基于灰产色链霉菌基因组信息发掘抗革兰氏阴性菌的天然“木马分子”

Multidrug-resistant Gram-negative bacterial infections have emerged as one of the greatest health threats all over the world. Gram-negative bacteria are surrounded by the outer membrane, which is a permeability barrier as one of the major mechanisms of antibiotic resistance. The use of siderophore-antibiotic conjugates as "Trojan Horse" antibiotics (i.e., sideromycins) can bypass membrane associated resistant mechanisms and has attracted particular interest in recent years. There are plenty of secondary metabolite biosynthesis gene clusters in the genome of actinomycetes, which are the main sources of clinically used antibiotics. Based on the bioinformatic analyses of over ten draft genomes of actinomycetes，hundreds of biosynthetic gene clusters were uncovered. Interestingly, in the Streptomyces griseochromogenes, we found five siderophore-related biosynthetic gene clusters. In this study, the fine genome sequence has been determined and subsequent genome mining using antiSMASH predicts that four of them might encode sideromycins. The production of possible sideromycins will be detected using different fermentation strategies, such as iron deficient medium, OSMAC strategy, chemical elicitors, and co-culturing. When the target silent biosynthetic gene clusters can not be activated with above methods, overexpression of cluster-situated regulators or heterologous expression of the target cluster in a Streptomyces strain with clear genetic background will be performed. The produced sideromycins will be isolated and the structures of the purified new compounds will be determined by nuclear magnetic resonance spectroscopy etc. The antibacterial activities will be evaluated against Gram-negative bacteria and multidrug-resistant bacteria. The discovery of novel natural sideromycins will be useful to provide new lead compounds combating multidrug-resistant Gram-negative bacterial infections. The novel structure of natural sideromycins will also provide some new clues for the molecular design of chemically synthesized sideromycins.

“外膜屏障”是导致革兰氏阴性菌抗生素耐药的主要原因之一。“木马策略”是将抗生素与铁载体偶联获得“木马分子”，利用细菌细胞（外）膜上的铁载体通道进入细胞的抗菌策略，可突破革兰氏阴性菌的“外膜屏障”。本课题组前期通过对十余株放线菌基因组序列的生物信息学分析和次级代谢产物预测，发现灰产色链霉菌基因组中存在5个与铁载体相关的生物合成基因簇，对这些基因簇上下游基因进行功能分析，发现其中4个基因簇可能编码潜在的“木马分子”。本课题将通过改变培养条件（如贫铁条件、OSMAC策略、化学诱导和共培养等）或对产生菌进行遗传操作等方法来激活潜在“木马分子”生物合成基因簇的表达，并分离纯化获得天然“木马分子”，解析其结构，测定其抗革兰氏阴性菌及耐药菌活性。本课题的实施有望发现新型的天然“木马分子”，为发现抗多药耐药革兰氏阴性菌的药物先导化合物提供物质基础，还可为化学合成多种结构新颖的“木马分子”提供新思路。

抗耐药菌药物缺乏是引起临床死亡的重要原因，是迫切需要解决的民生问题。细菌细胞“外膜屏障”是引起耐药的关键因素，天然“木马分子”因其独特的化学结构，以及细胞外膜上广泛存在特异性通道，成为寻求解决“外膜屏障”耐药的重要有机有机结构。本项目以分析药用微生物基因组数据为切入点，重点分析铁载体与抗生素杂合生物合成基因簇，并借助于分子生物学方法对目标基因进行研究，期望获得抗革兰氏阴性菌的天然“木马分子”。通过本项目研究已获得以下结果：（1）完成了十株药用菌株的基因测序，并借助于antiSMASH等技术对二十株菌株基因组信息进行了系统生物信息学分析，初步建立实验室内部小型基因组生物信息库；（2）应用OSMAC策略，对二十余株药用微生物次级代谢产物进行定向诱导，确定了铁载体类化合物的检测方法；（3）建立了基于分子网络化关键聚类方法分析微生物次级代谢产物的方法；（4）确定了目标菌株的遗传操作条件，并对目标基因簇中的调控基因进行了过表达，获得了10株不同基因的过表达菌株，8株对照菌株。（5）获得单体化合物12个，一个铁载体类化合物表现出了抗流感病毒活性。本研究结果中的药用微生物生物信息学数据库的建立，为进一步发掘其它功能有机分子，提供了重要数据。本研究遗传操作构建的生物元件，将为借助组合生物学合成新型抗生素分子奠定基础。