Novel Benzene-Containing Polyketides and Modified Diketopiperazine from Actinomycete of the Genus Cryptosporangium

Academic Background

Microbial natural products hold significant value in drug development, particularly actinomycetes, which provide a rich source of various therapeutic compounds, such as aminoglycosides, macrolides, and tetracyclines. However, the frequent re-isolation of known compounds has become a major obstacle in natural product-based drug discovery in recent years. To address this issue, researchers have attempted various new methods, such as co-culturing, adding secondary metabolic signaling molecules, or applying high temperatures during cultivation. The authors of this study chose to focus on less-studied actinomycete genera to discover new compound scaffolds.

The subject of this study is the genus Cryptosporangium, which was first described in 1998 and currently includes eight known species. Although bioinformatics analysis has identified biosynthetic gene clusters (BGCs) for polyketides and non-ribosomal peptides in this genus, only two novel tetronate-class polyketides had been reported previously. This paper reports the isolation of two new benzene-containing polyketides (cryptoic acids A and B) and a new acylated diketopiperazine (cyclocryptamide) from Cryptosporangium sp. YDKA-T02, along with studies on their structures, biological activities, and biosynthetic potential.

Source of the Paper

This paper was co-authored by Md. Julkar Nime, Hideki Yamamura, Masayuki Hayakawa, Nobuyasu Matsuura, Naoya Oku, and Yasuhiro Igarashi. The authors are affiliated with the Biotechnology Research Center at Toyama Prefectural University, the Faculty of Life and Environmental Sciences at the University of Yamanashi, Yamanashi Prefectural University, and the Department of Bioscience at Okayama University of Science. The paper was submitted on October 2, 2024, revised on November 20, 2024, accepted on November 21, 2024, and published in The Journal of Antibiotics with the DOI 10.1038/s41429-024-00794-4.

Research Process and Results

1. Strain Isolation and Identification

The study began with the isolation of an actinomycete strain, YDKA-T02, from agricultural field soil collected on the campus of the University of Yamanashi, Japan. Through 16S rRNA gene sequence analysis, the strain showed 98.11%-99.58% similarity to known species of the genus Cryptosporangium, with the closest match being Cryptosporangium eucalypti EURKPP3H10T. Scanning electron microscopy revealed that the strain exhibited typical morphological features of the genus Cryptosporangium, such as the formation of sporangia with motile spores.

2. Fermentation and Compound Isolation

Preliminary fermentation experiments using three different media identified A11 medium as the most productive. High-performance liquid chromatography (HPLC) and UV spectral analysis revealed three prominent peaks whose UV spectra did not match any known microbial secondary metabolites in the database. Subsequently, large-scale fermentation was conducted using V-22 liquid medium for seed culture over seven days, followed by fermentation in A11 liquid production medium for 14 days. The culture broth was extracted with 1-butanol, and the extract was fractionated using silica gel and ODS column chromatography, followed by HPLC purification to yield three compounds: cryptoic acid A (1), cryptoic acid B (2), and cyclocryptamide (3).

3. Structural Identification

The molecular formulas of the three compounds were determined using high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry (HR-ESI-QTOF-MS) and nuclear magnetic resonance (NMR) analysis. The molecular formula of cryptoic acid A (1) was determined to be C22H26O3, cryptoic acid B (2) as C22H24O4, and cyclocryptamide (3) as C18H22N2O4. Further structural elucidation was achieved through 1D and 2D NMR spectroscopy. The absolute configuration of cyclocryptamide (3) was determined to be L-leucine and L-proline using Marfey’s method.

4. Biological Activity Testing

The researchers tested the cytotoxicity and antimicrobial activity of the three compounds. Results showed that cryptoic acid A (1) and B (2) exhibited moderate cytotoxicity against P388 murine leukemia cells, with IC50 values of 27 μM and 29 μM, respectively, while cyclocryptamide (3) showed no cytotoxicity even at 100 μM. Additionally, compounds 1 and 2 showed no antimicrobial activity at 200 μg/mL against various microorganisms, including Gram-positive bacteria, Gram-negative bacteria, yeast, and fungi. However, compounds 1 and 2 demonstrated peroxisome proliferator-activated receptor γ (PPARγ) agonistic activity in the concentration range of 6.25-100 μM.

Conclusions and Significance

This study isolated two novel benzene-containing polyketides and a new acylated diketopiperazine from Cryptosporangium sp. YDKA-T02, providing detailed insights into their structures and biological activities. The PPARγ agonistic activity of cryptoic acids A and B offers potential candidates for the development of new drugs targeting metabolic diseases. Additionally, the structural novelty of cyclocryptamide provides new perspectives for the biosynthesis of diketopiperazine derivatives.

Research Highlights

1. Novelty: This study is the first to isolate benzene-containing polyketides and acylated diketopiperazines from the genus Cryptosporangium, enriching the chemical diversity of natural products.
2. Biological Activity: The PPARγ agonistic activity of cryptoic acids A and B opens new avenues for research into metabolic disease treatments.
3. Methodological Innovation: The study employed advanced separation and structural identification techniques, such as HR-ESI-QTOF-MS and 2D NMR, providing reliable technical support for natural product research.

Additional Valuable Information

This study further supports the potential of understudied actinomycete genera as sources of novel secondary metabolites, offering new directions for natural product-based drug discovery. Additionally, the exploration of compound biosynthetic pathways provides an important theoretical foundation for future related research.