一株红树林链霉菌所产抑菌活性化合物的分离及其生物合成基因簇的研究

Isolation and identification of antifungal compound from a mangrove-derived Streptomyces sp.and its biosynthetic gene cluster analysis

目的分离鉴定一株红树林来源具有拮抗真菌活性的链霉菌MCCG 2008,分析其对植物病原真菌香蕉尖孢镰刀菌热带4号小种(Fusarium oxysporum f.sp.cubense tropical race 4)、苹果链格孢菌(Alternaria alstroemeriae)、珍珠李葡萄座腔菌(Botryosphaeria dothidea)和芒果炭疽菌(Colletotrichum fructicola)的抑制作用,对其所产的抑菌活性化合物进行了分离纯化,并通过基因扩增测序分析与活性化合物生产相关的生物合成基因簇,为进一步调控活性化合物的生产以及结构优化提供依据。方法通过形态观察、生理生化特性和16S rRNA基因序列分析对MCCG 2008进行初步鉴定;通过发酵液原液、发酵液乙酸乙酯粗提取物和菌丝体丙酮浸提物针对不同植物病原真菌进行了活性测试与效用组成分析;采用液液萃取和柱色谱分离该菌株所产生的具有抑菌活性的次级代谢产物,通过高效液相色谱质谱联用仪和核磁共振仪对活性化合物结构进行解析;设计引物使用PCR扩增该菌中与活性化合物生物合成相关的基因。结果菌株MCCG 2008可形成茂盛的气生菌丝,并产生黄色的可扩散色素,16S rRNA基因序列系统发育分析显示菌株MCCG 2008与微小链霉菌Streptomyces parvulus NBRC 13193T的相似度为100%;在拮抗活性试验中,菌株MCCG 2008对珍珠李葡萄座腔菌的抑制活性最强,抑制率为48.0%,其发酵液乙酸乙酯粗提物和菌丝体丙酮浸提物仅对香蕉尖孢镰刀菌热带4号小种有抑制活性;质谱(MS)和核磁(1H NMR)数据分析表明从菌株MCCG 2008的发酵液中分离得到的活性化合物为放线菌素D;以放线菌素D生产菌为参照,通过序列比对发现MCCG 2008中的同源序列,并通过PCR扩增获得菌株MCCG 2008基因组中与放线菌素D生物合成相关的基因。结论分离自广西红树林根际土壤的链霉菌MCCG 2008初步鉴定为微小链霉菌,其具有拮抗植物病原真菌的生防潜力,所产的活性化合物为放线菌素D。

Objective A strain of Streptomyces sp.MCCG 2008 with antifungal activity from mangroves was isolated and identified,and its inhibitory effect on Fusarium oxysporum f.sp.cubense tropical race 4,Alternaria alstroemeriae,Botryosphaeria dothidea and Colletotrichum fructicola were determined.The structure and biosynthetic gene cluster of antifungal compounds were analyzed using chemical analysis and gene amplification sequencing,which provided the basis for further regulation of the production of the active compounds and structural optimization.Methods Preliminary identification of MCCG 2008 was carried out through morphological observation,physiological and biochemical characteristics and 16S rRNA gene sequence analysis.Activity test and effective composition analysis of different plant pathogenic fungi were performed by fermentation broth,ethyl acetate extraction of fermentation broth and acetone crude extraction of mycelium.The antifungal compound produced by this strain were separated by liquid-liquid extraction and column chromatography.The structure was elucidated based on the chromatograms of HPLC-MS and NMR.Primers were designed to amplify genes related to antifungal compound biosynthesis by polymerase chain reaction(PCR).Results Strain MCCG 2008 has lush aerial hyphae and produce yellow diffusible pigment.The phylogenetic analysis of the 16S rRNA of strain MCCG 2008 showed 100%sequence similarity with strain Streptomyces parvulus NBRC 13193T.As compared to other plant pathogens,the results demonstrated that strain MCCG 2008 showed the strongest inhibition to L.theobromae(48.0%inhibition rate).The ethyl acetate extract of the fermentation broth and the acetone extract of its mycelium only exhibited activities against F.oxysporum f.sp.cubense tropical race 4.Based on the MS and 1H NMR spectroscopy analysis,the antifungal compound was identified to be actinomycin D.With actinomycin D producing bacteria as reference,the homologous sequences of MCCG 2008 were found through sequence alignment,and genes related to actinomycin D biosynthesis were obtained by PCR amplification.Conclusion Streptomyces sp.MCCG 2008 isolated from the rhizosphere soil of Guangxi Mangrove forest was preliminarily identified as Streptomyces parvulus NBRC 13193T,producing antifungal compound actinomycin D.