1. ORIGINAL ARTICLE1
2
Nutritional Control of Antibiotic Production by3
Streptomyces platensis MA7327: Importance of L-4
Aspartic Acid5
Maria Falzone, Emmanuel Crespo, Klarissa Jones, Gulaba Khan, Victoria L.6
Korn, Amreen Patel, Mira Patel, Krishnaben Patel, Carrie Perkins, Sana7
Siddiqui, Drew Stenger, Eileen Yu, Michael Gelber, Robert Scheffler, Vasyl8
Nayda, Ariela Ravin, Ronica Komal, Jeffrey D. Rudolf, Ben Shen, Vincent9
Gullo and Arnold L. Demain1
10
Streptomyces platensis MA7327 is a bacterium producing interesting antibiotics which11
act by the novel mechanism of inhibiting fatty acid biosynthesis. The antibiotics12
produced by this actinomycete are platensimycin and platencin plus some minor related13
antibiotics. Platensimycin and platencin have activity against antibiotic-resistant14
bacteria such as methicilin-resistant Staphylococcus aureus (MRSA) and vancomycin-15
resistant Enterococcus (VRE); they also lack toxicity in animal models. Platensimycin16
also has activity against mice in a diabetes model. We have been interested in studying17
the effects of primary metabolites on production of these antibiotics in our chemically-18
defined production medium. In the present work, we tested 32 primary metabolites for19
their effect. They included 20 amino acids, 7 vitamins and 5 nucleic acid derivatives. Of20
these, only L-aspartic acid showed stimulation of antibiotic production. We conclude21
that the stimulatory effect of aspartic acid is due to its role as a precursor involved in22
the biosynthesis of aspartate-4- semialdehyde, which is the starting point for the23
biosynthesis of the 3-amino-2,4-dihydroxy benzoic acid portion of the platensimycin24
molecule.25
________________________________________________________________26
__27
1
Research Institute of Scientists Emeriti (RISE), Drew University, Madison, New Jersey28
07940, USA; Correspondence: Dr. A.L. Demain, Research Institute of Scientists Emeriti29
(RISE), Drew University, Madison, New Jersey 07940, USA; E-mail: ademain@drew.edu30
31
32
33
2. INTRODUCTION34
The world is in desperate need for new antibiotic compounds. Resistance to35
currently available drugs is growing and rapidly spreading.1
Every class of36
antibiotic drugs has at least one, and often more than one, pathogen that shows37
resistance against it; moreover, every major pathogen is resistant to at least one38
type of antibiotic.2
This situation is problematic because a myriad of diseases39
develop that are difficult to treat and are often easily spread. Two examples of40
problematic resistant pathogens are methicillin-resistant Staphylococcus aureus41
(MRSA) and vancomycin-resistant Enterococcus (VRE), each of which kills42
tens of thousands of people per year.3,4
Because of resistant pathogens like these,43
infectious diseases remain a leading cause of death worldwide, even in44
developed countries.45
Two compounds that have shown promising activity against resistant46
pathogens are platensimycin and platencin. These compounds work by47
inhibiting FabF and FabH, two key elongation enzymes in the fatty acid48
synthesis II (FASII) pathway. This is an attractive antibiotic target due to a lack49
of similarity between the prokaryotic and eukaryotic fatty acid synthesis50
enzymes.5
Accordingly, platensimycin and platencin potentially have low51
cytotoxicity in humans, adding to their promise. Both compounds are natural52
products made by the actinomycete Streptomyces platensis MA7327.53
Platensimycin was discovered by Merck & Co. in 2006 in a soil sample from54
South Africa6
, using a target-based whole-cell antisense differential sensitivity55
assay for inhibitors of fatty acid biosynthesis. It is characterized by a 3-amino-56
2,4-dihydroxybenzoic acid and C-17 pentacyclic enone with an ether ring joined57
by an amide bond and a hydrophobic region at the latter end of the molecule58
(Figure 1). Platencin was discovered using the same59
approach on soil samples from Spain in 2009. Its structure is very similar to60
platensimycin; the main structural difference is the presence of a ketolide61
portion lacking the cyclic ether ring (Figure 1).62
In addition to their activity against resistant pathogens (MRSA and VRE),63
both antibiotics exhibit potent antibacterial activity (MIC=0.06-2 ug/ml) against64
Gram-positive pathogens7
, including penicillin-resistant Streptococcus65
pneumoniae (PRSP).8
They have also shown activity against mycobacteria such66
as Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium67
smegmatis.9
Platensimycin has the potential to be an effective treatment for68
tuberculosis because it inhibits biosynthesis of fatty acids and mycolic acid in M.69
tuberculosis.9,10
. Of further interest, platensimycin has shown potential as an70
agent for treatment of diabetes in mouse models and has potential as an agent71
for treatment of type II diabetes mellitus.11
Despite problems with their72
pharmokinetic properties, platensimycin and platencin are promising because of73
their effective and non-toxic mode of action.74
These antibiotics have been reviewed by Singh12
and Martens and75
Demain.13
Also produced by S. platensis are analogs such as platensic acid, the76
3. 2,4-diaminobutyric acid amide derivative14
, platensimycin A1 (hydroxylated77
platensimycin) and its methyl ester, hydroxyplatensic acid and its methyl ester15
,78
and platensimycin B4 and its methyl ester.79
Following the discovery of platensimycin and platencin, Merck scientists80
produced these antibiotics in the laboratory by culturing S. platensis in a crude,81
insoluble production medium. Since that time, we developed a semi-defined16
,82
and then a completely chemically-defined production medium (PM7).17
Using83
PM7, we have been investigating the nutritional control of antibiotic production84
by S. platensis MA 7327, which could provide valuable information about the85
biosynthesis and regulation of the biosynthetic pathways of platensimycin and86
platencin.87
88
MATERIAL AND METHODS89
Media and fermentation90
All media were made using distilled water, and the pH was adjusted to 7 using91
concentrated potassium hydroxide. The stock culture medium contained 30g/L92
Difco Trypticase Soy Broth. The seed medium developed by Merck and93
employed in this study contained (g/L): 20 soluble starch, 10 dextrose, 5 NZ94
Amine type A, 3 beef extract, 5 Bacto-Peptone, 5 yeast extract and 1 CaC03. All95
flasks were autoclaved at 121°C for 30 minutes prior to use. The stock culture96
medium was inoculated with a sample of S. platensis MA7327, and incubated97
on the gyratory shaker at 220 rpm at 28°C for two days followed by storage in98
the refrigerator until use. The seed medium was inoculated with 0.5 mL of S.99
platensis stock culture and shaken at 220 rpm and 28°C for five days. The five-100
day seed culture was spun down in a sterile 50-mL conical tube using a clinical101
one-speed centrifuge for 10 minutes. The supernatant fluid was discarded,102
replaced with an equal volume of sterile water, and the pellet was re-suspended.103
This washing process was repeated two more times. The culture was stored as a104
liquid in the refrigerator after the third addition of water. Fermentations were105
carried out in 125-mL Erlenmeyer flasks containing 25 mL of fermentation106
broths on a rotary shaker at 28o
C and 250 rpm.107
108
Compound testing109
Flasks with chemically-defined medium PM7 (g/L: 30 glucose, 25 lactose, 0.5110
ammonium sulfate and 5 MOPS buffer), with and without added compounds,111
were inoculated with 0.5 mL of washed seed culture per 25 mL of media. Each112
experimental condition was evaluated using duplicate flasks. Vitamins tested113
were ascorbic acid, folic acid, thiamine, inositol, d-biotin, riboflavin and114
vitamin B12. They were tested at 10 ug/ml, except for vitamin B12 which was115
tested at 1 ug/ml. Nucleic acid components, such as purines (adenine and116
guanine) and pyrimidines (cytosine, thymine, uracil ), were examined at 0.5117
mg/ml. L-Amino acids were alanine, aminoadipic acid, arginine, asparagine,118
4. aspartic acid, citrulline, cysteine, glutamine, glycine, histidine, isoleucine,119
leucine, methionine, ornithine, phenylalanine, threonine, serine, tryptophan,120
tyrosine, and valine. They were tested at 1 mg/ml. After adding the additives,121
the pH was adjusted to 7.0. To evaluate effects of the additives, their zones of122
inhibition were compared directly to the PM7 control.123
124
Agar diffusion assay125
To test for antibiotic activity, zone of inhibition assays against S. aureus,126
Bacillus subtilis, and/or Bacillus megaterium were carried out in Petri plates127
(Aluotto et al.16
). Stock cultures of these test organisms were made by128
inoculating 0.5 mL of a liquid culture into 25 mL of stock culture medium (see129
above) and incubating on the shaker at 35ºC for two days. 37 g/L of LB agar130
Miller was suspended in de-ionized water and autoclaved at 121°C for 30131
minutes. After autoclaving, the mixture was allowed to cool for a period of132
time and was then inoculated with 2.5 mL of stock culture for every 100 mL of133
agar. The inoculated agar was distributed into sterile Petri plates at 10 mL per134
plate.135
To assay, a small amount of the fermentation broths were aseptically136
removed from each experimental flask and placed into individual tubes. A137
paper disk, 6 mm in diameter, was dipped into each tube, dried briefly, and138
placed onto the agar plates. Plates were inverted and placed in the incubator at139
35°C for 2 days. Clear zones of inhibition around the disks were measured in140
millimeters across the diameter on two different sides and averaged. Each plate141
was read by two to three individuals, and the readings were averaged to142
generate a value for each flask. Values for identical flasks were also averaged,143
generating one value for each experimental condition. Conditions were144
compared by making a general comparison of the zone of inhibition size145
throughout the fermentation period, as well as by comparing the highest zone of146
inhibition observed, regardless of which day it occurred. Assays were conducted147
every one to three days, and zone sizes were compared throughout the length of148
the experiment.149
150
151
RESULTS152
Table 1 shows the results obtained with the 32 compounds tested (20 amino153
acids, 7 vitamins and 5 nucleic acid derivatives). The only active compound154
was L-aspartic acid. Addition of L-aspartic acid at 1 mg/mL consistently155
increased the zone of inhibition size compared to the PM7 control, an example156
of which is shown in Figure 2.157
After consistently observing the stimulation by aspartic acid at 1 mg/mL,158
we tested it at other concentrations, both higher and lower, to find its optimal159
stimulatory concentration. Higher concentrations of aspartic acid, i.e. 3 mg/mL160
and 5 mg/mL did not increase the zone of inhibition size relative to PM7 plus 1161
5. mg/ml aspartic acid. Lower concentrations, i.e. 0.25 and 0.5 mg/ml were also162
tested. The optimal result was obtained with 0.5 mg/ml. aspartic acid as163
compared with all concentrations tested.164
165
DISCUSSION166
Using agar diffusion assays, we have shown that addition of aspartic acid to167
chemically- defined production medium PM7 increases antibiotic production by168
S. platensis. The optimal concentration for stimulation was 0.5 mg/ml.169
The aspartic acid stimulation is interesting, given what we know about the170
biosynthesis of platensimycin and platencin. Aspartate-4-semialdehyde, which171
is synthesized from aspartic acid, is the starting material for the biosynthesis of172
the 3-amino-2,4-dihydroxybenzoic acid (AHDBA) portion of both molecules, as173
shown in Figure 3. Isotopic labeling studies have also connected this synthesis174
to the citric acid cycle, which is connected to aspartic acid synthesis.18,19
175
Between glycolysis and the citric acid cycle, pyruvate is converted to acetate,176
which enters the citric acid cycle.20
In the citric acid cycle, two molecules of177
acetyl CoA are incorporated into two molecules of oxaloacetate, which is178
involved in the synthesis of aspartic acid. Aspartic acid is then converted to179
aspartate-4-semialdehyde to serve as starting material for AHDBA synthesis.180
The link of the citric acid cycle to this synthesis highlights the importance of181
aspartic acid, which is reflected in our experimental studies. These results are182
helpful in better understanding the biosynthesis and nutritional control of183
antibiotic formation. These studies are also helpful in working towards a184
production medium that yields higher levels of antibiotic.185
Now that we have successfully evaluated the effects of added primary186
metabolites on antibiotic production, we will use chemically-defined PM7 plus187
aspartic acid to test the effects of inorganic salts. These additional studies will188
allow us to further investigate the biosynthesis and nutritional regulation of189
antibiotic production by S. platensis.190
191
192
CONFLICT OF INTEREST193
The authors declare no conflict of interest.194
195
ACKNOWLEDGEMENTS196
Authors include (a) former and present undergraduate students at Drew197
University: MF, EC, KJ, GK, VK, AP, MP, KP, SS, DS, RS, VN and RK, (b)198
visiting scientist CP, and (c) high school students MG, AR, and EY. Co-authors199
Jeffrey D. Rudolf and Ben Shen are from Scripps Institute, Florida. Vincent200
Gullo and Arnold L. Demain are Research Fellows of the Research Institute for201
Scientists Emeriti (RISE) at Drew University. We acknowledge the202
6. encouragement by RISE Director Dr. Jon Kettenring, and the assistance of203
RISE Administrative Assistant Miriam Donohue and Nilya Schaber,204
RISE/ResMed Program Coordinator.205
------------------------------------------------------------------------------------------------206
----207
208
1 Laxminarayan, R. et al. Antibiotic resistance-the need for global solutions.209
The Lancet Inf. Dis.13, 1057-1098 (2013).210
2 Allahverdiyev, A. M. et al. The use of platensimycin and platencin to fight211
antibiotic resistance. Infect. Drug Res. 6, 99-114 (2013).212
3 Klevens, R. M. et al. Invasive methicillin-resistant Staphylococcus aureus213
infections in the United States. JAMA 298, 1763-1771 (2007).214
4 Courvalin, P. Vancomycin resistance in gram-positive cocci. Clin. Infect. Dis.215
42 Suppl 1, S25-S34 (2006).216
5 Wang, J. et al. Discovery of platencin, a dual FabF and FabH inhibitor with217
in vivo antibiotic properties. Proc. Natl. Acad. Sci. USA, 104, 7612-7616218
(2007).219
6 Wang, J. et al. Platensimycin is a selective FabF inhibitor with potent220
antibiotic properties. Nature 441, 358-361 (2006).221
7 Singh, S.B., & Young, K. New antibiotic structures from fermentations.222
Expert Opin. Ther. Patents 20,1359-1371 (2010).223
8 Tiefenbacher K. & Mulzer J. A nine-step total synthesis of (-)-platencin. J.224
Org. Chem. 74, 2937-2941 (2009).225
9 Brown, A. K., Taylor, R. C., Bhatt, A., Fütterer, K., & Besra, G. S.226
Platensimycin activity against mycobacterial -ketoacyl-ACP synthases.227
PLoS One 4(7), 1-10 (2009).228
10 Ashforth, E.J. et al. Bioprospecting for antituberculosis leads from microbial229
metabolites. Nat. Prods. Repts. 27, 1709-1719 (2010).230
11Wu, M. et al. Antidiabetic and antisteatotic effects of the selective fatty acid231
synthase (FAS) inhibitor platensimycin in mouse models of diabetes. Proc.232
Natl. Acad. Sci. USA, 108, 5378-5384 (2011).233
7. 12 Singh, S.B. Discovery and development of platensimycin and platencin. In234
Drug Discovery from Natural Products (eds. Genilloud, O. & Vicente, F.);235
chapter 12 in RSC Drug Discovery Series No. 25. 249-277; (Royal Society of236
Chemistry, 2012).237
13 Martens, E., & Demain, A. L. Platensimycin and platencin: promising238
antibiotics for future application in human medicine. J. Antibiot., 64, 705–239
710 (2011).240
14 Herath, K.B. et al. Structure and semisynthesis of platensimide A, produced241
by Streptomyces platensis. Org. Lett., 10, 1699-1702 (2008).242
15 Singh, S.B. et al. Isolation, enzyme-bound structure, and activity of243
platensimycin A1 from Streptomyces platensis. Tetrahedron Lett., 50, 5182-244
5185 (2009).245
16 Aluotto, S. et al. Development of a semi-defined medium supporting246
production of platensimycin and platencin by Streptomyces platensis. J.247
Antibiot., 66, 51-54 (2013).248
17 Falzone, M. et al. Development of a chemically defined medium for the249
production of the antibiotic platensimycin by Streptomyces platensis. Appl.250
Microbiol. Biotechnol., 97, 9535-9539 (2013).251
18 Herath, K., Attygalle, A., & Singh, S. B. Biosynthetic studies of platencin.252
Tetrahedron Lett., 49, 6265-6268 (2008).253
19 Herath, K. B., Attygalle, A. B., & Singh, S. B. Biosynthetic studies of254
platensimycin. J. Am. Chem. Soc., 129, 15422-15423 (2007).255
20 Berg, J.M, Tymoczko, J.L., & Stryer, L. Biochemistry, 7 ed. W. H.256
Freeman and Company. NewYork, NY, pp. 499-511; 710-714 (2012).257
21 Smanski, M.J., Peterson, R.M., & Shen, B. Platensimycin and platencin258
biosynthesis in Streptomyces platensis, showcasing discovery and259
characterization of novel bacterial diterpene synthases. Methods Enzymol.260
515:163-186 (2012).261
22 Dairi, T. Studies on the biosynthetic genes and enzymes of isoprenoids262
produced by actinomycetes. J. Antibiot. 58:227-243 (2005).263
264
265
266
