Sustainable enhancements to crop productivity and increased resilience to adverse conditions are critical for modern agriculture, and application of plant growth promoting rhizobacteria (PGPR) is a promising method to achieve these goals. However, many desirable PGPR traits are highly regulated in their native microbe, limited to certain plant rhizospheres, or insufficiently active for agricultural purposes. Synthetic biology can address these limitations, but its application is limited by availability of appropriate tools for sophisticated, high-throughput genome engineering that function in environments where selection for DNA maintenance is impractical. Here we present an orthogonal, Serine-integrase Assisted Genome Engineering (SAGE) system, which enables iterative, site-specific integration of up to 10 different DNA constructs at efficiency on par or better than replicating plasmids. SAGE does not require use of replicating plasmids to deliver recombination machinery, and employs a secondary serine-integrase to excise and recycle selection markers. Furthermore, unlike the widely utilized pBBR1 origin, DNA transformed using SAGE is stable without selection. We highlight SAGE′s utility by constructing a 287-member constitutive promoter library with a ~40,000-fold dynamic range in P. fluorescens SBW25. We show that SAGE functions robustly in diverse γ- and α-proteobacteria, thus providing evidence that it will be broadly useful for engineering industrial or environmental bacteria.

中文翻译：

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SAGE遗传工具包可实现细菌中高效，重复的位点特异性基因组工程

可持续提高作物生产力和增强对不利条件的适应能力对现代农业至关重要，应用促进根瘤菌生长的植物（PGPR）是实现这些目标的一种有前途的方法。但是，许多理想的PGPR性状在其天然微生物中受到高度调节，仅限于某些植物根际，或对于农业目的活性不足。合成生物学可以解决这些局限性，但是其应用受到用于复杂，高通量基因组工程的适当工具的可用性的限制，而这些工具在无法选择DNA维持的环境中起作用。在这里，我们介绍了正交的丝氨酸整合辅助基因组工程（SAGE）系统，该系统可进行迭代，位点特异性整合最多10种不同的DNA构建体，其效率与复制质粒相当或更好。SAGE不需要使用复制质粒来传递重组机制，而是使用二级丝氨酸整合酶来切割和回收选择标记。此外，与广泛使用的pBBR1来源不同，使用SAGE转化的DNA无需选择即可稳定。我们通过构建一个具有40,000倍动态范围的287个成员组成型启动子文库来突出SAGE的效用。荧光假单胞菌SBW25。我们表明，SAGE在各种γ-和α-变形杆菌中均具有强大的功能，因此提供了证据，表明它对工程化工业或环境细菌具有广泛的用途。