Biosynthesis and mechanism of action of a sideromycin classantibiotic from Streptomyces sp
Shumpei ASAMIZU
Graduate School of Agricultural and Life Sciences, The University of Tokyo
Research aims
Infectious diseases caused by multidrug-resistant bacteria are anticipated to become amajor cause of death within the next 30 years; therefore, there is a strong demand for thedevelopment of antibiotics with new mechanisms of action. Microbes such as Actinomycetes
are important resources for the production of antibiotics, and their metabolites havecontributed to the fight against human infectious disease for nearly 80 years. Furthermore,with the rapid development of genome analysis technology since 2000, it is strongly
suggested that a huge number of untapped potential secondary metabolites still exist.
In an approach to activate the potential secondary metabolic ability of Actinomycetes,studies demonstrating the efficiency of using the interaction between microorganisms(co-culture) have gradually accumulated. In this study, we screened for antibiotics
specifically produced in a combined-culture of Actinomycetes and mycolic acid-containing
bacteria (MACB), and characterized a newly discovered sideromycin class antibiotic.
Methods and results
In 2014, soil samples were collected from the Forest of the University of Tokyo in
Furano, Hokkaido, and 59 species of Actinomycetes were isolated. A combined-culture ofthese strains and the MACB, Tsukamurella pulmonis TP-B0596, was performed. Culture
extracts showing growth inhibition against Staphylococcus aureus, which was specificallyinduced during the combined-culture, were investigated. As a result of screening, theHOK021 strain, identified as Streptomyces hygroscopicus by 16S rDNA analysis, was foundto produce a substance exhibiting growth inhibition against S. aureus specifically during
combined-culture.
Based on the data obtained by ultra-high performance liquid chromatography-quadrupole
time-of-flight mass spectrometry (MS) analysis, the active substance contained in the
combined-culture extract was estimated, and the bacterial fatty acid synthesis inhibitor

platensimycin (PTM) 1) was identified. Furthermore, MS network analysis revealed thatmany unknown PTM analogues were included; thus new analogues were purified using MS
information as an index. From 10.8 L of the combined-culture, 8.7 g of crude extract wasobtained by ethyl acetate extraction, and 16.7 mg of the target compound was purified with alow-pressure octadecylsilyl column and two cycles of preparative HPLC. As a result ofstructural analysis using 1H, 13C, and 2D NMR data, the purified compound was revealed tobe a substance in which the antibiotic PTM was linked to the siderophore enterobactin(ENT) 2) dehydrated dimer (DED) through a thioester bond. This novel sideromycin-likeantibiotic was named harundomycin (HDM). 3)
To obtain knowledge on the mechanism of HDM synthesis, especially the thioester
bond formation that links PTM and the DED, the newly purified thioPTM 4) and DED wereincubated under physiological buffer conditions, but HDM formation could not be confirmed.
This suggested that some intracellular catalyst was required for bond formation. Further, thedraft genome sequence of the HOK021 strain was acquired and analyzed. Specific genes
with high homology to known PTM biosynthetic genes and ENT biosynthetic genes wereidentified in the HOK021 genome. Future plans involve acquiring more accurate genome
sequence data so that the identification of cellular factors involved in bond formation cancontinue.
In collaboration with Prof. Yoshichika Arakawa, Graduate School of Medicine,
Nagoya University, MIC measurements on various bacteria were performed to clarify theantibacterial spectrum of HDM. HDM showed an MIC of 16-32 µM against
methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci(VRE). 3) However, it did not show antibacterial activity against E. coli, which produces
ENT as a siderophore, or other Gram-negative bacteria. This was consistent with the fact thatPTM itself does not exhibit antibacterial activity against Gram-negative bacteria. In addition
to HDM having an ENT dimer unit, new analogs that bound an ENT monomer or trimer
were recently isolated. Currently, MIC measurements of these analogs under conditions
where iron is depleted from the medium are planned to investigate whether there is a change
in antibacterial activity against Gram-positive bacteria, such as MRSA, in iron-depletedconditions.
Conclusion
In this study, our group discovered a novel sideromycin-like antibiotic, HDM, from a

combined-culture of S. hygroscopicus HOK021 and T. pulmonis, and analyzed theproduction mechanism and biological activity. "Trojan horse" antibiotics are a group ofantibiotics that enter the cell using the transporters of target bacteria and exert antibacterial
activity through cleavage of the antibiotic unit, mostly by hydrolysis. Among them, a groupof antibiotic conjugants using a siderophore as a “carrier cargo” are called sideromycin classantibiotics, which when connected to an antibiotic warhead, are transported into the cell via
a siderophore-specific transporter, resulting in enhanced antibacterial activity. 5)
The presence of the “Trojan Horse” natural antibiotics can be thought to reflect theexistence of a natural logic that actively attacks the competitor in a microbial interaction. As
HDM is produced specifically in combined-culture, the regulation of the mechanism ofproduction would be also interesting. I would like to analyze the biosynthetic mechanismand mechanism of action of HDM in more detail at the molecular level, which could lead tothe development of new research areas. In the future, using methodologies such as MS
network analysis, I will screen for new sideromycin antibiotics in the extracts ofActinomycetes and MACB combined-culture to identify naturally-occurring sideromycinclass compounds and explore a new direction for drug discovery.
References
1) Wang, J. et al. (2006) Platensimycin is a selective FabF inhibitor with potent antibiotic
properties. Nature. 441: 358-361.
2) Reitz, ZL., Sandy, M., Butler, A. (2017) Biosynthetic considerations of triscatechol
siderophores framed on serine and threonine macrolactone scaffolds. Metallomics. 9:824-839.
3) Asamizu, S., Sugai, Y., Onaka, H. (2018) A new sideromycin-like antibiotic fromcombined-culture of Streptomyces hygroscopicus HOK021 and Tsukamurella pulmonis.
IFO Res. Commun. 32: 117-127.
4) Dong, LB. et al. (2018) Biosynthesis of thiocarboxylic acid-containing natural products.
Nat Commun. 9: 2362.
5) Braun, V., Pramanik, A., Gwinner, T., Köberle, M., Bohn, E. (2009) Sideromycins: tools
and antibiotics. Biometals. 22: 3-13.