PNNL

Abstract

Efficient genome engineering is critical to understand and utilize 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 ten 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 over 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.