs were then carefully placed on the surface of bioassay medium seeded with indicator strain. After incubation for 20 h at 37ºC, inhibition zone around each disc was recorded. Similarly, inhibition zones around standard concentrations of Rap were recorded. Plotting the relation between logarithms of Rap concentration against inhibition zone showed straight line whose linear equation was used to get Rap concentrations from inhibition zone readings. The results were expressed as the arithmetic mean of the experiments performed in triplicates and standard errors were shown as error bars.

2.7 Preparation of different nano-sized soybean meal:

Rap production medium consists mainly of Soybean meal. Different Nano-sized soybean meal were prepared by being blended mechanically for 15 h with ball-to-powder ratio (BPR) equals to 1:2, and the diameters of balls were 10 mm. Then, that resulting powder was milled for 2, 5, 10h in a planetary ball mill (type MTI SFM (QM-3SP2) with rotation speed equals to 350 rpm and ball-to-powder (BPR) weight ratio was 10:1. The milling was performed using Al₂O₃ vial and balls. Milling process was conducted in a cycle of 2h and paused for 2h.After that, morphology and particle size of the milled powders were examined by transmission electron microscopy (TEM), (type JEOL JEM-1230).

2.8. Effect of increasing the strength of production medium:

The effect of increasing the concentration of all components in the production medium at the same time to be many-fold as that in the normal control production medium was studied. This allowed the increase in nutritional components of the medium up to stressing levels while maintaining the carbon/nitrogen ratio as the same in all tested strengths. Normal production medium strength (control) was the one-fold strength medium while other media of different strengths were prepared as indicated in Table 1. Fermentation process was conducted as described before and Rap production was assayed.

Table (1) Composition of prepared different folds of Rap production medium.

Medium Components	1-fold (g/L) (control)	1.5-folds (g/L)	2 folds (g/L)	3-folds (g/L)
Soybean meal	20.00	30.00	40.00	60.00
D(+) mannose	20.00	30.00	40.00	60.00
KH₂PO₄	5.00	7.50	10.00	15.00

2.9 Effect of incubation at successive high and low temperatures:

After inoculation of the production medium with the seed culture of S. hygroscopicus strain, the flask cultures were incubated at different temperatures during the incubation period. At the first day, the culture was incubated at 30˚C, then the temperature was dropped to 20°C at the second day. At the third day, the temperature was elevated up to 25 ˚C where it maintained in such degree up to the end of incubation period at the fifth day.

2.10. Impact of salinity on Rap production:

Sodium chloride, in different concentrations(1, 3, 7 and 10 g/L),was added to production medium, then Rap concentration was determined at the end of fermentation as described previously.

2.11. Effect of repeated inoculation of fermentation flasks:

Under this aim, each fermentation flask was further inoculated with three extra doses of inoculum. All doses were containing the same amount of inoculum culture (3 ml) and applied to fermentation flasks at constant intervals of one day from each other starting after 24 hour of the initial principal inoculum.

2.12.Production of Rapunder conditions of co-culturing the producing strain with another competing rapamycin-sensitive microbe:

In order to study the stimulatory effect of co-culturing a competing microbe, which is sensitive to Rap, simultaneously with the producing strain, Candida albicans ATCC 10231 was inoculated into theproduction medium at different times of fermentation (after 24, 48 and 72 hours of inoculating the producing strain). A suspension of 24-hour old culture of Candida albicans ATCC 10231 was prepared under sterile conditions (in a concentration of nearly 0.5 McFarland) and used to inoculate Rap fermentation flasks (0.5 ml/flask) after different times of inoculating the Rap producing strains.

2.13. Production of Rap in medium containing rapamycin-adsorbent matrix (silica gel-G60):

To fermentation flasks containing the normal production medium, 1 gm silica gel (G60) was added to each flask before autoclaving. Then, the flasks were inoculated with the producing strain and the fermentation was developedin typical standard conditions described previously in materials and methods section.

2.14. Production of Rap in medium enriched with camel milk:

In order to enrich production medium with camel milk, typical fermentation was done using camel milk as a dissolving medium for all components of the production medium instead of tap water. The pH value of the medium was adjusted at 6 before autoclaving and then all the subsequent steps of fermentation were carried outaccording tostandard conditions.

3. RESULTS:

3.1. Production of Rap using different nano-sized soy meal:

Studying the impact of using nano-sized soy meal on Rap production revealed that the average particle sizes of soybean meal powder decreases with increase in milling times (Fig.1 a, b, and c). The calculated average particle sizes of the milled soybean meal powder are 89, 59.75, and 49 nm for powders milled for 2, 5 and 10h, respectively. On the other hand, a slight increase in Rap titer could be attained when using soya meal in size of 89 nm as shown in Fig.1 (d), and Rap concentration has increased from 29.8±2.5 mg/l in control to 34.3±1.4 mg/l in soy meal size of 89 nm. Soy meal of smaller nano-sizes (59.75 and 49 nm)could not potentiate more increase in Rap concentration which was comparable to that in control sample. The results also showed an obvious decrease in microbial growth upon using nano-sized soy meal.

3.4. Production of Rap under different treatments:

Different treatments were tested for their ability to stimulate Rap production. The treatments included fluctuation in incubation temperature, co-culturing producer strain together with Candida albicans, enriching medium with camel milk, production in presence of silica gel (G60), and repeated inoculation of producing strain. The results represented in Fig. 4 showed that great activation of Rap production was attained under the influence of fluctuation in incubation temperature. The drug yield has elevated to 68.2±17.5 mg/l which was about 132% increase in yield comparing with control sample (29.4±2.5mg/l). Growth measurements showed that the increase in Rap yield was associated by an increase in PCV% from 25±1.4% in control to 31.7±1.4% under fluctuated temperature. Co-culture of producing strain with competing Candida albicans could not potentiate an obvious increase in drug yield. Moreover it was deleterious if competing microbe was introduced after 48 hours of starting fermentation. The results in Fig. 4 showed also that other treatments yielded amounts of Rap that were closely comparable to that in control sample. The exception was in case of media enriched with camel milk where strong suppression in the drug production (0.8±0.1mg/l) and growth of producing strain (PCV%=17.30±0.82%) was observed. The significant drop in final pH of the production medium enriched with camel milk may explain the suppressive effect of such medium.

4. DISCUSSION:

Since the discovery of Rap, and due to its various medical applications as a monotherapy or as part in a combination therapy, studies have been conducted in order to increase its production yield. Mostly in all optimization studies on rapamycin production, searching for optimum conditions was conducted via classical or statistical approaches. On completely different side, the current work intended to screen many non-conventional aids, which were thought to have a physical or nutritional effect, to realize their role in the production of Rap. Modifying the physical structure of soy meal, as one of key nutrients in Rap production, by nano-sizing was employed. Using different particle sizes of soybean meal powder slightly increased Rap production as shown in Fig.1 (d), especially when using soya meal in size of 89 nm. Noticeable decrease in microbial growth during using nano-sized soy meal was also observed, which suggested that soy meal under nano-sizing conditions could exert antimicrobial activity as a result of increased surface area-to-mass ratio^[29]. One of the mechanisms by which nanomaterials exhibit the antimicrobial action is the production of reactive oxygen species^[29]. As the protective role of Rap against oxidative stress and reactive oxygen species was well established^[30,31], it was proposed that increased Rap yield under nano-sized soy meal was a spontaneous response of the Rap producer strain to protect itself against oxidative stress created by nanoparticles of soy meal.

Another modification in production medium was tested by using many-fold concentrated production media. The Rap production on 1.5-fold concentrated production medium as shown in Fig. 2 was 129.7% higher than that in case of using the regular one fold medium (control medium). Regarding the previous sophisticated work of Zhao et al.^[32], Kim et al.^[33] and Dutta et al.^[34] for optimizing the medium for Rap production which led to an increase in the production by 56.6%, 45% and 72% respectively, hence the current 129.7% increase in yield represented an interesting record in Rap optimization studies. Many investigators employed different strategies for getting the optimal composition of Rap production medium^[23,25,35] however, they did not study the effect of concentrating the optimum medium. As such, this is the first report about the importance of medium strength and the ability to achieve more than two-fold increase in Rap yield by manipulating the production medium strength.

Great number of factors affecting Rap production was surveyed in previous literatures^[^1,^16,23,²⁵^,^28,35,36,³⁷^] however, salinity was rarely included in studied factors. Therefore, the effect of different NaCl concentration was investigated. As a result, using 1% NaCl concentration yielded an elevationof 56.4% in the drug yield. Further rise in salinity caused stability in production yield during using 3% NaCl, while a dramatic reduction in Rap yield and PCV% was observed during using 7% and 10% NaCl concentrations which reflected the deep inhibitory effect of such concentrations on growth and drug production.

In an attempt to stimulate Rap production, fermentation was carried out under different treatments. Repeated inoculation of the fermentation flasks with the producer microbe and enriching the production medium with camel milk were tested. Stressing the producer strain with fluctuated temperatures across fermentation course was also studied. Promoting the productivity through managing the feedback inhibitory mechanisms that halter overproduction was tested by addition of Rap adsorbent matrix (silica gel G60) which was thought to reduce free drug in medium, the principal cause of regulatory feedback cascades. Induction through stimulating the natural role of Rap as secondary metabolite produced to inhibit the growth of competing microbes was validated through inoculation of strain of Candida albicans to be co-cultured with Rap producing microbe. The inoculation of Candida albicans was performed after delay time of starting growth of Rap producer to ensure sufficient growth of the producer before introduction of the competitor. All tested treatments were compared to control fermentation representing non-treated sample. The results depicted in Fig. 4 showed that great activation up to 132 % increase of Rap production was attained under the influence of fluctuation in incubation temperature. On the contrary, co-culturing of producer strain with a competing strain, Candida albicans, failed to increase Rap yield. Moreover it was deleterious if competing microbe was introduced after 48 hours of starting fermentation. The importance of co-culture as an efficient aid for increasing the production of secondary metabolites was reported previously^[38,-41]. In the study of Luti and Mavituma^[42], 6-fold increase in the yield of immunosuppressant undecylprodigiosin was attained when the producing strain of Streptomyces was co-cultured with Escherichia coli. The current result of the inability of Candida albicans to stimulate overproduction of Rap was in agreement with findings of Slattery et al.^[38] who found that only 12 out of 53 tested microbial strains were able to increase production of istamycin in co-cultures. Therefore, screening more microbial species for inducing Rap production in co-cultures is a reasonable target in future studies. Other treatments did not have a noticeable influence on yield of Rap except for using camel milk which exhibited a strong suppress or to both Rap production and growth of producing strain. The significant drop in final pH of the production medium enriched with camel milk may explain the suppressive effect of such medium.

Upon using the adsorbent matrix, silica gel G60, no change was observed on Rap production, which may indicate that the concentration of yielded Rap under studied conditions was below the threshold of turning on feedback inhibitory mechanisms.

5. CONCLUSION:

In the present study, different strategies were employed to increase Rap production. Superior increase in the yield of Rap production (about 132%) was achieved under fluctuated incubation temperature, followed by accomplishing 129.7% increase in the yield in case of using 1.5-fold concentrated medium, and finally 56.4% increase in the yield has been attained during using 1% NaCl. The current results encourage searching for innovative conditions to elevate the production of drugs as important as Rap. Additionally, the current study highlighted many ways and strategies that could inspire novel trend in optimization studies of secondary metabolites production.

6. FUNDING:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

7. CONFLICT OF INTEREST:

The authors declare that they have no competing interests.

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Received on 21.03.2019 Modified on 18.04.2019

Research J. Pharm. and Tech 2019; 12(9):4197-4204.

DOI: 10.5958/0974-360X.2019.00722.4