多元基因组编辑技术是实现细菌基因组高效挖掘的必要条件。基于迭代Red/ET重组工程的CoS-MAGE技术具有不引起DNA损伤、操作简单等优点，但由于筛选方法和工具酶的限制，CoS-MAGE的应用主要集中在大肠杆菌中。伯克氏菌基因组中蕴含着巨大的生物合成基因簇资源，但因缺乏有效的多元基因组编辑技术导致挖掘效率很低。本项目通过引入新工具酶，改变筛选策略以及优化编辑效率等措施在CoS-MAGE原理上建立了能够对伯克氏菌进行多元编辑的技术-RaMGE。RaMGE既保留了CoS-MAGE优势又解决了宿主范围窄、多筛选标记依赖、编辑位置限制和筛选效率低等问题。初步优化的RaMGE在伯克氏菌DSM7029中通过一轮重组可实现基因组上任意位置4个位点的高效编辑（效率10%），实现了抗肿瘤化合物glidobactinA的高效表达和新天然产物的获得，为伯克氏菌基因组挖掘提供了技术支持。

Efficient and accurate multiplex genome editing technology is critical for bacterial genome mining and natural products acquisition.. CoS-MAGE based on Red/ET recombineering which has the advantages such as without causing DNA damage, independence of long homologous sequence and sgRNAs expression vectors is currently the most mature multiplex genome editing technology. However, due to the deficient of enzyme function and the low sensitivity of the screening method, the application of CoS-MAGE was mainly concentrated in E. coli, the multiplex genome editing of most bacteria still facing technical barriers.. Burkholderiales strains are an important source of bacterial natural products. Short of efficient multiplex genome editing technologies resulted in low genome mining efficiency of Burkholderiales strains.. Based on the principle of CoS-MAGE, this project will establish a novel multiplex genome editing technology RaMGE, which has significantly improved the application scope and editing/screening efficiency of CoS-MAGE by extra tool enzymes introduction, screening strategies change and efficiency optimization. RaMGE technology could avoid ssDNA dependence and target site position limitation of CoS-MAGE. Pre-experimental results showed that RaMGE can achieve simultaneous editing of 4 loci through a single round of recombination in Burkholderiales strains DSM 7029 with the efficiency of 10 %, the high-expression of glidobactin A and the acquisition of new natural products were achieved by multiplex genome editing mediated by RaMGE. RaMGE can be applied in Burkholderiales strains for the improvement of genome mining efficiency.