Abstract
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.
| Original language | English |
|---|---|
| Article number | eade1285 |
| Journal | Science Advances |
| Volume | 9 |
| Issue number | 10 |
| DOIs | |
| State | Published - Mar 2023 |
Funding
Acknowledgments:W ethankC.HarwoodandY .Odaforpro viding R.palustrisCGA009andfor helpfulinformation regarding cultivation of thestrain. W e also thank E.Yeung and Y . Farris for providingplasmidpEYF2K.Funding:ThisworkwassupportedinpartbytheU.S.Department of Energy (DOE), Office of Biological and Environmental Research (BER), as part of BER’s GenomicScienceProgram(GSP),andisacontributionofthePa cific NorthwestNational Laboratory(PNNL)SecureBiosystemsDesignScienceFocusArea“PersistenceControlof EngineeredFunctionsinComplexSoilMicrobiomes. ”Additionalsupportwasprovidedbythe DARPASynergisticDiscoveryandDesignprogr am (contract#HR0011045664).PNNLisa multiprogramnationallabora tory operatedbyBattellefortheDOEundercontractDE-A C05-76RL01830.ThisworkwasauthoredinpartbytheOakRidgeNationalLaboratory,whichis managedbyUT-BattelleLLC,fortheU.S.DOEundercontractDE-A C05-00OR22725. Funding waspro vided inpartbytheU.S.DOE,OfficeofEnergyEfficiencyandRenewableEnergy BioenergyT echnologies Office(BETO)totheAgileBioFoundry.Thisworkwasinpartsupported bytheCenterforBioenergyInnovation,U.S.DOEBioenergyResearchCenter,supportedbythe OfficeofBiologicalandEnvironmentalResearchintheDOEOfficeofScience.Author contributions:Conceptualization:J.R.E.,A.M.G.,andR.G.E.Methodology:J.R.E.,G.N.D.,L.A.R., A.M.G.,G.L.P ., T .S., andD.C.-D.Investiga tion: J.R.E.,G.N.D.,R.F ., J.D.H.,H.B.,L.A.R.,andG.L.P . Validation:R.F ., H.B.,andJ.M.-B.Datacuration:J.R.E.andR.G.E.Formalanalysis:J.R.E.andR.G.E. Writing(originaldraft):J.R.E.Writing(reviewandediting):J.R.E.,A.M.G.,andR.G.E.Funding acquisition:J.R.E.,A.M.G.,andR.G.E.Projectadministration:J.R.E.,A.M.G.,andR.G.E.Competing interests:Theauthorsdeclarethattheyhav enocompetinginterests.Dataandmaterials availability:Alldataneededtoevaluatetheconclusionsinthepaperarepresentinthepaper and/ortheSupplementaryMaterials.RawsequencingdatacanbefoundattheNCBISequence ReadArchive(BioProjectPRJNA841687;www.ncbi.nlm.nih.gov/bioproject/PRJNA841687/). CustomcodeusedfordataprocessingandanalysisispubliclyavailableatZenodo(https://doi. org/10.5281/zenodo.7388406)orGitHub(https://github.com/PerConSFA/SAGE_RTP).The materials presented in this study can be provided by R.G.E. ’s pending scientific review and a completed material transferagreement. Requests for materials should be submitted at robert. [email protected].