基因和通路大小的DNA盒的基因组整合通常是构建强大和高效的微生物细胞工厂的不可或缺的方式。对于一些不常见的微生物宿主,如分枝杆菌和分枝杆菌,然而,这是一个挑战。这里,我们提出了一种多重整合酶辅助位点特异性重组(miSSR)方法,以精确和迭代地将具有可控拷贝的基因/途径整合到分枝杆菌染色体中,以开发细胞工厂。首先,通过结合应用分枝杆菌噬菌体L5整合酶和两步等位基因交换策略,在新牛分枝杆菌中建立了一步多拷贝整合方法,对于不超过三拷贝整合事件,其效率为100%,对于五拷贝整合事件,其效率急剧下降至20%。第二,选择R4,Bxb1和ΦC31噬菌体Att/Int系统,以扩展用于多重基因整合事件的可用整合工具箱.第三,使用重建的霉菌Xer重组酶(Xer-cise)系统回收基因重组的选择标记,以促进迭代基因操作。作为概念的证明,新分枝杆菌ATCC25795中麦角硫因(EGT)的生物合成途径是通过用miSSR系统重塑其代谢途径来实现的。EGT和磷酸戊糖异构酶(PRT)的生物合成基因簇(BGC)的六个拷贝,在30mL摇瓶中,5天内所得菌株的EGT滴度提高到66mg/L,是野生菌株的3.77倍。改进表明,miSSR系统是一种有效的,灵活,和方便的工具来设计分枝杆菌以及分枝杆菌科中其他菌株的基因组,因为它们之间的近邻进化关系。
Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell factories. For some uncommon microbial hosts, such as Mycolicibacterium and Mycobacterium species, however, it is a challenge. Here, we present a multiplexed integrase-assisted site-specific recombination (miSSR) method to precisely and iteratively integrate genes/pathways with controllable copies in the chromosomes of Mycolicibacteria for the purpose of developing cell factories. First, a single-step multi-copy integration method was established in M. neoaurum by a combination application of mycobacteriophage L5 integrase and two-step allelic exchange strategy, the efficiencies of which were ∼100% for no more than three-copy integration events and decreased sharply to ∼20% for five-copy integration events. Second, the R4, Bxb1 and ΦC31 bacteriophage Att/Int systems were selected to extend the available integration toolbox for multiplexed gene integration events. Third, a reconstructed mycolicibacterial Xer recombinases (Xer-cise) system was employed to recycle the selection marker of gene recombination to facilitate the iterative gene manipulation. As a proof of concept, the biosynthetic pathway of ergothioneine (EGT) in Mycolicibacterium neoaurum ATCC 25795 was achieved by remodeling its metabolic pathway with a miSSR system. With six copies of the biosynthetic gene clusters (BGCs) of EGT and pentose phosphate isomerase (PRT), the titer of EGT in the resulting strain in a 30 mL shake flask within 5 days was enhanced to 66 mg/L, which was 3.77 times of that in the wild strain. The improvements indicated that the miSSR system was an effective, flexible, and convenient tool to engineer the genomes of Mycolicibacteria as well as other strains in the Mycobacteriaceae due to their proximate evolutionary relationships.