Skip to main content
ARS Home » Pacific West Area » Albany, California » Plant Gene Expression Center » Research » Publications at this Location » Publication #392580
Research Project: Discovery of Plant Genetic Mechanisms Controlling Microbial Recruitment to the Root Microbiome

Location: Plant Gene Expression Center

Title: High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration

Author
item ELMORE, JOSHUA - Pacific Northwest National Laboratory
item DEXTER, GARA - Oak Ridge National Laboratory
item BALDINO, HENRY - Pacific Northwest National Laboratory
item HUENEMANN, JAY - Oak Ridge National Laboratory
item FRANCIS, RYAN - Pacific Northwest National Laboratory
item PEABODY, GEORGE - Oak Ridge National Laboratory
item MARTINEZ-BAIRD, JESSICA - Oak Ridge National Laboratory
item RILEY, LAUREN - Oak Ridge National Laboratory
item SIMMONS, TUESDAY - University Of California
item Coleman-Derr, Devin
item GUSS, ADAM - Oak Ridge National Laboratory
item EGBERT, ROBERT - Pacific Northwest National Laboratory

Submitted to: Science Advances
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/1/2023
Publication Date: 3/10/2023
Citation: Elmore, J., Dexter, G., Baldino, H., Huenemann, J., Francis, R., Peabody, G.L., Martinez-Baird, J., Riley, L.A., Simmons, T., Coleman-Derr, D.A., Guss, A., Egbert, R. 2023. High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration. Science Advances. 9(10). Article eade1285. https://doi.org/10.1126/sciadv.ade1285.
DOI: https://doi.org/10.1126/sciadv.ade1285

Interpretive Summary: Genome engineering is a vital tool to understand and utilize microbial functions. While recently established genome engineering approaches, such as CRISPR-Cas gene editing, are available for an increasing number of microbes, efficient integration of exogenous DNA with well-characterized functions remains limited to model bacterial hosts. Here we report on a new technology that addresses this issue, Serine integrase-Assisted Genome Engineering (SAGE). We anticipate SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput systems and synthetic biology research.

Technical Abstract: Here we describe Serine integrase-Assisted Genome Engineering, or SAGE, which enables iterative, site-specific genome integration of up to 8 independent DNA constructs at efficiency on par with or better than replicating plasmids. SAGE integration does not require maintenance of replicating plasmids and selection markers are easily removed by transformation and transient expression of a secondary serine integrase, enabling multi-cycle, marker-free integrations. We demonstrate the value of SAGE by characterizing genome integration efficiency and gene expression activity for a library of 287 phylogenetically diverse promoter elements for five bacterial hosts that include metabolic engineering hosts used for bioconversion of diverse feedstocks into valuable chemicals, a plant growth promoting rhizobacterium, an environmental bioremediation host, and an undomesticated sorghum root endophyte. From these data we curated a set of at least 95 promoters for each organism with expression profiles that are independent of environmental condition and genetic context. We anticipate SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput systems and synthetic biology research.