Appl Microbiol Biotechnol (2013) 97:9535–9539 DOI 10.1007/s00253-013-5201-6
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Development of a chemically defined medium for the production of the antibiotic platensimycin by Streptomyces platensis Maria Falzone & Evan Martens & Heather Tynan & Christian Maggio & Samantha Golden & Vasyl Nayda & Emmanuel Crespo & Gregory Inamine & Michael Gelber & Ryan Lemence & Nicholas Chiappini & Emily Friedman & Ben Shen & Vincent Gullo & Arnold L. Demain
Received: 12 June 2013 / Revised: 14 June 2013 / Accepted: 14 August 2013 / Published online: 11 September 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract The actinomycete Streptomyces platensis produces two compounds that display antibacterial activity: platensimycin and platencin. These compounds were discovered by the Merck Research Laboratories, and a complex insoluble production medium was reported. We have used this medium as our starting point in our studies. In a previous study, we developed a semidefined production medium, i.e., PM5. In the present studies, by varying the concentration of the components of PM5, we were able to develop a superior semi-defined medium, i.e., PM6, which contains a higher concentration of lactose. Versions of PM6, containing lower concentrations of all components, were also found to be superior to PM5. The new semi-defined production media contain dextrin, lactose, MOPS buffer, and ammonium sulfate in different concentrations. We determined antibiotic production capabilities using agar diffusion assays and chemical assays via thin-layer silica chromatography and high-performance liquid chromatography. We reduced crude nutrient carryover from the seed medium by washing the cells with distilled water. Using these semi-defined media, we determined that addition of the semi-defined component M. Falzone : E. Martens : H. Tynan : C. Maggio : S. Golden : V. Nayda : E. Crespo : G. Inamine : M. Gelber : R. Lemence : N. Chiappini : E. Friedman : V. Gullo : A. L. Demain (*) Charles A. Dana Research Institute for Scientists Emeriti (R.I.S.E.), Drew University, Madison, NJ 07940, USA e-mail:
[email protected] B. Shen Department of Chemistry, Scripps Institute, Jupiter, FL 33458, USA B. Shen Department of Molecular Therapeutics, Scripps Institute, Jupiter, FL 33458, USA
soluble starch stimulated antibiotic production and that it and dextrin could both be replaced with glucose, resulting in the chemically defined medium, PM7. Keywords Antibiotic production . Platensimycin . Streptomyces platensis . Media
Introduction The world is in a crisis today with respect to antibioticresistant pathogenic microbes and the lack of new antibiotics (Spizek et al. 2010). Platensimycin is a promising antibiotic produced by Streptomyces platensis strain MA7327 (Martens and Demain 2011). Microorganisms, specifically bacteria, carry out fatty acid synthesis using the type II system, whereas eukaryotes, such as humans, carry it out employing the type I system (Payne et al. 2001). Platensimycin interferes with the action of FabF enzyme, which occurs only in the type II pathway of microorganisms (Wang et al. 2007). Platensimycin has shown the ability to inhibit resistant strains including linezolid- and macrolide-resistant bacteria and vancomycin-resistant enterococci. It is also active against many gram-positive pathogens. Platensimycin has shown activity against Mycobacterium tuberculosis (Potera 2006) and is characterized by its 3-amino-2,4-dihydroxybenzoic acid and C-17 pentacyclic enone with an ether ring joined by an amide bond and a hydrophobic region at the latter end of the molecule (Martens and Demain 2011). Since all previous work by others on the fermentative production of platensimycin has been done in complex media (Singh et al. 2006; Smanski et al. 2009), nothing is known
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about nutritional control of its biosynthesis. In our previous publication (Aluotto et al. 2013), we reported the development of a soluble semi-defined production medium (PM5) containing dextrin, lactose, ammonium sulfate, and MOPS buffer. The present publication describes our experiments using PM5 and improved semi-defined media as well as our development of the first chemically defined production medium for S. platensis.
Materials and methods
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deionized water. The mixture was autoclaved and allowed to cool before inoculating with 2.5 mL of a S. aureus stock culture. The inoculated mixture was placed into Petri dishes at 10 mL per dish. To test for antibiotic production, paper disks (6 mm in diameter) were dipped into the S. platensis MA7327 fermentation broths, laid out to dry on aluminum foil, placed on the S. aureus agar plate, and incubated overnight at 35 °C. Two disks per fermentation flask were used; all fermentations were run in duplicate. The disk assay was carried out for a number of days throughout the course of fermentation.
Stock and seed cultures S. platensis strain MA7327 (ATCC #PTA-5316), which produces platensimycin, was obtained from Merck & Co., Inc. It was grown in a stock culture medium containing 30 g/L Difco Trypticase Soy Broth (TSB) solids in deionized water. The medium was distributed into a 250-mL flask at 50 mL. The flask was autoclaved and inoculated with S . platensis MA7327. The inoculated TSB flask was placed on a gyratory shaker at 220 rpm and 36 °C for 5 days and then used to inoculate a seed medium. The Merck seed medium, which we used, consists of 20 g/L soluble starch, 10 g/L glucose, 5 g/L NZ-Amine type A, 3 g/L Difco beef extract, 5 g/L Difco Bacto peptone, 5 g/L Difco yeast extract, and 1 g/L CaCO3. Before the addition of CaCO3, the pH was adjusted to 7 (Singh et al. 2006). The medium was transferred to 250-mL flasks at 50 mL per flask and autoclaved. Once autoclaved, each seed flask was inoculated with a wire loopful of the S. platensis MA7327 stock culture and incubated on the gyratory shaker at 220 rpm and 28 °C for a period of 5 days. Antibiotic production The original insoluble, complex production medium developed by Merck (which we call PM1) consists of 5 g/L Amberex, 40 g/L yellow cornmeal, and 40 g/L lactose. The semi-defined medium which we developed, i.e., PM5, contains 40 g/L dextrin, 40 g/L lactose, 20 g/L MOPS buffer, and 2 g/L ammonium sulfate. Experiments were conducted in 250-mL Erlenmeyer flasks containing 50 mL of the medium and in 125-mL flasks containing 25 mL of medium. The pH of all production media was adjusted to 7, and flasks were autoclaved for 30 min at 121 °C. The flasks were inoculated with the S. platensis seed culture and incubated on a gyratory shaker at 220 rpm and 28 °C. Agar diffusion assay In order to test for antibiotic production, Difco LB agar plates inoculated with Staphylococcus aureus (Wards Natural Science commercial strain 85 W1178) were used. The plates were made by combining 3.7 g of LB agar solids with 100 mL of
Chemical assay For quantitative chemical assay, a 25-mL portion of fermentation broth was centrifuged in each of four 50-mL centrifuge tubes. To the supernatant fluid, 1.2 M hydrochloric acid was added to adjust the pH to approximately 5. It was then extracted three times with an amount of ethyl acetate equal to the volume of the supernatant. Both the extract and spent aqueous solution were tested for antibacterial activity by the agar diffusion assay. To test the mycelial layer of the centrifuged fermentation broth for the presence of antibiotic activity, 15 mL of acetone was added to the cells and the mixture was stirred for 30 min. It was then centrifuged to remove the cells, and the acetone was evaporated from the supernatant using a rotary evaporator. The aqueous solution was then extracted three times with an equal volume of ethyl acetate. The extract was tested for antibacterial activity. Early procedure (for media PM5 and PM6) Thin-layer silica chromatography (TLC) was used to detect the presence of platensimycin in the samples. A control sample of platensimycin was spotted along with ethyl acetate extracts from the mycelia and the supernatant. The plate was developed with 90 % dichloromethane and 10 % methanol. The TLC plate was then tested for antibacterial activity using bioautography to elute the compounds into agar plates inoculated with S. aureus. The plates were incubated overnight (as described above) and observed for zones of inhibition. High-performance liquid chromatography (HPLC; Agilent 1100 HPLC) was used. Platensimycin control dissolved in methanol and ethyl acetate extracts of the supernatant and mycelia from the fermentation were chromatographed on a C18 column (Phenomenex 5-μm C18, 150×4.6 mm) and eluted with a linear gradient from 15 % acetonitrile/aqueous 0.01 % trifluoroacetic acid to 95 % acetonitrile/aqueous 0.01 % trifluoroacetic acid. All samples were run for 20 min at a flow rate of 1 mL/min. Detection of platensimycin was carried out at 254 nm. The quantity of platensimycin in the fermentation extracts was determined by comparing the concentrations to the reference standard.
Appl Microbiol Biotechnol (2013) 97:9535–9539 Table 1 Effect of the volume of seed added to medium PM5 on antibiotic production
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Amount of inoculum per flask (mL)
Zone diameter at 11 days (mm)
1 3 5
12 14 26
Later procedure (for medium PM7) Liquid chromatography– mass spectrometry was performed on a Waters Micromass LCT spectrometer interfaced with an Agilent 1100 HPLC. Chromatography was performed on an analytical Echelon, C18 4 μm, 100×4.6-mm column using a gradient system from 10 % acetonitrile/aqueous 0.1 % formic acid to 75 % acetonitrile/aqueous 0.1 % formic acid. All samples were run for 15 min at a flow rate of 1.5 mL/min. Detection of the compound was performed using an Agilent 1100 HPLC diode array detector. Peaks were identified by retention time and the presence of the platensimycin molecular ion. Concentrations were determined by comparing the UV peak area to a platensimycin reference standard.
Washing of seed Seed culture at 25 mL was placed in a sterile centrifuge tube and centrifuged for 10 min. After centrifugation, the supernatant fluid was poured off, replaced with sterile deionized water, and centrifuged again. This was repeated once more, and finally, the washed cells were suspended in 25 mL of sterile deionized water.
Results Effect of seed volume In our previous publication (Aluotto et al. 2013), we reported that production of platensimycin occurred in semi-defined
Table 2 Chemical assay of platensimycin produced in production media PM5 and PM6 after growth for 6 days
Medium
Platensimycin concentration (μg/mL)
PM5 PM6
1.40 2.26
medium PM5, as measured by agar diffusion and chemical assays. We confirmed this in the present work and studied the effect of seed volume on production. The amount of seed inoculum was varied between 1 and 5 mL. Table 1 shows that production in semi-defined medium PM5 was considerably affected by the level of seed added per flask, with 5 mL showing the best results and 1 mL the poorest. Improved semi-defined medium PM6 We were interested in determining whether the addition of any single ingredient of the complex, insoluble production medium PM1 to semi-defined medium PM5 would have an effect on antibiotic production. The three ingredients of PM1 were added at the same concentration as that in PM1. We did this experiment using a very small amount of seed (0.5 mL per flask) to lessen the possible effect of carryover of crude seed medium components into the production medium. Assays were observed for up to 6 days. Results are shown in Fig. 1. Of all the nutrients added to PM5, yellow cornmeal showed the greatest stimulation of antibiotic production and Amberex the least. Lactose was the second most active of the three materials added and is the only chemically defined component. We increased its concentration to 60 g/L in a new semidefined medium, PM6, which contains 40 g/L dextrin, 60 g/L lactose, 2 g/L ammonium sulfate, and 20 g/L MOPS buffer. To determine whether the new medium PM6 is better than PM5, we compared antibiotic production in these two media. We used 0.5 mL of inoculum in this experiment. Zone sizes were compared on day 6 with PM6 showing higher production, i.e., 21 mm as compared to 16 mm for PM5. Thus, the additional lactose in PM6 had led to a higher final production than observed in PM5. In the same experiment, quantitative Table 3 Composition of semi-defined media
Fig. 1 Effects of components of complex PM1 on antibiotic production in semi-defined medium PM5. YCM yellow cornmeal
Medium
Dextrin (g/L)
Lactose (g/L)
MOPS buffer (g/L)
Ammonium sulfate (g/L)
PM5 PM6 PM6A PM6B PM6C
40 40 10 10 10
40 60 25 20 50
20 20 5 5 5
2 2 0.5 0.5 0.5
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Appl Microbiol Biotechnol (2013) 97:9535–9539 Table 5 Effect of glucose on replacement of both dextrin and soluble starch
Fig. 2 Comparison of antibiotic production in complex insoluble PM1 and in semi-defined, soluble PM6A inoculated with 5 mL of washed cells
chemical assays of platensimycin in broths were done on the 6-day fermentation samples. The antibiotic was not found in the mycelial sample. The chemical data for the culture supernatants are shown in Table 2 confirming the superiority of PM6 overPM5. Modifications of semi-defined medium PM6 Several dilute versions of semi-defined medium PM6 were also studied. These media, PM6A, PM6B, and PM6C, have the same ingredients as PM6 but in lower amounts (Table 3). Like PM6, they were found to support antibiotic production as measured by agar diffusion (data not shown). Elimination of crude nutrient carryover from the seed medium It is clear that we had been carrying over complex materials from the seed medium to semi-defined production media when we inoculated the culture. The Merck seed medium contains soluble starch, glucose, NZ-Amine type A, beef extract, peptone, yeast extract, and CaCO3. We conducted an experiment to eliminate the carryover by washing the cells. After washing the cells twice with distilled water, we used the washed cell suspension to inoculate experimental flasks containing complex medium PM1 and semi-defined medium
Medium and additives
Largest zone of inhibition (mm)
PM6C PM6C+20 g/L soluble starch PM6C+20 g/L of glucose PM6C without dextrin PM6C without dextrin+30 g/L glucose
17 19 20 no zone 24
PM6A. The results are shown in Fig. 2. As expected, the insoluble complex medium 1 showed earlier and higher production than PM6A. However, it is clear that washed seed is effective in producing antibiotic in both semi-defined and complex media.
Chemically defined medium PM7 We next studied the effects of the components of the complex seed medium on production in semi-defined medium PM6C. We thus tested addition of soluble starch, glucose, NZ-Amine type A, beef extract, Bacto peptone, yeast extract, and CaCO3. We found that soluble starch markedly stimulated production in medium PM6C. Since starch, like dextrin, is semi-defined but not chemically defined, we conducted an experiment to determine if simple chemically defined sugars could replace the semidefined component starch when added to PM6C. The results in Table 4 showed that glucose was as active as soluble starch. In a further experiment, we were interested in whether glucose could replace the dextrin in medium PM6C. Table 5 shows that medium PM6C without dextrin was unproductive and that glucose was again stimulatory and could replace dextrin. Since all of the components in this experimental condition are soluble and chemically defined, we have thus
Table 4 Effect of sugars at 20 g/L on antibiotic production as compared to soluble starch in semi-defined medium PM6C Medium and additives
Largest zone size (mm)
PM6C PM6C+soluble starch PM6C+maltose PM6C+cellobiose PM6C+glucose PM6C+lactose
14 28 15 15 28 13
Fig. 3 Comparison of antibiotic production in soluble chemically defined medium PM7 and insoluble complex medium PM1
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developed the first chemically defined medium (PM7) able to support antibiotic production by S. platensis. Chemically defined medium PM7 contains 30 g/L glucose, 50 g/L lactose, 0.5 g/L MOPS buffer, and 0.5 g/L ammonium sulfate. In a comparison between chemically defined PM7 and complex, insoluble medium PM1 (Fig. 3), it can be seen that although the production in PM7 is less than that observed in PM1, it is effective in support of antibiotic production.
9539 Acknowledgments We thank Dr. Sheo Singh of Merck & Co., Inc. for the S. platensis culture and for platensimycin. The S. aureus test culture was a gift from Dr. Joanna Miller of Drew University. The work was supported by the Drew Summer Science Institute (DSSI) and R.I.S.E. We acknowledge the encouragement of the R.I.S.E. Director Dr. Jon Kettenring and R.I.S.E. Administrator Miriam Donohue. Authors MF, EM, HT, CM, SG, VN, EC, RL, and NC were or are Drew University undergraduates. MG is a high school student. EF is a graduate of Syracuse University.
References Discussion Up to this point, there has been little to no information in the literature about the nutritional control of platensimycin biosynthesis. Such information is necessary to improve production and to learn something about the precursors, biosynthesis and control of the biosynthetic process. The original Merck production medium (PM1) is very complex and could not be used to assess the effect of added compounds on the production of platensimycin. We have spent considerable time on medium development. We were successful in developing a semi-defined medium (PM6) for the production of antibiotic (Aluotto et al. 2013). This medium contained one semidefined component, i.e., dextrin. We noted in the present work that starch is stimulatory when added to a semi-defined medium. Since starch and dextrin are only semi-defined, we were pleased to find out that both could be replaced by glucose. Thus, the important result of this work was the development of the first chemically defined medium capable of supporting the production of platensimycin by S. platensis. We are now ready to examine the effects of primary metabolites (i.e., amino acids, vitamins, and nucleic acid components) and inorganic salts on production. We hope this will provide information about precursors, the biosynthetic pathway, and its regulation.
Aluotto S, Tynan H, Maggio C, Falzone M, Mukherjee A, Gullo V, Demain AL (2013) Development of a semi-defined medium supporting production of platensimycin and platencin by Streptomyces platensis. J Antibiot 66:51–54 Martens E, Demain AL (2011) Platensimycin and platencin: promising antibiotics for future application in human medicine. J Antibiot 64: 705–710 Payne DJ, Warren PV, Holmes DJ, Ji Y, Lonsdale JT (2001) Bacterial fatty-acid biosynthesis: a genomics-driven target for antibacterial drug discovery. Drug Disc Today 6:537–544 Potera C (2006) Novel pentacyclic from soils blocks bacterial fatty acid synthesis. Microbe 1:350–351 Singh SB, Jayasuriya H, Ondeyka JG, Herath KB, Zhang C, Zink DL, Tsou NN, Ball RG, Basilio A, Genilloud O, Diez MT, Vicente F, Pelaez F, Young K, Wang J (2006) Isolation, structure, and absolute stereochemistry of platensimycin, a broad spectrum antibiotic discovered using an antisense differential sensitivity strategy. J Am Chem Soc 128:11916–11920 Smanski MJ, Peterson RM, Rajski SR, Shen B (2009) Engineered Streptomyces platensis strains that overproduce antibiotics platensimycin and platencin. J Antimicrob Ag Chemother 53:1299–1304 Spizek J, Novotna J, Rezanka T, Demain AL (2010) Do we need new antibiotics? The search for new targets and new compounds. J Ind Microbiol Biotechnol 37:1241–1248 Wang J, Kodali S, Lee SH, Galgoci A, Painter R, Dorso K, Racine F, Motyl M, Hernandez L, Tinney E, Colleti SL, Herath K, Cummings R, Salazar O, Gonzalez I, Basilio A, Vicente F, Genilloud O, Pelaez F, Jasasuriya H, Young K, Cully DF, Singh SB (2007) Discovery of platencin, a dual FabF FabH inhibitor with in vivo antibiotic properties. Proc Natl Acad Sci U S A 104:7612–7616
