类芽孢杆菌B69中脂肽类化合物Pelgipeptins的分离鉴定及相关基因簇分析
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摘要
脂肽类抗生素是一类重要的抗生素,具有抗菌谱广,抗菌效果佳,不容易使病原菌产生耐药性等特点。随着达托霉素、棘白霉素等新一代脂肽类抗生素的上市,越来越多的科研工作者将目光转移至对该类抗生素的研发上。本室在微生物资源筛选时获得一株对所有测试过的致病菌均有明显抑制作用的类芽孢杆菌B69,盘栽试验表明该菌所产化合物具有良好的生物防治效果。前期的研究结果表明,该菌所产抗生素可能为脂肽类化合物。本研究围绕着该菌所产抗菌活性物质展开系统研究,包括对活性物质进行分离、纯化、结构鉴定及生物活性测定。同时,利用全基因组测序获得该类次级代谢产物生物合成基因簇信息；并通过异源表达A结构域蛋白的方法对其功能进行验证。具体的研究内容与结果如下：
1、脂肽类抗生素pelgipeptins的分离纯化与结构鉴定。通过培养基和发酵条件优化筛选出最适的发酵工艺；该菌发酵液中的活性物质经大孔吸附树脂、固相萃取、高效液相等多种手段的分离,获得了4种不同组分。ESI-CID-MS质谱分析和核磁共振技术分析表明,这4种化合物(pelgipeptins A、B、C和D)是结构非常相似的脂肽类化合物,其分子量分别为1072Da、1100Da、1086Da和1086Da。其中化合物A和D被鉴定为国内外首次发现的新次级代谢产物。
2、脂肽类抗生素pelgipeptins的生物活性测定。对pelgipeptins A-D纯品进行抗菌活性检测,结果表明：四种化合物对病原微生物大肠杆菌、绿脓菌等革兰氏阴性细菌以及枯草芽孢杆菌、金黄色葡萄球菌、粪肠球菌等革兰氏阳性菌均有很强的抗菌活性；其中包括多重耐药金黄色葡萄球菌ATCC43300,暗示了该类化合物在医药领域应用的潜力。此外,pelgipeptins对假丝酵母、尖镰孢菌、禾谷镰孢菌、串珠镰孢菌、立枯丝核菌和亚麻刺盘镰孢菌等植物致病真菌也有明显的抑制作用。盘栽实验表明,pelgipeptins粗提物对水稻纹枯病、黄瓜灰霉病、黄瓜白粉病、小麦白粉病和黄瓜霜霉病均有一定的生防作用,其中对水稻纹枯病的防治效果最佳。杀菌曲线表明该类抗生素的杀菌效果具有浓度依赖性。在外膜通透性实验中,pelgipeptin B具有与polymyxin B类似的细菌外膜破坏作用,推测该类抗生素的作用机制可能与polymyxin B相似,即均通过对细菌内、外膜的破坏达到快速杀菌的效果。
3、基因组水平挖掘pelgipeptins生物合成plp基因簇。为研究pelgipeptins生物合成途径,本实验室测定了该菌株的全基因组草图。生物信息学分析表明,B69基因组中至少包含2个完整的NRPS家族基因簇、3个PKS/NRPS杂合基因簇以及一个trans-PKS基因簇。其中一个至少包含8个ORF的plp基因簇(plp A-H)可能对应于已分离获得的pelgipeptins;根据相关基因的比对分析以及功能预测,推测该基因簇编码蛋白催化合成的次级代谢产物与pelgipeptins的结构完全吻合,暗示pelgipeptins是通过NRPS途径催化合成的。
4、plp基因簇的功能验证。通过PCR技术扩增plp基因簇部分模块中的A结构域基因序列,克隆至大肠杆菌表达载体pET-28a,并在大肠杆菌BL21(DE3)中进行异源表达。体外活性分析表明,所测3个A结构域的底物特异性与pelgipeptins对应位点的氨基酸基本一致,最终间接证明了plp基因簇通过NRPS途径负责pelgipeptins在细菌体内的合成。
Lipopeptide antibiotic is one important kind of antibiotics with broad spectrum and good antibacterial effect, and usually it is not easy to make pathogens resistant. As the launch of daptomycin and echinocandin recently, much more attention was focused on the development of this kind of compounds. The strain, isolated and identified as Paenibacillus elgii B69in our lab, showed potent antimicrobial activity to the tested indicator strains, including gram-positive, gram-negative bacteria, and fungi, which attracted our attention for further study. In this study, the work was mainly carried out on the separation, purification, structure elucidation and biological activity of the bioactive compounds isolated from this strain. Meanwhile, according to the genome information, we obtained the gene cluster possibly responsible for these bioactive antibiotics. Further study on the functional relationship between the compounds and the gene cluster was performed, including gene knock-out and heterologous expression The results were shown as follows:
1. The purification and identification of lipopeptide antibiotics-pelgipeptins. Under the appropriate medium composition and culture conditions, four unobserved lipopeptide antibiotics, named pelgipeptins A, B, C and D, were isolated by macroporous resin adsorption and RP-HPLC separation in the B69fermentation culture. The molecular mass of these four compounds were1072Da,1100Da,1086Da,1086Da, respectively. Mass-spectrometry and NMR spectroscopy indicated that all these active compounds belonged to the polypeptin family, and pelgipeptins A and D were the novel antibiotics to date.
2. The biological characterization of pelgipeptins. The biological activities of pelgipeptins were tested as follows:pelgipeptins A-D showed potent antibacterial activity to the tested gram-negative bacteria and gram-positive bacteria, including MRSA, suggests application of such compounds in the potential field of medicine. In addition, pelgipeptins also showed strong inhibition on the fungi, such as Candida albicans, Fusarium oxysporium, Fusarium graminearum. Fusarium maniliforme, Rhizoctonia solani. Colletotrichum lini. Also, in plant experiments, Rhizoctonia solani, Botrytis cinerea. Sphaerotheca fuliginea, Blumeria graminis and Pseudoperonospora cubensis were effectively inhibited during its growth under the pelgipeptin with the concentration of250μg/mL. Among these tests, the biocontrol property in Rhizoctonia solani was the best. In vitro time kill curve, the bactericidal effect of these antibiotics performed in a concentration-dependent manner. Through the outer membrane permeability test, we found that pelgipeptin B might be with the similar bactericidal action of polymyxin B, suggesting that the targets of the class of antibiotics may be associated with the inner and outer membrane of bacteria to achieve rapid bactericidal effect.
3. The pip gene cluster responsible for pelgipeptins biosynthesis. In order to analyze pelgipeptins biosynthetic pathway in strain B69, we obtained the draft genome sequence through the Solexa paired-end sequencing technology. Several unknown giant gene clusters were found in B69genome, including two nonribosomal peptide synthetases (NRPS), one polyketide synthetase (PKS) and three hybrid NRPS-PKS synthetases gene. Further inspection revealed that one NRPS gene cluster (plp) harboring8open reading frames (plp A-H) could be assigned to pelgipeptins biosynthesis. According to the gene blast and functional perdition, the proposed structure of this secondary metabolite was in agreement with that of pelgipeptins, indicating that pelgipeptins might be catalyzed through plp gene cluster in NRPS synthesis pathway.
4. The functional analysis of plp gene cluster. Three A domains in plp gene cluster were cloned by PCR. Then, the genes were cloned into plasmid pET-28a and expressed in E.coli BL21(DE3). In vitro biochemical analysis showed that the measured substrate specificities of tested A domain proteins were consistent with the corresponding amino acids in pelgipeptins. These results provided the indirect evidence that the plp gene cluster was responsible for the catalytic synthesis of pelgipeptins.
1、脂肽类抗生素pelgipeptins的分离纯化与结构鉴定。通过培养基和发酵条件优化筛选出最适的发酵工艺；该菌发酵液中的活性物质经大孔吸附树脂、固相萃取、高效液相等多种手段的分离,获得了4种不同组分。ESI-CID-MS质谱分析和核磁共振技术分析表明,这4种化合物(pelgipeptins A、B、C和D)是结构非常相似的脂肽类化合物,其分子量分别为1072Da、1100Da、1086Da和1086Da。其中化合物A和D被鉴定为国内外首次发现的新次级代谢产物。
2、脂肽类抗生素pelgipeptins的生物活性测定。对pelgipeptins A-D纯品进行抗菌活性检测,结果表明：四种化合物对病原微生物大肠杆菌、绿脓菌等革兰氏阴性细菌以及枯草芽孢杆菌、金黄色葡萄球菌、粪肠球菌等革兰氏阳性菌均有很强的抗菌活性；其中包括多重耐药金黄色葡萄球菌ATCC43300,暗示了该类化合物在医药领域应用的潜力。此外,pelgipeptins对假丝酵母、尖镰孢菌、禾谷镰孢菌、串珠镰孢菌、立枯丝核菌和亚麻刺盘镰孢菌等植物致病真菌也有明显的抑制作用。盘栽实验表明,pelgipeptins粗提物对水稻纹枯病、黄瓜灰霉病、黄瓜白粉病、小麦白粉病和黄瓜霜霉病均有一定的生防作用,其中对水稻纹枯病的防治效果最佳。杀菌曲线表明该类抗生素的杀菌效果具有浓度依赖性。在外膜通透性实验中,pelgipeptin B具有与polymyxin B类似的细菌外膜破坏作用,推测该类抗生素的作用机制可能与polymyxin B相似,即均通过对细菌内、外膜的破坏达到快速杀菌的效果。
3、基因组水平挖掘pelgipeptins生物合成plp基因簇。为研究pelgipeptins生物合成途径,本实验室测定了该菌株的全基因组草图。生物信息学分析表明,B69基因组中至少包含2个完整的NRPS家族基因簇、3个PKS/NRPS杂合基因簇以及一个trans-PKS基因簇。其中一个至少包含8个ORF的plp基因簇(plp A-H)可能对应于已分离获得的pelgipeptins;根据相关基因的比对分析以及功能预测,推测该基因簇编码蛋白催化合成的次级代谢产物与pelgipeptins的结构完全吻合,暗示pelgipeptins是通过NRPS途径催化合成的。
4、plp基因簇的功能验证。通过PCR技术扩增plp基因簇部分模块中的A结构域基因序列,克隆至大肠杆菌表达载体pET-28a,并在大肠杆菌BL21(DE3)中进行异源表达。体外活性分析表明,所测3个A结构域的底物特异性与pelgipeptins对应位点的氨基酸基本一致,最终间接证明了plp基因簇通过NRPS途径负责pelgipeptins在细菌体内的合成。
Lipopeptide antibiotic is one important kind of antibiotics with broad spectrum and good antibacterial effect, and usually it is not easy to make pathogens resistant. As the launch of daptomycin and echinocandin recently, much more attention was focused on the development of this kind of compounds. The strain, isolated and identified as Paenibacillus elgii B69in our lab, showed potent antimicrobial activity to the tested indicator strains, including gram-positive, gram-negative bacteria, and fungi, which attracted our attention for further study. In this study, the work was mainly carried out on the separation, purification, structure elucidation and biological activity of the bioactive compounds isolated from this strain. Meanwhile, according to the genome information, we obtained the gene cluster possibly responsible for these bioactive antibiotics. Further study on the functional relationship between the compounds and the gene cluster was performed, including gene knock-out and heterologous expression The results were shown as follows:
1. The purification and identification of lipopeptide antibiotics-pelgipeptins. Under the appropriate medium composition and culture conditions, four unobserved lipopeptide antibiotics, named pelgipeptins A, B, C and D, were isolated by macroporous resin adsorption and RP-HPLC separation in the B69fermentation culture. The molecular mass of these four compounds were1072Da,1100Da,1086Da,1086Da, respectively. Mass-spectrometry and NMR spectroscopy indicated that all these active compounds belonged to the polypeptin family, and pelgipeptins A and D were the novel antibiotics to date.
2. The biological characterization of pelgipeptins. The biological activities of pelgipeptins were tested as follows:pelgipeptins A-D showed potent antibacterial activity to the tested gram-negative bacteria and gram-positive bacteria, including MRSA, suggests application of such compounds in the potential field of medicine. In addition, pelgipeptins also showed strong inhibition on the fungi, such as Candida albicans, Fusarium oxysporium, Fusarium graminearum. Fusarium maniliforme, Rhizoctonia solani. Colletotrichum lini. Also, in plant experiments, Rhizoctonia solani, Botrytis cinerea. Sphaerotheca fuliginea, Blumeria graminis and Pseudoperonospora cubensis were effectively inhibited during its growth under the pelgipeptin with the concentration of250μg/mL. Among these tests, the biocontrol property in Rhizoctonia solani was the best. In vitro time kill curve, the bactericidal effect of these antibiotics performed in a concentration-dependent manner. Through the outer membrane permeability test, we found that pelgipeptin B might be with the similar bactericidal action of polymyxin B, suggesting that the targets of the class of antibiotics may be associated with the inner and outer membrane of bacteria to achieve rapid bactericidal effect.
3. The pip gene cluster responsible for pelgipeptins biosynthesis. In order to analyze pelgipeptins biosynthetic pathway in strain B69, we obtained the draft genome sequence through the Solexa paired-end sequencing technology. Several unknown giant gene clusters were found in B69genome, including two nonribosomal peptide synthetases (NRPS), one polyketide synthetase (PKS) and three hybrid NRPS-PKS synthetases gene. Further inspection revealed that one NRPS gene cluster (plp) harboring8open reading frames (plp A-H) could be assigned to pelgipeptins biosynthesis. According to the gene blast and functional perdition, the proposed structure of this secondary metabolite was in agreement with that of pelgipeptins, indicating that pelgipeptins might be catalyzed through plp gene cluster in NRPS synthesis pathway.
4. The functional analysis of plp gene cluster. Three A domains in plp gene cluster were cloned by PCR. Then, the genes were cloned into plasmid pET-28a and expressed in E.coli BL21(DE3). In vitro biochemical analysis showed that the measured substrate specificities of tested A domain proteins were consistent with the corresponding amino acids in pelgipeptins. These results provided the indirect evidence that the plp gene cluster was responsible for the catalytic synthesis of pelgipeptins.
引文
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