四株深海放线菌抗菌活性次级代谢产物的研究

Deep sea actinomycetes have been considered to be a promising microorganism resource for drug discovery recently. In our effort to search for antibacterial lead compounds, about 82 deep-sea actinomycetes isolated from marine sediment samples were preliminarily screened for their antibacterial activity using paper disc diffusion method. Among these strains, four actinomycetes showed significant antibacterial actitvity. Subsequent HPLC-DAD analyses indicated that the fermentation extract displayed particular UV absorbance. In this research, the secondary metabolites from these four actinomycetes will be investigated extensively in order to search for new antibacterial lead compounds. The culture medium will be optimized. Then the four strains will be fermented in large scale using the optimized medium. The secondary metabolites will be isolated by chromatography guided by antibacterial tests against Escherichia coli, Bacillus Subtillis, Staphylococcus aureus, Bacillus thuringensis. The structures of the obtained compounds would be determined by 1D, 2D NMR, MS spectra, IR and UV spectra and so on. The new compounds with antibacterial activities will be further tested aganist drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus. The production conditions of the new compounds will be optimized by single factors, orthogonal design and linear regression. It is of a great value to search the antibiotics with our own intellectual property right, especially to the antibiotics that could be used to treat drug-resistant bacterial disease.

近年来，深海放线菌被认为是一种极具开发前景的药源微生物。前期我们对82株分离自中国南海1000m以下海底沉积物的放线菌进行了筛选，以滤纸片法抗菌活性和化学HPLC-DAD相结合，发现其中4株放线菌的发酵产物具有良好的抑菌活性和鲜明的紫外吸收特征。本项目拟对这4株菌株，以对枯草芽孢杆菌、大肠杆菌、金黄色葡萄球菌等四株革兰氏细菌的抗菌活性为目标，通过培养基筛选、大规模发酵、代谢产物的提取和分离，获得目标化合物，通过核磁、质谱、红外、紫外等技术解析其化学结构。对结构新颖、抗菌活性显著的化合物，进一步对耐甲氧西林金黄色葡萄球菌等耐药性细菌进行抑菌活性测试。对活性良好的新化合物，进行发酵条件的单因子、正交、线性回归优化，提高其产率。以期为具有我国自主知识产权的新型抗生素、特别是抗耐药性细菌的抗生素的研发奠定基础。

随着细菌耐药性的日益严重，新型抗生素的研发已经迫在眉睫。本项目在前期以及实施过程对来自1000m以下的深海放线菌进行抗菌活性的筛选中，先后发现了具有良好抗菌活性的潜力菌株10余株，包括SCSIO 2904、3503、2703、0206、3406、ZJ46、5604、11594、ZJ46、10141、04777，项目先后对潜力菌株SCSIO 3406、 ZJ46、11594、5604、10141、ZJ49、04777进行了研究。从中分离鉴定活性化合物41个，包括5个天然产物的衍生物，合计得到新化合物21个。发表论文7篇，含SCI论文5篇，申请专利5项，授权4项。已经超额完成了项目任务。发现的代表性活性化合物包括从菌株SCSIO 3406中分离得到的具有抗耐甲氧西林金黄色葡萄球菌（MRSA）的新型先导化合物marfuraquinocins A-D，其对耐甲氧西林表皮葡萄球菌（MRSE）和金黄色葡萄球菌的MIC均达到6μg/mL。从菌株SCSIO ZJ46中分离得到的对肺炎球菌、（MRSE）具有抑制活性的新型环六肽先导化合物desotamides B-D，发现色氨酸基团为其功能基团。从SCSIO 10141中发现了对藤黄微球菌、丙酸杆菌具有抑制活性的新型抗感染环肽类先导化合物marformycins，个别化合物对藤黄微球菌的抑制活性达到0.063μg/mL等。本项目通过目标菌株活性次级代谢产物的研究，发现了系列结构新颖的抗菌化合物，不但为具有我国自主知识产权的抗生素奠定了基础，也揭示了深海放线菌作为新型抗生素药物资源的巨大开发潜力。