嗜麦芽窄食单胞菌次级代谢资源开发

A large number of antibiotics were repeatedly discovered in recent years, asking for the expansion of microbial resources. Gram-negative bacteria, Xanthomonadales in particular, contain rich natural product resources to be discovered. Xanthomonadales include genus Xanthomonadaceae , Lysobacter and Stenotrophomonas. Applicant focused on the secondary metabolism of Lysobacter enzymogenes C3 during graduate study in US and has initiated the investigation in natural product resources within Stenotrophomonas maltophilia at current time. Fifteen S. maltophilia strains were identified and HPLC analysis suggests that they contain rich natural product resources. This project plans to systematically analyze the complete genome sequence of two S. maltophilia strains-13637 and K297a; screen all S. maltophilia strains using primers designed for amplifying PKS, NRPS and PKS-NRPS hybrid gene cluster and knock out relevant genes in strains with positive screening results; isolate desired natural product through fermentation optimization, compare the differences in total metabolites between wild-type and mutant strains. This project aims at exploring novel strategy for mining natural products and is significant in developing novel drugs.

近年来，大量抗生素被重复发现，要求我们拓展微生物资源。革兰氏阴性细菌，尤其是黄单胞菌科蕴藏丰富的次级代谢资源，但是尚待深入发掘。黄单胞菌科主要有黄单胞菌、溶杆菌和窄食单胞菌等属。申请者在美国攻读博士学位期间，主要研究产酶溶杆菌C3菌株的次级代谢。回国后开展了窄食单胞菌的资源研究，选择性分离鉴定了15个菌株，HPLC分析结果表明它们的次生代谢产物丰富。本申请项目拟系统分析数据库中嗜麦芽窄食单胞菌13637和K279a的全基因组序列；选择3条聚酮合成酶（PKS）、非核糖体合成酶（NPRS）和杂合PKS-NPRS基因簇设计引物，PCR筛选三株实验室现有代谢谱复杂的嗜麦芽窄食单胞菌菌株，并对阳性菌株进行相关基因的敲除突变；通过发酵条件优化、比较野生型和突变株的代谢谱差异，定向分离次级代谢产物。本申请项目探索次级代谢产物发掘的新策略，对新药研发有重要的意义。

微生物来源的天然药物及其衍生物在对抗微生物感染中占据着很大的比例。微生物在长期进化中保留下来的抗生素具有很强的靶位点特异性和很高的生物活性。近年来，抗生素的普遍使用在全球范围内导致一些具有抗生素抗药性的人类致病菌的产生，给临床治疗带来了新的挑战。而新批准的抗生素大部分具有与原有抗生素相似的骨架结构，极易造成靶向致病菌再次培育完善的抗药机制以及抗生素的二次失效。耐药型致病菌株的快速传播，已经导致临床上出现能对抗多种抗生素的“超级细菌”，给病人带来了致命威胁。充分挖掘已有的抗生素主要来源菌细胞内的次生代谢物资源、开发全新的抗生素菌种资源迫在眉睫。. Stenotrophomonas maltophilia是一种广泛分布于各种生态位中的革兰氏阴性菌。我们研究发现它的滑动行为及其产生代谢产物的关系。研究证实4%甘油和0.25%酵母提取物发酵条件下，粗提物中检测出含有抗金黄色葡萄球菌活性的代谢产物。为大规模挖掘革兰氏阴性微生物抗生素合成潜力提供了依据。