Department Microbial Natural Products, Actinobacteria Metabolic Engineering Group, Saarland University, Campus C2.3, 66123 Saarbrücken, Germany.
Department Microbial Natural Products, Actinobacteria Metabolic Engineering Group, Saarland University, Campus C2.3, 66123 Saarbrücken, Germany; i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
Metab Eng. 2018 Sep;49:299-315. doi: 10.1016/j.ymben.2018.09.010. Epub 2018 Sep 18.
We present a random rational approach enabling the construction of overproducing strains in two steps. The approach first involves creating a library of clusters of interest, in which native promoters are substituted with randomly generated constitutive synthetic promoters, and then expressing this library in an appropriate host strain. This strategy is fast, easy to use, accounts for the architecture of a cluster and completely decouples the expression of a gene cluster from complex native regulatory networks. The strategy was applied to improve the production of a macrocyclic peptide, bottromycin, which possesses antibacterial activity against multidrug-resistant bacteria and is a blueprint for a new class of antibacterials. We successfully optimized the expression of genes in operons and created several variants of the bottromycin gene cluster that provide 5-50 fold higher titres of bottromycin than the natural one, thus resulting in the identification of several new bottromycin derivatives not previously described. Moreover, due to the higher bottromycin yield, bottromycin derivatization was performed via the biosynthetic engineering of the gene cluster. The abovementioned features make this generic strategy a promising tool for the overproduction of known secondary metabolites and the activation of silent secondary metabolites in Actinobacteria.
我们提出了一种随机理性的方法，能够分两步构建高产菌株。该方法首先涉及创建一个感兴趣的簇库，其中将天然启动子替换为随机生成的组成型合成启动子，然后在合适的宿主菌株中表达该文库。该策略快速、易于使用，考虑到簇的结构，并将基因簇的表达与复杂的天然调控网络完全解耦。该策略被应用于提高大环肽博特霉素的产量，博特霉素对耐多药细菌具有抗菌活性，是一类新型抗菌药物的蓝图。我们成功地优化了操纵子中基因的表达，并创建了博特霉素基因簇的几个变体，这些变体提供的博特霉素产量比天然基因簇高 5-50 倍，从而鉴定了几种以前未描述的新博特霉素衍生物。此外，由于博特霉素产量的提高，通过基因簇的生物合成工程对博特霉素进行了衍生化。上述特征使这种通用策略成为一种很有前途的工具，可用于已知次生代谢物的大量生产和放线菌中沉默次生代谢物的激活。
