抗细菌药物默诺霉素生物合成关键机制的解析

Moenomycins are classified as phosphoglycolipids based on their chemical building blocks. They are potent anti-bacterial agent by directly inhibiting peptidoglycan glycosyltransferases (PDTs) involved in the penultimate step of bacterial cell wall biosynthesis. Most of the antibiotics found in moenomycin family are displayed biological activity against bacteria. It prompts continuing efforts to study their chemistry and biology. . In this project, we will comprehensively investigate the biosynthetic mechanism of moenomycin by exploring five related enzymes. Firstly, the catalytic characteristics of the free radical mediated methyltransferase MoeK5, and its distinctive recognition of phosphorylated substrates will be studied to increase our understanding of the proteins in this family. Secondly, four enzymes with broad substrate specifity including two glycosyl transferases (MoeGT3 and MoeGT5) and two modification enzymes (MoeM5 and MoeN5) in biosynthetic mid-term will be explored by elucidating their catalytic characteristics, and determining their natural substrates, respectively. Furthermore, the limited factors in the pathway will be revealed based on comparing the kinetic constants of these enzymes and verification of intermediate feeding experiments. These results will facilitate our construction of effective biosynthetic system in engineered strain, leading to increasing the production of moenomycin.

默诺霉素属于磷糖脂寡糖类抗生素，其生物活性机制是通过抑制细菌细胞壁生物合成中的糖基转移酶而实现阻止细菌细胞壁的合成，在已经发现的结构衍生物中，许多成员都具有显著的抗菌活性，具有重要的研究价值。. 本项目拟以默诺霉素生物合成的关键机制解析为切入点，在了解自由基介导的甲基转移酶MoeK5催化特征的基础之上，深入研究其催化机制及识别磷酸化底物的独特性质，增加对该家族蛋白成员催化机理的理解。然后，针对默诺霉素生物合成中期阶段底物宽泛性的2个糖基转移酶和2个修饰酶，开展相关酶学催化特征解析，明确各蛋白的催化特征及最佳天然底物，结合计算机模拟设计，尝试进行底物专一性优化。最后，基于各蛋白的酶促反应动力学常数的比较和中间体的喂养的确证实验，全面解析默诺霉素生物合成机制，并系统发掘和疏通默诺霉素生物合成途径中的限制因素，利用高效的基因编辑手段，实现其高效生物合成系统的构建。

默诺霉素属于磷糖脂类抗生素，其生物活性机制是通过抑制细菌细胞壁生物合成中的糖基转移酶而实现阻止细菌细胞壁的合成，在已经发现的结构衍生物中，许多成员都具有显著的抗菌活性，具有重要的研究价值。. 本项目以默诺霉素生物合成的关键机制解析及高效合成的系统优化为切入点，在全面阐明默诺霉素生物合成的机制及关键酶催化特征的基础之上。通过整合代谢工程、合成生物学和发酵优化策略，系统发掘了默诺霉素生物合成的限速因子。在研究过程中，我们首先克隆了24 kb 的nosokomycin 的生物合成基因簇(nosa-BGC)，并将其组装成了一个杂合的默诺霉素A的生物合成基因簇（moe-BGC），大小为 34 kb。然后，将该杂合的moe-BGC在白色链霉菌J1074中成功实现了异源合成，产量为12.1 ± 2 mg/L。进一步通过该基因簇中启动子的替换，默诺霉素A的产量提高了58%，进而导致了菌株生长受到了抑制。接着，通过在底盘菌株中整合一个拷贝salb-PBP2 (P238N&F200D)的编码基因，菌株的生长状态得到了恢复，进一步提升了默诺霉素A的产量。最后，综合各种机制的解析和相关的高产因素，利用代谢工程和合成生物学方法构建默诺霉素的高产工程菌株，为默诺霉素工业化生产和产品的优化升级奠定坚实的基础。