Streptomyces tsukubaensis Fermentation Using Brazil Nut Oil to Enhance Tacrolimus Production

Tacrolimus, also known as FK-506, is a drug first isolated in 1984, that has been widely used to prevent the rejection of transplanted organs, and to also treat dermatoses and eye diseases.1-3 It is a 23 membered polyketide macrolide produced during fermentation through different Streptomyces species. The most commonly used is the Streptomyces tsukubaensis.4 This medication is interesting to dermatology because it is rapidly absorbed by the epidermis and has less side effects than commonly used drugs.5 Besides, as an immunosuppressant agent it is 10-100 times more potent than cyclosporine.6 Nevertheless, the treatment cost using tacrolimus is high because of its low productivity.7 Up to now, different strategies have been used to solve this problem, including the use of mutant bacteria,8-11 and exogenous feeding of precursors in the biosynthesis.12-14 Literature reports that S. tsukubaensis is capable of metabolizing different carbon sources, such as glucose, maltose, dextrin, soybean oil, cottonseed oil and groundnut oil.14-16 Exogenous feeding of oil has played an important role in this bioprocess, since it is a complex source of nutrients which has increased the tacrolimus production.12,14,17 The Brazil nut oil is rich in fatty acids and are broken in order to produce coenzymes (acetyl or propionyl) which are known as precursors for the polyketide production.17 This study has investigated the influence of the Brazil nut (Bertholletia excelsa) oil on the tacrolimus production. The fermentative process was carried for 168 h using S. tsukubaensis.

for 20 minutes, inoculated with a Petri dish colony and incubated at 28 o C on a temperature controlled orbital shaker at 130 rpm for 24 hours in a 250-mL Erlenmeyer baffled flask. The 24 hour-old culture was used as pre-inoculum, and 10% pre-inoculum was inoculated in a seed culture medium solution, and incubated on a temperature controlled orbital shaker at the same conditions previously described for 24 hours. This culture was used as inoculum. Actually, 10% inoculum was inoculated in the production medium, incubated at 28 o C on a temperature controlled orbital shaker at 130 rpm for 168 hours in a 500-mL Erlenmeyer baffled flask. The fermentation batch was performed in duplicates. The Brazil nut (B. excelsa) oil was acquired from Sinergia Cientifica. In order to quantify the tacrolimus production along the fermentative process, 10 mL of the culture broth were collected each for 24 hours.

Biomass Quantification
After collecting 10 mL of the culture broth, 10 mL of acetone was added to these samples in order to end the fermentation. The resulting content was filtered in pre-weighted filter paper (Whatman n o 1). The cells were washed with deionized water and kept at 80 o C for 24 hours for drying (Sterilifer -SX1.0).

HPLC Analysis
The tacrolimus quantification method was based on consolidated literature. [14][15][16] After filtration, culture broth samples were purified by evaporating at reduced pressure until the formation of an oily compound. It was added 3.5 mL of mobile phase (acetonitrile-water 70:30) to the oily compound, which was centrifuged at 4 o C for 10 minutes. After centrifugation, a two phases mixture was formed. The supernatant was taken and analysed by HPLC (Shimadzu -SPD20A) using a C18 column. The flow rate was maintained at 1 mL/min and the column temperature at 60 o C. Acetonitrilewater (70:30) was used as mobile phase and the UV detector was kept at 210 nm. The injection sample volume was 20 µL.

Calibration Method
The samples used in the calibration method were prepared by adding FK506-standard to 3.5 mL of mobile phase (acetonitrile-water 70:30). These samples concentrations were 30, 60, 90, 120 and 150 mg.L -1 . The FK506-standard was purchased from Sigma-Aldrich (CAS109581-93-3). The quantification of these samples was made using the same conditions cited in the previous item.

Fermentable Sugars and Proteins Quantification
In order to quantify fermentable sugars and proteins, Somogyi-Nelson and Bradford methods were used, respectively. 18,19 Results Tacrolimus and Biomass Production By aiming to enhance the tacrolimus production, the fermentative process was conducted based on previous literature. 15 The tacrolimus production increased along the fermentation, reaching a maximum concentration of 41.87 g.L -1 at 96 hours. After this period of time, the drug concentration reduced until 5.02 mg.L -1 (168 hours). The biomass production was low and reached the highest concentration at 120 hours. The specific tacrolimus production is the ratio between the real drug production and the biomass concentration. The maximum specific production was 4.06 mg.g -1 , which is near the specific production reached by Singh and Behera in a fermentation using cottonseed oil as the carbon source. 14 These results are shown in the Figure 1.
Proteins and Fermentable Sugars Production According to Figure 2, the amount of proteins produced during the fermentative process raised. At the beginning of fermentation (0 h), the proteins concentration in the broth culture was 83.46 mg.L -1 which reached 120.94 mg.L -1 at the end of fermentation. The biggest increase occurred between 96 and 120 hours. After 120 hours, the proteins concentration did not vary significantly. Figure 3 shows the fermentable sugars behavior during fermentation. The amount of sugars was consumed along the process. The initial concentration was 522.04 g.L -1 and the final was 254.56 g.L -1 . The greatest decrease occurred in the first 72 hours.

Discussion
The use of vegetable oils as an alternative carbon source has been an efficient alternative to boost the tacrolimus production during fermentation. 12,14,16,17 In the present research, the Brazil nut (B. excelsa) oil was used because it contains sugars, amino acids, proteins, vitamins, lipids and fatty acids, 20,21 that may be tacrolimus precursors. Besides, the concentration of 30 g.L -1 was used based on a previous study conducted by Singh and Behera,14 which achieved reasonable tacrolimus production through fermentation of S. tsukubaensis, using this oil concentration. Also, literature says that high carbon concentrations in the fermentation medium may inhibit the secondary metabolites production. 22 Therefore, concentrations higher than 30 g.L -1 are not recommended. Figure 1 shows that the highest concentration of tacrolimus obtained was 41.87 mg.L -1 , which is greater than the concentration reported on literature, reached in a fermentation conducted using glucose as the carbon source. 15 This result was expected and agreed with the results of other papers which used other vegetable oils as carbon sources. [12][13][14] It is believed that one of the factors which regulates the bacterial production of secondary metabolites, such as FK506, is the response to nutrients. 12 By analyzing previous research, it is possible to realize that all oils that were successful at improving the tacrolimus production were rich in linoleic acid, 14,23,24 which is an unsaturated fatty acid. 25 This fatty acid is also a major component of the Brazil nut oil, what may indicate that the linoleic acid is a FK506 precursor. It is worth mentioning that the tacrolimus concentration decrease at 120 hours is not predicted by literature. 11,13,15 The biomass production reached its highest values from 120 to 168 hours, which probably corresponds to the death phase, characterized by the unavailability of nutrients resulting in cellular death. 26,27 From 48 to 96 hours, the biomass production almost remained constant because biomass formation competes with secondary metabolites biosynthesis. 28 The amount of proteins increased during the tacrolimus biosynthesis. It is actually known that proteins are consumed and produced along the fermentative process, 12 so in the present study the producing rate was higher than the consuming rate, which may be considered as a problem to this drug purification. On the other hand, the amount of fermentable sugars decreased along the process. These sugars were mainly supplied to the S. tsukubaensis through the culture broth and are catabolized to produce energy. The production of secondary metabolites, such as tacrolimus, is a bacterial response to a stress condition (reduction of nutrients, for example). 28 Figures 1 and 3 show that the greatest tacrolimus production occurred at 96 hours, when the amount of fermentable sugar available in the culture broth had already diminished, what is in agreement with the literature.

Conclusions
The usage of Brazil nut oil (B. excelsa) as a carbon source in S. tsukubaensis fermentation increased the tacrolimus production and at the same time did not stimulate the biomass increase, which makes this vegetable oil a feasible option to replace traditional carbon sources. Future studies should investigate the influence of the linoleic acid in the FK506 production, since all fermentations using oils containing this substance were successful at enhancing tacrolimus production. Also, the proteins production and the fermentable sugars consumption occurred as expected. These results may be useful for pharmaceutical industries focused on enhancing tacrolimus production, particularly in Brazil, since this vegetable oil is an affordable native product.