一个用于表达黏细菌天然产物的底盘菌的优化及应用

Myxobacteria are an important source of bioactive natural products, more than 100 novel scaffolds including the marketed anticancer drug lead epothilones. The genome sequencing also revealed there are still a huge number of compounds waiting for mining in myxobacteria. However, the yields of myxobacterial natural products are usual low in the native producers, and most of myxobacteria are slow-growing and genetically hard-to-manipulate strains. Thus transferring biosynthetic gene clusters into a simple chassis for heterologous expression is a feasible strategy of myxobacterial genome mining. Our preliminary study showed that epothilones were robustly produced in a heterologous host strain DSM7029, and its growth rate, cultivation and genetic manipulation are superior to myxobacteria. Thus this strain have the potential to be a general chassis. This project intends to construct and optimize the Red/ET recombineering system based on the native phage protein pairs for the rapid genome manipulation in DSM7029, to delete non-essential genes and insert some useful genes, thus to optimize the growth and fermentation characteristics and decrease the metabolite profile. Then to screen of several strong promoters by transcriptome sequencing for improving the yields of target compounds. In summary, this project is to use synthetic biology strategy to build a general chassis of artificial expression system of natural products for mining of unknown (myxobacterial) natural products and yield improvement of valuable natural products, such as epothilones.

黏细菌是重要的天然活性物质来源，发现的新骨架天然化合物超过100种，其中埃博霉素已经作为抗癌药物上市。基因组测序表明黏细菌中仍然有大量化合物有待发掘。但是黏细菌天然产物在本源菌中产量通常很低，生长缓慢，难于操作和遗传改造。因此将其生物合成基因转移到底盘菌中应用异源表达策略是黏细菌基因组挖掘的可行方法。前期研究发现埃博霉素能在异源宿主菌株DSM7029中稳定产生，该菌在生长培养和遗传操作等方面均优于黏细菌，具有成为表达黏细菌天然产物通用底盘菌的潜力。本项目拟建立并优化基于该菌自身噬菌体蛋白所介导的Red/ET同源重组系统，用于快速的基因组大片段删除和插入，实现基因组简化和改造，从而优化菌株的生长发酵属性、降低代谢背景；并利用转录组测序筛选高强度的启动子，通过提高转录水平增加目标化合物产量；最终使其成为人工表达天然产物系统的通用底盘菌，用于挖掘未知天然化合物和提高高价值化合物产量。

黏细菌和伯克氏菌等革兰氏阴性细菌也是活性天然产物的重要来源，而且将基因簇的异源表达是挖掘细菌天然产物的重要方法，但是革兰氏阴性细菌天然产物底盘的匮乏阻碍了后基因组时代的天然产物挖掘。本项目对一个能表达黏细菌天然产物的伯克氏菌DSM7029进行了一系列优化。首先，通过挖掘新型噬菌体重组酶构建了基于重组工程技术的基因组编辑技术系统，并利用该技术快速精准的插入启动子在该菌中通过基因组挖掘获得了十余个新天然产物，还应用于其它伯克氏菌基因组编辑和挖掘，获得了十余个新天然产物。其次，利用基因组编辑技术结合点特异性重组酶逐步无痕删除了伯克氏菌DSM7029基因组中的次级代谢产物合成基因簇以及与转座子、原噬菌体、基因组岛相关的序列，实现基因组的简约化，显著提升了生长状态，延缓了自溶现象，多种异源天然产物如黏细菌来源埃博霉素和副伯克氏菌来源的根菌素的产量具有明显提高；利用基因组简约化底盘，挖掘和鉴定了几丁质单胞菌来源的未知基因簇产物chitinimide；从黏细菌中克隆了十个基因簇，其中一个基因簇的产物已经成功异源表达。再次，利用转录组测序结合报告基因测试，获得一系列高活性组成型启动子，使得异源天然产物的产量具有较大提高。最后，利用基因组编辑技术和底盘对根际副伯克氏菌来源的非核糖体脂肽rhizomide和holrhizin的脂链进行改造，通过结构替换和点突变能够实现了脂链的大幅度改变，建立了脂肽的组合生物合成新方法。该底盘生长速度、遗传操作优于黏细菌底盘，代谢网络前体供应优于假单胞菌底盘，有望成为表达革兰氏阴性细菌基因簇的通用底盘，应用于细菌天然产物的挖掘、高产和改造，为天然药物发现和优化奠定使能技术的基础。项目资助发表论文15篇，申请中国发明专利4项，获得教育部自然科技奖二等奖一项，培养研究生6名。