Food Microbiology (FM) | Bioactive Compounds or Metabolites: Function and Application
Microbiol. Biotechnol. Lett. 2021; 49(1): 32-38
https://doi.org/10.48022/mbl.2010.10001
Hyesook Hyun1, Juo Choi1, 2, Daun Kang2, Yungpil Kim2, Pilgoo Lee 2, Gregory Chung 2 and Kyungyun Cho1*
1Department of Biotechnology, Hoseo University, Asan 31499, Republic of Korea 2MECOX CureMed Co., Seoul 06744, Republic of Korea
Correspondence to :
Kyungyun Cho,
kycho@hoseo.edu
Tubulysins are a group of secondary metabolites produced by myxobacteria that inhibit the function of the eukayotic cytoskeleton. We developed a pair of PCR primers that specifically amplified tubulysin biosynthetic genes. Using these primers, eight out of the eighty-one strains of myxobacteria belonging to the Cystobacteraceae family that harbored putative tubulysin biosynthetic genes were screened through PCR analysis. The selected strains included two Archangium gephyra, two Stigmatella sp., two Vitiosangium cumulatum, and two unidentified myxobacteria. LC-MS analysis of the culture extracts from the selected strains revealed that A. gephyra KYC4066 produced putative tubulysin A and B.
Keywords: Archangium gephyra, myxobacteria, secondary metabolite, tubulysin
Myxobacteria are delta-proteobacteria that produce diverse bioactive secondary metabolites. More than 600 different metabolites have been isolated from myxobacteria [1−3]. Among these metabolites isolated from myxobacteria, seven groups of metabolites exhibit inhibitory activity against the function of the cytoskeleton in eukayotic cells. Substances inhibiting the function of the cytoskeleton possess the potential to be developed as anticancer drugs. For example, epothilones are a group of the substances produced by myxobacteria that inhibit cytoskeleton function [4]. The United States Food and Drug Administration approved Ixabepilone, a semisynthetic derivative of epothilone B, as an anticancer treatment in 2007, as it exhibits excellent effects on cancer cells that possess multi-pharmaceutical resistance [5].
Tubulysins are another group of substances that inhibit the function of the cytoskeleton and have been studied for its superior anticancer activity [6−9]. Tubulysins promote the depolymerization of tubulin polymers [10], while epothilones promote the polymerization of tubulin [4]. Tubulysins are attractive lead structures for development as antineoplastic agents; however, tubulysin-producing strains have not been isolated and reported in Korea.
Tubulysins were previously reported to be produced by the myxobacteria
The Myxobacteria Bank at Hoseo University possesses approximately 2,600 myxobacterial strains that were isolated in Korea. In this study, we attempted to identify tubulysin-producing strains among the myxobacterial strains in the Myxobacteria Bank by screening strains carrying tubulysin biosynthetic genes.
All myxobacterial strains used in this study were isolated from Korean soil by the Myxobacteria Bank at Hoseo University [14, 15]. Among the strains used for this study, another strain number of KYC2615 was MEHO 001, and the accession number for this strain provided by the International Depositary Authority was KCTC 13929BP. Another strain number of KYC4066 was MEHO 003, and the accession number was KCTC 14258BP. VY/3 medium [16] was used for the vegetative growth of the strains, and CYS medium [16] was used for the preparation of culture extracts. All strains were cultured at 32℃. Synthetic tubulysin (TAM1446) was purchased from Tub Pharmaceuticals GmbH, Austria.
Two oligonucleotides, 5′-GTGCAGAAYCTGCGGCTCT T-3′ (H403) and 5′-AGCTGCTCCTGGAACCACA-3′ (H404) were used to detect tubulysin bioisynthetic genes using PCR. Two oligonucleotides, 5′-GAGTTTGATCCTGGCTCAG- 3′ (27f) and 5′-AGAAAGGAGGTGATCCAGCC-3′ (1525r) that were reported previously [17] were used for PCR amplification of 16S rRNA genes. PCR was performed using 2X
DNA sequences of the PCR products were determined by Macrogen (Korea) and were analyzed using the BLAST program [18]. The phylogenetic analysis of the strains based on 16S rRNA sequences was performed using the MEGA-X program [19] and the 16S rRNA database from EZBioCloud [20].
Myxobacterial strains were cultured in CYS liquid media with 1% Amberlite XAD-16 resin (Sigma, USA) for 7 days. The resin was harvested and extracted using methanol. Methanol was evaporated from the extract, and the residue was dissolved in a mixture of ethyl acetate and water in a 1:1 ratio (v/v). After the ethyl acetate and water layers were separated, the ethyl acetate layer was recovered and dried. The dried residue was dissolved in methanol.
HPLC was performed using an HPLC system (1260 VL Infinity Series; Agilent, USA) equipped with a Zorbax SB-C18 column (4.6 × 150 mm, 5 μm) (Agilent). Mobile phase A was water and mobile phase B was acetonitrile, and both mobile phases contained 0.1% formic acid. The gradient elution at a flow rate of 0.5 ml/min was performed as follows: 0−5 min 30% B (isocratic), 5−25 min 30−60% B (linear gradient), 25−30 min 60− 100% B (linear gradient), and 30–35 min 100% B (isocratic).
Liquid chromatography mass spectrometry (LC-MS) was performed using an Accelar UHPLC (Thermo Scientific, USA) equipped with an Acquity UPLC® BEH C18 column (2.1 × 150 mm, 1.7 μm) and an LTQ-Orbitrap XL high-resolution mass analyzer located at Gyeonggi Bio-Center. Mobile phase A was water, mobile phase B was acetonitrile, and both mobile phases contained 0.1% formic acid. The gradient elution at a flow rate of 0.4 ml/min was performed as follows: 0−1 min 20% B (isocratic), 1−20 min 20−80% B (linear gradient), 20−24 min 80− 100% B (linear gradient), and 24−27 min 100% B (isocratic).
In this study, we used PCR analyses in an attempt to select a tubulysin-producing strain by screening strains with tubulysin biosynthetic genes from among the 81 strains of
To develop PCR primers specific for the tubulysin biosynthesis genes, the nucleotide sequences of the tubulysin biosynthesis genes from
When PCR was performed on 81 strains using the H403 and H404 primer pair, it was found that a PCR product of approximately 1.4 kb in size was generated from 8 strains (KYC1032, KYC2615, KYC2714, KYC2827, KYC2836, KYC2837, KYC2840, and KYC4066). Analysis of the nucleotide sequences of the resulting PCR products indicated that all of the PCR products possessed the end of the
Six of the 8 selected strains (KYC1032, KYC2615 (= MEHO 001), KYC2714, KYC2836, KYC2837, and KYC4066 (= MEHO 003)) were pure isolated strains, while KYC2827 and KYC2840 were not. The 27f and 1525r primers were used to amplify the 16S rRNA gene of 6 pure isolated strains by PCR. The nucleotide sequences of the resulting PCR products were determined and then used to identify the strains according to phylogenetic analysis. The nucleotide sequences of the 16S rRNA genes from the KYC2615 and KYC4066 strains were 99.11% and 99.79% identical, respectively, to that of DSM 2261T, which is the type strain of
To test if the 8 strains selected through PCR detection actually produce tubulysins, culture extracts were prepared and analyzed by HPLC. When the culture extract of the KYC4066 strain was analyzed, the peak substances detected at 22.58 and 25.21 min exhibited a PDA spectrum that was highly similar to that of TAM1446, a synthetic tubulysin (Fig. 3). Subsequently, these peak substances were separated by HPLC and then analyzed by mass spectrometry. The 22.58 min peak substance exhibited an m/z value of 830.4334 ([M+H]+) that was highly similar to the theoretical molecular weight of tubulysin B (830.4368 ([M+H]+)), and the 25.21 min peak substance possessed a m/z value of 844.4487 ([M+H]+) that was highly similar to the theoretical molecular weight of tubulysin A (844.4525 ([M+H]+)) (Fig. 4). When all the possible chemical formulas possessing a molecular weight within Delta (ppm) ±5 were analyzed, no other substances produced by myxobacteria other than tubulysin A and B were found. Therefore, the 22.58 min and 25.21 min peak substances detected in the culture extract from the KYC4066 strain were highly likely to be tubulysin B and tubulysin A, respectively.
Searching for microorganisms that produce specific substances requires a great deal of time and effort. Even more efforts are required when the microorganisms that produce a specific target substance are not common or when the substance is very difficult to analyze due to small production amounts. Tubulysins inhibit the cytoskeleton and exhibit excellent anticancer activity. Therefore, many studies examining this substance have been conducted. However, only a few of the strains producing trubulysins are known worldwide. Additionally, the trubulysin producing strains produce trace amounts of tubulsysins. In this study, we developed a primer pair that specifically detects the tubulysin biosynthesis genes, and used it in PCR analyses to select 8 strains containing the tubulysin biosynthesis gene. By analyzing the culture extracts of these strains, we confirmed that
Of the 81 strains used in this study, 24 were presumed to be
Tubulysins were first reported to be produced by the myxobacteria
The authors have no financial conflicts of interest to declare.
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