利用细胞色素P450单加氧酶理性改造抗生素结构的研究

The macrolide antibiotics belong to type I polyketide natural products. Due to excellent wide-spectrum antibacterial activity, this class of antibiotics has been one of the most widely used antimicrobial agents in clinical applications. Nowadays, bacterial strains resistant to 14-membered ring macrolide antibiotics are becoming more and more common, while the 16-membered ring macrolide antibiotics such as tylosin and mycinamicin display good activity against many drug-resistant pathogens. However, tylosin's defects in pharmakinetics and mycinamicin's neurotoxicity have limited further application of these two macrolides. In comparison, these two antibiotics have similar chemical structures, biosynthetic gene clusters and hence biosynthetic pathways. Our preliminary study has demonstrated that the cytochrome P450 monooxygenase MycG from mycinamicin gene cluster is capable of converting the tylosin derivative descomycin into hydroxylated and epoxidized products. Based on the structural analysis of the new products, they may possess the characteristics of enhanced antibacterial activity and reduced neurotoxicity. Thus, in this project we propose to introduce the MycG encoding gene to the chromosome of tylosin producing strain Streptomyces fradiae. It is anticipated that the heterologous expression of MycG would reprogram the tylosin biosynthetic pathway to generate novel hybrid antibiotics with improved drug-like properties and lower toxicity. This work will provide a new strategy for exploring new antibiotics against drug resistant bacteria or other diseases. Moreover, the idea developed from this project would allow the transfer of such logic to the discovery of new bioactive natural products and optimization of the structure and activity of other exsiting anti-infectives.

大环内酯属于I型聚酮类化合物，具有良好的广谱抗菌活性，是临床应用最广泛的抗菌药物之一。目前具有14元环大环内酯类抗生素耐药性的细菌已越发普遍，而16元环大环内酯类抗生素如泰乐霉素与麦新米星对许多耐药菌显示出良好活性，但却分别因药代动力学缺陷和神经毒性导致其应用受限。基于两类抗生素化学结构相似，生物合成途径和基因簇同源性高的特点，本项目前期研究已经证实麦新米星基因簇中的细胞色素P450单加氧酶MycG可以在体外催化泰乐霉素衍生物descomycin形成羟基化和环氧化产物。因此，我们提出通过生物合成途径重组技术将P450 MycG整合到泰乐霉素产生菌弗氏链霉菌染色体上进行异源表达，对泰乐霉素类抗生素的化学结构进行改造，理性地设计和创造具有更好成药性和更低毒性的新型抗生素，为开发新的抗耐药菌或其他疾病的抗生素提供新思路。本研究对新活性天然产物的发掘和现有抗生素的改良也具有重要的理论和应用价值。

大环内酯类化合物具有良好的广谱抗菌活性，是临床应用最广泛的抗菌药物之一。但常见的14元环大环内酯类抗生素耐药性的细菌已越发普遍，而16元环大环内酯类抗生素如泰乐霉素与麦新米星对许多耐药菌具有良好抗菌活性，但却因其各自的缺陷导致应用受限。基于两类抗生素化学结构相似，生物合成途径和基因簇同源性高的特点，青年基金项目证实麦新米星基因簇中的细胞色素P450单加氧酶MycG与还原伴侣RhFRED的融合蛋白可以在体外催化泰乐霉素衍生物descomycin形成羟基化及环氧化产物；借助链霉菌接合转移技术将P450 MycG-RhFRED融合基因整合到泰乐霉素产生菌弗氏链霉菌染色体，利用该P450对descomycin的化学结构进行改造，成功获得新型16元环大环内酯——12,13-expoxydesmycosin和14-hydrodesmycosin。这两个新型抗生素从结构上看，避免了神经毒性基团的影响的同时保留了抗菌活性单元，其生物活性正在进一步评价中，并获得青岛市应用研究基础项目资助。本项目的顺利实施为利用生物合成途径重组技术开发新的抗耐药菌或其他疾病的抗生素提供了新的思路，对新活性天然产物的发掘和现有抗生素的改良也具有重要的理论和应用价值。.本项目在研究P450 MycG催化天然底物麦新米星和非天然底物descomycin的过程中，发现MycG和RhFRed在融合和分离状态下，MycG能够以两种完全不同的作用方式对麦新米星进行修饰，融合状态下MycG负责催化麦新米星C14 位的羟基化和C12/13 位的环氧化；而在分离状态下，MycG不仅能催化羟基化和环氧化反应，更能够催化脱甲基反应。这一发现是对P450领域传统认知的一个挑战，也是对P450作用方式认识的新的补充和完善，相关成果发表在J. Am. Chem. Soc.杂志，并两次被推荐为“Faculty 1000论文”。