Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China.
School of Pharmacy, Fudan University, Shanghai 201203, China; Department of Biochemistry, Shanghai Institute of Pharmaceutical Industry, Shanghai 201210, China.
Metab Eng. 2019 Mar;52:153-167. doi: 10.1016/j.ymben.2018.12.001. Epub 2018 Dec 5.
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.
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尤为重要。然而，长 DNA 片段（例如，大基因簇）的稳定、多拷贝整合仍然具有挑战性。在这里，我们描述了一种基于“多个整合酶-多个 attB 位点”的创新概念的工具包，该工具包允许在放线菌中高效、一步、多位点整合天然产物（NP）生物合成基因簇（BGC），基于“多个整合酶-多个 attB 位点”的创新概念。该工具包由 27 个合成模块化质粒组成，其中包含源自五个正交位点特异性重组（SSR）系统的单个或多整合模块（从两个到四个）。多整合模块可以通过 Gibson 组装轻松连接到含有大 BGC 的质粒中，然后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 羧化酶基因来促进链霉菌中红色素的生物合成，证明了该工具包的适用性。此外，我们使用该工具包，通过多基因座整合整个 5-氧代米尔贝霉素 BGC（72kb）（高达四个拷贝），使吸水链霉菌的 5-氧代米尔贝霉素产量提高了 185.6%（从 2.23 到 6.37g/L）。与之前报道的方法相比，高级多重位点特异性基因组工程（aMSGE）方法在扩增目标基因或 BGC 之前不需要对宿主基因组进行任何修饰，这将大大简化和加速提高 NP 产量的努力。考虑到 SSR 系统广泛分布于各种工业微生物中，这项新技术也有望成为提高其他高价值生物制品生物合成的有价值的工具。
