基于组合生物学和交流结构域的新脂肽类抗生素罗克霉素非线性合成分子机制的研究

Locillomycins are a novel family of cyclic lipopeptides with a molecular structure not observed in other lipopeptides, including a unique nonapeptide sequence and macrocyclization, and active against bacteria and viruses with potential therapeutic applications. Particularly, the locillomycins are synthesized by a non-linear nonribsomal peptide synthetases (NRPSs) pathway which a hexamodular NRPSs conducted a cyclic nonapeptides biosynthesis. Given the nonlinear NRPSs rare and precious in the nature and its broad application in the combinatorial biosynthesis, it is very important to elucidate the detail molecular mechanism of a nonapeptide assembled by a hexamodular NRPSs. On the basis of the elucidated structures and the functional analysis biosynthetic genes of the locillomycins, this project is aim to set up a excellent nonlinear NRPSs assemble theoretical model and constructed a series of hybrid NRPSs used to synthesize new lipopeptide compounds by performing the following studies. Firstly, this research horizontal transfer of locillomycins operon, loc, to Bacillus amyloliquefaciens FZB42 and conversion into a locillomycins producer. Secondly, heterologous expression of the four genes (LocA-D) in the loc operon and taking advantage of the recombinant enzymes to biosynthesize the locillomycins and intermediates in vitro. Thirdly, construction of a series of hybrid new NRPSs by modules and domains exchange to biosynthesize of a series of locillomycins analogues. Finally, this research further study on the corresponding functions of short communication domains in the locillomycins’ NRPSs by fusion the short communication domains with peptide synthesis modules and detected their catalysis functions. In summary, fulfill this project will help us further understand the nonlinear NRPSs for the locillomycins and provide a raw nonlinear NRPSs models in the nature. In addition, elucidation the functions of the short communication domains of the locillomycins will provide novel means to rationally construct NRPSs to synthesize new peptide antibiotics.

罗克霉素是由含有6个模块的非核糖体合成酶系经非线性途径合成的含有9个氨基酸残基的脂肽类抗生素，具有较高抗细菌和病毒活性。鉴于非线性合成途径在自然界中的稀有和在组合生物学中的广泛用途，探明罗克霉素非线性合成途径具有重要的科学意义和应用价值。在阐明罗克霉素的结构、鉴定罗克霉素合成基因簇和初步提出其合成途径的基础上，本项目拟进一步通过同源重组实现基因簇在模式芽胞杆菌菌株中异源表达；采用基因簇中关键基因的表达产物建立体外合成罗克霉素及中间产物的催化体系；特别是通过组合生物学对基因簇关键结构域、模块进行替换，理性设计合成多个新型基因簇，从而创制出系列罗克霉素结构类似物；最后通过生物信息学预测基因簇中交流结构域，并阐明交流结构域在催化合成多肽化合物中的通讯功能。本项目旨在建立一个相对完善的非线性生物合成罗克霉素的理论模型并为组合生物学理性设计非核糖体合成途径的基因簇提供理论指导和可供选择的功能元件。

脂肽类抗生素是由亲水性环状短肽头部和长链疏水性脂肪酸尾部组成的杂合抗生素，具有广谱抗真菌、细菌、卵菌和病毒活性，具有广泛的应用价值。罗克霉素是本团队从枯草芽孢杆菌鉴定到的一种新型环脂肽抗生素，具有较高生物活性和潜在应用价值，存在一种非线性合成途径。为进一步探究罗克霉素合成途径和潜在价值，本项目取得如下进展：1）开展了罗克霉素的全基因簇在模式菌株B. velezensis FZB42中进行异源高效表达工作，通过构建基因组文库，更换诱导表达强启动子等方法在B. velezensis FZB42中成功实现了罗克霉素基因簇的高效异源表达，进一步证实了罗克霉素非线性合成途径，同时获得了表达量相对野生型菌株提高了15倍的工程菌株；2）构建了芽孢杆菌双载体基因编辑系统，开展了采用该基因编辑的系统对罗克霉素基因簇中的A3氨基酸激活结构域更换为多粘菌素基因簇中的A3氨基酸激活结构域，通过重组载体构建，转化等方法已经成功构建突变株，并检测到突变株产罗克霉素的结构类似物，但活性和结构还需进一步确认；3）对罗霉素四个亚基的通讯交流结构域进行预测和功能研究发现，通讯交流结构域并不能按预期指导多肽的合成，罗克霉素的非线性机制可能并不依赖预测的通讯交流结构域，推测可能形成五个亚基（LocA，LocB，locB， LocC和LocD）的复合酶来指导合成；4）开展了罗克霉素四个亚基重组表达研究，虽然已经重组表达三个蛋白LocA， LocC和LocD，但LocB一直未能成功表达，因此关于罗克霉素体外重组合成的未能实现。通过对本项目的实施，成功实现罗克霉素的异源高效表达，构建了芽孢杆菌双载体基因编辑系统并成功编辑了罗克霉素基因簇，提出了罗克霉素非线性合成机制，目前已经发表SCI论文3篇，获得授权专利1项，申请专利3项。对该项目的后续研究，有望继续发表SCI文章1篇和申请专利1项。