Screening Of Actinomycetes Possessing Halogenase Gene And Study Of Their Metabolites With Bioactivity

Halogenated compounds with various structures have been successfully used in agriculture, industry, and the field of human health. Traditional synthetic methods of integrating halogens into complex molecules are often complicated by lack of specificity and regioselectivity. On the other hand, more and more halogenated compounds have been isolated from microorganism. The halogenases, mostly FADH2-dependent halogenase, are thought to be involved in the biosynthesis of these halometabolites. In this study, a PCR-guided method using primers derived from conserved regions of halogenase genes was applied to screening of microorganisms with potential to produce halometabolites. The results were as follows:(1) The degenerate primers derived from conserved regions of FADH2-dependent halogenase sequences were used for PCR screening of 57 strains, which were randomly selected from the soil samples. Ten PCR fragments were obtained with the expected size of approximately 500-700 base pairs. Amng these screened strains, HBDN08 showed the highest antibacterial activities towards Micrococcus luteus ATCC 9341 and Bacillus subtilis ATCC 6633. The PCR product (halA) from HBDN08 was then sequenced and submitted to the GenBank database under accession number FJ875940. The sequence of halA showed a high identity of > 90% to the genes of tryptophan halogenase PrnA from Burkholderia cepacin, Burkholderia pyrrocinia, and Pseudomonas fluorescences (the accession number of the prnA in GenBank are AF161183, AF161186, and AF161184, respectively). Comparison of the amino acid sequence of HalA with tryptophan 7-halogenase RebH from Lechevalieria aerocolonigenes and PrnA from Pseudomonas fluorescens BL915, with the tryptophan 5-halogenase PyrH from Streptomuces rugosporus showed high degrees of sequence identity of 36%, 38% and 35%, respectively. Furthermore, HalA possessed three conserved motifs. Therefore, the product of halA might be a part of a putative tryptophan halogenase, and it was deduced that actinomycete HBDN08 had the potential to biosynthesize halogenated compounds. The analysis of 16S rDNA identified the strain HBDN08 as a novel strain of Actinoplanes sp. The 16S rDNA sequence of this strain has been submitted to the GenBank database under accession number FJ770217. The strain Actinoplanes sp. HBDN08 was deposited at the China General Microbiological Culture Collection Center (accession number CGMCC2944).(2) A new chlorinated isoflavone 3',8-dichlorogenistein, along with 8-chlorogenistein were isolated from the fermentation broth of Actinoplanes sp. HBDN08. The origin of the two compounds was also investigated by high-performance liquid chromatography analysis. The results demonstrated that they were not biosynthesized but derived from the biotransformation of genistein by Actinoplanes sp. HBDN08. The antioxidant activities of 3',8-dichlorogenistein and 8-chlorogenistein were evaluated by using the lipid peroxidation assay. The results indicated that 3',8-dichlorogenistein (IC50 = 5.2μM) and 8-chlorogenistein (IC50 = 7.5μM) showed stronger antioxidant activities than genistein (IC50 = 13.6μM). The antitumor activities of these three compounds were calculated according to the inhibitory rate of cell proliferation against human lung adenocarcinoma cell line A549, mouse melanoma cell line B16, and human breast cancer cell line MDA-MB-231. The results demonstrated that genistein, 8-chlorogenistein and 3',8-dichlorogenistein all have antitumor activity. The IC50 for the three compounds against A549 cells were 23.13, 20.98 and 27.94μg/ml, respectively. The IC50 against B16 cells were 4.96, 3.95 and 8.26μg/ml, respectively. The IC50 against MDA-MB-231 cells were 19.24, 7.20 and 3.15μg/mlμg/ml, respectively. These results indicated that the chlorination at the site 8 of genistein had little effect on the antitumor activities of genistein against A549 and B16, but the chlorination of the sites of 8 and 3', could decreased the antitumor activities against A549 and B16. However, the chlorination could enhance the antitumor activities against MDA-MB-231, and in comparison with the antitumor activities of genistein, those of 3',8-dichlorogenistein and 8-chlorogenistein increased 6.1 and 2.7-fold, respectively. Although no chlorinated metabolites were yet isolated from the fermentation broth of Actinoplanes sp. HBDN08, it did not mean that any bioactive compound was produced. The supernatant from fermentation broth showed high antibacterial activities towards Micrococcus luteus and Bucillus subtillis. However, the activities disappeared when the supernatant was processed under 45℃for 1 h. This feature is similar to chlorinated glycopeptide antibiotics, such as vancomycin and teicoplanin. The further isolation and identification of the active components from the supernatant are still ongoing.(3) The interaction mechanism of 3′,8-dichlorogenistein and bovine serum albumin (BSA) was investigated by ultraviolet absorbance, fluorescence, circular dichroism (CD) and molecular docking methods. 3′,8-Dichlorogenistein caused a static quenching of intrinsic fluorescence of BSA, and the quenching data was analyzed by Stern-Volmer equation. There was one primary 3′,8-dichlorogenistein binding site on BSA with a binding constant of 2.07×105 M-1 at 300 K. Comparison the binding constant of 3′,8-dichlorogenistein with that of genistein showed a higher affinity of 3′,8-dichlorogenistein to BSA. Thermodynamic analysis by van't Hoff equation found enthalpy change (ΔH) and entropy change (ΔS) were -11.64 kJ mol-1 and 137.70 J mol-1 K-1, respectively, which indicated that the hydrophobic and electrostatic forces played important roles in the binding of 3′,8-dichlorogenistein to BSA. Competitive experiments revealed that binding site of 3′,8-dichlorogenistein was located at the drug site I (in subdomain IIA). The distance r = 4.59 nm between donor (Trp 213) and acceptor (3′,8-dichlorogenistein) was estimated based on the F?rster theory of non-radiative energy transfer. The synchronous fluorescence, three-dimensional fluorescence, UV-vis and CD spectral results demonstrated that the microenvironment and conformation of BSA were changed in the binding reaction. According to the molecular modeling, 3′,8-dichlorogenistein was located to the hydrophobic cavity of BSA, which matched exactly the corresponding experimental results. Furthermore, docking studies also implied that a halogen bond, which may influence the drug recognition and binding to BSA, was possibly formed between 3′,8-dichlorogenistein and amino acid residue Asp 450. The interaction between BSA and 3′,8-dichlorogenistein may be helpful for us to understand the bioactivity mechanism of chlorinated genistein derivatives at the biomolecular level.(4) In vitro and in vivo antifungal activity assay indicated that Actinoplanes sp. HBDN08 could produce antifungal metabolites. The antifungal components were found to be produced only at the later stages of the exponential growth phase and stationary phase during the fermentation. The antifungal metabolite was isolated using the bioactivity-guided method and identified as 5-hydroxyl-5-methyl-2-hexenoic acid on the basis of NMR and MS spectral analysis. This compound showed strong in vitro antifungal activity against Botrytis cinerea, Cladosporium cucumerinum and Corynespora cassiicola, with IC50 of 32.45, 27.17, and 30.66 mg/L, respectively; however, moderately inhibited the hyphal growth of Rhizoctonia solani with IC50 of 61.64 mg/L. Although 5-hydroxyl-5-methyl-2-hexenoic acid showed better inhibitory effect on the growth of cucumber pathogens than that of rice sheath blight pathogen on the basis of IC50, no significant differences were observed between their MFC against the test pathogens. A reasobable explanation for this observation could be that 5-hydroxyl-5-methyl-2-hexenoic acid had stronger fungistatic property against the test cucumber pathogens in contrast to R. solani, but equal fungicidal property against all the test pathogens. The in vivo antifungal activity under greenhouse conditions demonstrated that 5-hydroxyl-5-methyl-2-hexenoic acid could effectively control the plant diseases caused by B. cinerea, C. cucumerinum and C. cassiicola with 71.42%, 78.63% and 65.13% control values at 350 mg/L, respectively. To our knowledge, this is the first report on 5-hydroxyl-5-methyl-2-hexenoic acid possessing antifungal activity, which was generated by microorganism. Although the antifungal activity was less active than the commonly used fungicide pyraoxystrobin, the results obtained in this study suggested that 5-hydroxyl-5-methyl-2-hexenoic acid might be an interesting lead product for the further development of novel fungicides.