环脂肽多组分同系物生物合成的人工调节研究

The drug requirements of polymyxin and other lipopeptide antibiotics are increasing with the appearance of “superbugs” multidrug resistance and the rapid spread of antibiotic resistant bacteria. The multi-components homologues of the cyclic lipopeptide antibiotics lead to difficulties in the control of fermentation process. The multi-components homologues increase their cytotoxicity during clinical treatment. These problems limit the industrial production andclinical application of lipopeptide antibiotics, especially in treatment of drug-resistant “superbugs”.. In this work, to understand the low efficiency, poor controllability, and low yield of lipopeptide component homologues by combinatorial biosynthesis regulation of nonribosomal peptide synthetases (NRPS), we will focus on the key issue of different functional domain characteristics and lipopeptide precursors to artificially regulate the production of lipopeptide component homologues (e.g. polymyxin or surfactin) by the reconstruction of functional domains and precursors supplies. Firstly, synthetic biology technologies and Saccharomyces cerevisiae will applied to build the engineered strains containing NRPS structure domains and precursor synthesis pathways. Furthermore, we will build Engineering Microbial Consortia (EMC) with the engineered S. cerevisiae to artificially regulate the production of lipopeptide multi-component homologues. Understanding the characteristics of the NRPS gene cluster domain will help us to reveal the molecular mechanism of the domain integrity regulation of the lipid peptide homologues. Secondly, EMC containing quorum sensing system will be applied to regulate the precursor supplies of lipopeptide. The relationship between precursor supplies of lipopeptide and the complexity of lipopeptide multi-compound homologues by measuring their levels. Therefore these works will provide a theoretical basis for reconstituting stable and controllable lipopeptide synthesis system and for improving development of a new high standard lipid peptide drug. Additionally, it also provides new strategies for solve the lack of drug for treating the antibiotic resistant bacteria.

超级细菌多重耐药的出现，引发对多粘菌素等脂肽类抗生素的需求增大。环脂肽复杂的同系物组分导致发酵过程稳定性差难控、产品细胞毒性大等问题限制脂肽抗生素在应对超级细菌时其效能的发挥。. 本项目针对组合生物合成方法调控非核糖体肽合成酶(NRPS)脂肽同系物组分效率低，功能可控性差和合成能力低的问题，以多粘菌素（或表面活性素）同系物组分人工调控为例，从NRPS功能结构域改造和脂肽合成前体供给入手，利用合成生物技术重构NRPS功能结构域和前体物供给工程菌，构建工程化酿酒酵母进行脂肽同系物生物合成人工调控的混菌系统；解析NRPS基因簇结构域特性，揭示结构域完整性调控脂肽同系物组分的分子机制；利用群体效应系统调节前体物供给水平，探索前体物供给与脂肽同系物复杂性间的关系，为构建稳定可控的脂肽合成系统和新型高标准脂肽类药物的开发提供理论依据，为解决缺乏应对超级耐药菌的新型药物寻找新的策略。

超级细菌多重耐药的出现，引发对多粘菌素等环脂肽类抗生素的需求增大。环脂肽复杂的同系物组分导致其生产稳定难、产品细胞毒性大等问题限制其应用,本项目采用合成生物学策略开展环脂肽多组分生物合成的人工调节研究，结果表明：. 完成了产多粘菌素脂肽的多粘芽孢杆菌(CJX518)和产表面活性素等脂肽的解淀粉芽孢杆菌(HM618)的全基因组测序, CJX518菌能合成粘菌素B1、B2、B3和B1-1多种组分; 通过对亮氨酸特异性的腺苷酸活化结构域替换使B1从41.36%上升到62.69%，B3和B1-1降低到6.70%和3.32%；完成了产多粘菌素B的枯草芽孢杆菌工程菌构建，重组菌多粘菌素B产量达74.1mg/L。. 解淀粉芽孢杆菌(HM618) 含有合成脂肽（Surfactin, Fengycin等）的多个基因簇，构建了用于删除HM618中表面活性素生产负调节因子和在酿酒酵母中成功组装了HM618菌中的表面活性素合成酶基因srfAA, srfAB, srfAC和srfAD；利用响应面法优化培养基，HM618菌纯培养和其与产Vb2菌共培养时表面活性素产量显著增加。. 前体脂肪酸和氨基酸能促进脂肽合成，添加直链脂肪酸总脂肽产量增大了2.25倍，添加3种0.8% (w/v)的氨基酸(L-Ser、L-Pro、L-Asn)显著提高了菌株HM618的脂肽合成能力；HM 618与产脂肪酶真菌构巢曲霉共培养时，表面活性素的产量最高；菌株HM618与重组高产脯氨酸C. glu-proline菌共培养的接种时间和比例为4 h和2:0.5时的脂肽产量是纯培养的4.22倍；同时发现产脂肽解淀粉芽孢杆菌HM618可提高小麦耐盐和铜的抗胁迫能力。. 这些研究为揭示脂肽同系物组分调控的分子机制提供理论依据、为构建人工系统高效合成多粘菌素等脂肽提供了新思路。