一个新颖淡水蓝藻源羊毛硫肽天然产物的生物合成研究

Lanthipeptides comprise one major type of Ribosomally synthesized and post-translationally modified peptide (RiPPs) natural products, and usually possess potent anticancer and antibacterial activities. The structures of lanthipeptides are characterized by their lanthionine residues, as well as other post-translational modifications, which are the chemical foundations of their biological activities. Cyanobacteria, Gram-negative bacteria, are the only bacteria that can perform oxygenic photosynthesis. Lanthipeptide biosynthetic gene clusters are widely distributed in cyanobacteria, whose natural product biosynthetic potential was rarely investigated in China. In our previous studies, a strain library of freshwater cyanobacteria has been established. Degenerate PCR screening experiments revealed the presence of a lanthipeptide biosynthetic gene cluster that contains the class II LanM lanthionine synthetase. Detailed analysis of this lanthipeptide gene cluster showed the presence of a novel prenyltransferase homolog. Based on those findings, we hypothesize that this gene cluster can produce lanthipeptide with novel post-translationally modifications, and that this structural novelty may contribute to its potent biological activities. We have expressed several key enzymes from this gene cluster using a heterologous E. coli host, and reconstituted LanM enzymatic reactions in vitro. This project aims to elucidate the biosynthetic pathway of the lanthipeptide encoded by this gene cluster, and to use the obtained knowledge to perform combinatorial biosynthesis for structural derivatization, in order to provide novel lanthipeptide natural products for antimicrobial and anticancer drug development. This project will set a model example for future exploitation of fresh water cyanobacteria as a tremendous treasure of RiPP natural products.

羊毛硫肽是核糖体肽类天然产物的主要类型之一，具有良好的抗肿瘤和抗菌活性。其结构中含有标志性的羊毛硫氨酸基团及其它翻译后结构修饰，是其生物活性的关键基础。蓝藻有丰富的代谢能力，含有大量的羊毛硫肽类天然产物生物合成基因簇，我国有丰富的蓝藻资源，却鲜有团队关注蓝藻天然产物。前期研究中我们构建了一个小型淡水蓝藻菌种库，通过简并PCR发现了一个II型羊毛硫肽生物合成基因簇。基因组测序与生物信息学分析显示，该生物合成基因簇中含有独特的异戊烯基转移酶等基因，预示着羊毛硫肽类天然产物中发现了新颖的翻译后结构修饰，并可能具有良好的生物活性。我们用大肠杆菌异源表达了关键生物合成酶，并在体外初步重构了羊毛硫氨酸合成酶的功能。本项目拟结合体外生物化学实验与异源表达，解析关键生物合成酶的功能和催化机制，通过组合生物合成优化天然产物的结构，测试其抗肿瘤和抗菌活性，为进一步深入开发淡水蓝藻核糖体肽类天然产物提供模板。

本项目中，我们围绕着淡水蓝藻Microcystis aeruginosa NIES-88编码的一个新颖羊毛硫肽天然产物生物合成基因簇（mal）开展了系统的研究。该基因簇编码2个前体肽（MalA1和MalA2）和1个II型羊毛硫氨酸合成酶（MalM）。序列分析显示MalA1和MalA2含有保守的先导肽和多样化的核心肽序列，还具有典型的ABC转运蛋白偶联蛋白酶的识别位点，预示了MalM催化多样化底物生成羊毛硫肽的能力。基因共表达及多肽纯化与结构鉴定证明了MalM可以将MalA1和MalA2均转化为羊毛硫肽产物。为了进一步实现羊毛硫肽的结构多样化并探究合成酶的催化多样性，我们通过组合MalA和ProcA的先导肽和核心肽对前体肽进行工程化改造，并将它们与MalM和ProcM分别进行共表达，最终鉴定了11个羊毛硫肽及其类似物。即使底物多肽具有同样的核心肽序列，MalM和ProcM修饰产生的两组羊毛硫肽具有迥异的脱水和环化方式，证明了羊毛硫肽的翻译后修饰不仅由核心肽序列决定而且由合成酶的区域选择性控制。采用蓝藻素异戊烯基转移酶PagF作为工具酶，顺利实现部分羊毛硫肽的异戊烯基化，进一步扩展羊毛硫肽的结构多样性。生物信息学分析显示，蓝藻基因组中编码大量的MalM类杂泛性羊毛硫肽合成酶，附近伴随多个前体肽基因，这些前体肽与同源合成酶具有协同进化的关系。本项目鉴定了一个新颖的II型羊毛硫肽生物合成基因簇及其关键生物合成酶，为后续持续发现新颖羊毛硫肽天然产物奠定基础，并展示了一种羊毛硫肽结构多样化扩展的新策略。