aMSGE: advanced multiplex site-specific genome engineering with orthogonal modular recombinases in actinomycetes
aMSGE: advanced multiplex site-specific genome engineering with orthogonal modular recombinases in actinomycetes
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aMSGE：在放线菌中使用正交模块化重组酶进行先进的多重位点特异性基因组工程
DOI：
10.1016/j.ymben.2018.12.001
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发表时间：
2019-03-01
影响因子：
8.4
通讯作者：
Lu, Yinhua
中科院分区：
文献类型：
--
作者：
Li, Lei;Wei, Keke;Lu, Yinhua
基因和途径的染色体整合对于工业生物技术中的大规模和长期发酵特别重要。然而，长DNA片段（例如，大基因簇）仍然具有挑战性。在这里，我们描述了一个即插即用的工具包，允许高效率，单步，多位点的天然产物（NP）的生物合成基因簇（BGC）在放线菌中的整合，基于创新的概念“多个整合-多个attB位点”。该工具包由27个合成模块化质粒组成，其中包含来自5个正交位点特异性重组（SSR）系统的单或多整合模块（从2到4个）。通过吉布森组装，多整合模块可以容易地连接到含有大BGC的质粒中，其可以在单个步骤中同时插入多个天然attB位点。我们通过稳定扩增乙酰辅酶A羧化酶基因来促进天蓝色链霉菌中放线菌紫素的生物合成，证明了该工具包的适用性。此外，使用该工具包，我们通过多位点整合整个5-氧代米尔贝霉素BGC（72 kb）（最多4个拷贝），在吸水链霉菌中实现了5-氧代米尔贝霉素滴度增加185.6%（从2.23至6.37 g/L）。与以前报道的方法相比，先进的多重位点特异性基因组工程（aMSGE）方法不需要在扩增靶基因或BGC之前向宿主基因组中引入任何修饰，这将大大简化和加速提高NP生产的努力。考虑到SSR系统广泛分布在各种工业微生物中，这种新技术也有望成为增强其他高价值生物产品生物合成的有价值的工具。
Chromosomal integration of genes and pathways is of particular importance for large-scale and long-term fermentation in industrial biotechnology. However, stable, multi-copy integration of long DNA segments (e.g., large gene clusters) remains challenging. Here, we describe a plug-and-play toolkit that allows for high-efficiency, single-step, multi-locus integration of natural product (NP) biosynthetic gene clusters (BGCs) in actinomycetes, based on the innovative concept of "multiple integrases-multiple attB sites". This toolkit consists of 27 synthetic modular plasmids, which contain single- or multi-integration modules (from two to four) derived from five orthogonal site-specific recombination (SSR) systems. The multi-integration modules can be readily ligated into plasmids containing large BGCs by Gibson assembly, which can be simultaneously inserted into multiple native attB sites in a single step. We demonstrated the applicability of this toolkit by performing stabilized amplification of acetyl-CoA carboxylase genes to facilitate actinorhodin biosynthesis in Streptomyces coelicolor. Furthermore, using this toolkit, we achieved a 185.6% increase in 5-oxomilbemycin titers (from 2.23 to 6.37 g/L) in Streptomyces hygroscopicus via the multi-locus integration of the entire 5-oxomilbemycin BGC (72 kb) (up to four copies). Compared with previously reported methods, the advanced multiplex site-specific genome engineering (aMSGE) method does not require the introduction of any modifications into host genomes before the amplification of target genes or BGCs, which will drastically simplify and accelerate efforts to improve NP production. Considering that SSR systems are widely distributed in a variety of industrial microbes, this novel technique also promises to be a valuable tool for the enhanced biosynthesis of other high-value bioproducts.