Biological Science Division, Pacific Northwest National Laboratory, Richland, WA 99354, USA.
Biosciences Division, Oak Ridge National Laboratory, One Bethel Valley Road, Oak Ridge, TN 37831, USA.
Sci Adv. 2023 Mar 10;9(10):eade1285. doi: 10.1126/sciadv.ade1285.
Efficient genome engineering is critical to understand and use microbial functions. Despite recent development of tools such as CRISPR-Cas gene editing, efficient integration of exogenous DNA with well-characterized functions remains limited to model bacteria. Here, we describe serine recombinase-assisted genome engineering, or SAGE, an easy-to-use, highly efficient, and extensible technology that enables selection marker-free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. SAGE uses no replicating plasmids and thus lacks the host range limitations of other genome engineering technologies. We demonstrate the value of SAGE by characterizing genome integration efficiency in five bacteria that span multiple taxonomy groups and biotechnology applications and by identifying more than 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate that SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.
高效的基因组工程对于理解和利用微生物功能至关重要。尽管最近开发了 CRISPR-Cas 基因编辑等工具，但具有良好特性的外源 DNA 的有效整合仍然仅限于模式细菌。在这里，我们描述了丝氨酸重组酶辅助基因组工程（SAGE），这是一种易于使用、高效且可扩展的技术，可实现多达 10 个 DNA 构建体的无选择标记、定点基因组整合，其效率通常与复制质粒相当，甚至更高。SAGE 不使用复制质粒，因此没有其他基因组工程技术的宿主范围限制。我们通过在跨越多个分类群和生物技术应用的 5 种细菌中表征基因组整合效率，并在每个宿主中鉴定超过 95 个异源启动子，从而证明了 SAGE 的价值，这些启动子在环境和遗传背景下具有一致的转录。我们预计 SAGE 将迅速增加可与高通量遗传学和合成生物学兼容的工业和环境细菌的数量。
