PNAS:中科院微生物所娄春波与张立新课题组在合成生物学调控元

9月14号Proceedings of the National Academy of sciences上发表中国科学院微生物所娄春波与张立新课题组共同发表的一篇研究成果，文章是关于合成生物学调控元件方面的研究。张立新课题组白超弦及娄春波课题组博士生张洋、助理研究员赵学金为论文的共同第一作者。研究员张立新和娄春波为通讯作者，其他作者还有胡逸灵、向四海和苗靳。

合成生物学聚焦于各种生命系统工程化和模块化再造，并已经在能源、医药等领域取得了一些显著性成果。丝状微生物(真菌及放线菌等)是次级代谢产物的主要来源，约有90%的已知抗生素由这类微生物产生，其中三分之二以上是由以链霉菌为代表的放线菌产生的。原核的链霉菌基因组比真核的酵母菌还要大，基因还要多，其中三分之二的基因参与调控过程。然而由于链霉菌丝状生长的特性使其基因表达调控难以精确设计、定量和预测，成为合成生物学在该领域发展的一个巨大的技术瓶颈。由中国科学院微生物研究所研究员张立新担任首席科学家的“973”项目“合成微生物体系的适配性研究”正是试图解决这一科学问题，提升对链霉菌生命规律的认识层次，实现对链霉菌生命过程的定量预测、精准化设计、标准化合成与精确调控。

最近，微生物所娄春波课题组和张立新课题组合作首次针对链霉菌生物元件建立了基于流式细胞仪和报告基因(sfGFP)的单细胞精确定量方法。该方法优化了原生质体制备方案，替换了对流式细胞仪有损害的蔗糖缓冲体系，同时结合PI染色以分离出死细胞个体，解决了丝状微生物生长形态分化和程序性死亡不利于定量的缺陷，从而提高了测量精度。在高通量地刻画了覆盖范围足够大的启动子和RBS等调控元件后，又成功地利用RiboJ绝缘子在链霉菌中消除了启动子和RBS的干涉效应，提高了这些元件模块化性能。这些调控元件模块也被成功地应用于激活阿维链霉菌中沉默基因簇番茄红素的表达，并通过可预测地替换调控元件大大提高了番茄红素的产量和效率，达到工业生产的要求。该研究工作为应用合成生物学理念改造丝状微生物次级代谢产物及激活沉默基因簇奠定了重要基础。

原文链接：

Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces

原文摘要：

There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces.

doi: 10.1073/pnas.1511027112

作者：娄春波和张立新