Metabolic Engineering ( IF 8.4 ) Pub Date : 2020-08-20 , DOI: 10.1016/j.ymben.2020.08.007 Filipe Marques 1 , Andriy Luzhetskyy 2 , Marta V Mendes 3
The Gram-positive bacterium Corynebacterium glutamicum sustains the industrial production of chiral molecules such as L-amino acids. Through heterologous gene expression, C. glutamicum is becoming a sustainable source of small organic molecules and added-value chemicals. The current methods to implement heterologous genes in C. glutamicum rely on replicative vectors requiring lasting selection or chromosomal integration using homologous recombination. Here, we present a set of dedicated and transversal tools for genome editing and gene delivery into C. glutamicum. We generated a cosmid-based library suitable for efficient double allelic exchange, covering more than 94% of the chromosome with an average 5.1x coverage. We employed the library and an iterative marker excision system to generate the carotenoid-free C. glutamicum BT1-C31-Albino (BCA) host, featuring the attachment sites for actinophages ϕC31 and ϕBT1 for one-step chromosomal integration. As a proof-of-principle, we employed a ϕC31-based integration and a Cre system for the markerless expression of the type III polyketide synthase RppA, and a ϕBT1-based integration system for the expression of the phosphopantetheinylation-dependent non-ribosomal peptide synthetase BpsA in the C. glutamicum BCA host. The developed genomic library and microbial host, and the characterized molecular tools will contribute to the study of the physiology and the rise of C. glutamicum as a leading host for drug discovery.
中文翻译:
使用综合基因组文库和基于噬菌体的载体改造谷氨酸棒杆菌。
革兰氏阳性菌谷氨酸棒状杆菌维持手性分子(如 L-氨基酸)的工业生产。通过异源基因表达,谷氨酸棒杆菌正在成为有机小分子和附加值化学品的可持续来源。目前在谷氨酸棒杆菌中实施异源基因的方法依赖于需要使用同源重组进行持久选择或染色体整合的复制载体。在这里,我们展示了一组专门的横向工具,用于基因组编辑和基因传递到谷氨酸棒杆菌中. 我们生成了一个适用于高效双等位基因交换的基于粘粒的文库,覆盖了超过 94% 的染色体,平均覆盖率为 5.1x。我们采用文库和迭代标记切除系统来生成不含类胡萝卜素的谷氨酸棒杆菌 B T1- C 31- A lbino (BCA) 宿主,具有用于一步染色体整合的放线噬菌体 ϕC31 和 ϕBT1 的附着位点。作为原理证明,我们采用了基于 ϕC31 的整合和 Cre 系统用于 III 型聚酮化合物合酶 RppA 的无标记表达,以及基于 ϕBT1 的整合系统用于表达依赖于磷酸泛酰巯基乙胺化的非核糖体肽谷氨酸棒杆菌中的合成酶 BpsABCA 主机。开发的基因组文库和微生物宿主,以及特征化的分子工具将有助于生理学研究和谷氨酸棒杆菌作为药物发现的主要宿主的崛起。