title: Rapid and Efficient One-Step Metabolic Pathway Integration in E. coli.
abstract: Methods for importing heterologous genes into genetically tractable hosts are among the most desired tools of synthetic biology. Easy plug-and-play construction methods to rapidly test genes and pathways stably in the host genome would expedite synthetic biology and metabolic engineering applications. Here, we describe a CRISPR-based strategy that allows highly efficient, single step integration of large pathways in Escherichia coli. This strategy allows high efficiency integration in a broad range of homology arm sizes and genomic positions, with efficiencies ranging from 70 to 100% in 7 distinct loci. To demonstrate the large size capability, we integrated a 10 kb construct to implement isobutanol production in a single day. The ability to efficiently integrate entire metabolic pathways in a rapid and markerless manner will facilitate testing and engineering of novel pathways using the E. coli genome as a stable testing platform.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, ‡Department of Chemical and Biological Engineering, §Department of Chemistry and Biochemistry, University of Colorado Boulder , Boulder, Colorado 80303, United States.|Lawrence Berkeley National Laboratory, Physical Bioscience Division, ¶Department of Bioengineering, Berkeley , California 94720, United States.
authors.forename: Marcelo C
authors.initials: MC
authors.lastname: Bassalo
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, ‡Department of Chemical and Biological Engineering, §Department of Chemistry and Biochemistry, University of Colorado Boulder , Boulder, Colorado 80303, United States.|Lawrence Berkeley National Laboratory, Physical Bioscience Division, ¶Department of Bioengineering, Berkeley , California 94720, United States.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Garst
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, ‡Department of Chemical and Biological Engineering, §Department of Chemistry and Biochemistry, University of Colorado Boulder , Boulder, Colorado 80303, United States.|Lawrence Berkeley National Laboratory, Physical Bioscience Division, ¶Department of Bioengineering, Berkeley , California 94720, United States.
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Halweg-Edwards
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, ‡Department of Chemical and Biological Engineering, §Department of Chemistry and Biochemistry, University of Colorado Boulder , Boulder, Colorado 80303, United States.|Lawrence Berkeley National Laboratory, Physical Bioscience Division, ¶Department of Bioengineering, Berkeley , California 94720, United States.
authors.forename: William C
authors.initials: WC
authors.lastname: Grau
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, ‡Department of Chemical and Biological Engineering, §Department of Chemistry and Biochemistry, University of Colorado Boulder , Boulder, Colorado 80303, United States.|Lawrence Berkeley National Laboratory, Physical Bioscience Division, ¶Department of Bioengineering, Berkeley , California 94720, United States.
authors.forename: Dylan W
authors.initials: DW
authors.lastname: Domaille
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, ‡Department of Chemical and Biological Engineering, §Department of Chemistry and Biochemistry, University of Colorado Boulder , Boulder, Colorado 80303, United States.|Lawrence Berkeley National Laboratory, Physical Bioscience Division, ¶Department of Bioengineering, Berkeley , California 94720, United States.
authors.forename: Vivek K
authors.initials: VK
authors.lastname: Mutalik
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, ‡Department of Chemical and Biological Engineering, §Department of Chemistry and Biochemistry, University of Colorado Boulder , Boulder, Colorado 80303, United States.|Lawrence Berkeley National Laboratory, Physical Bioscience Division, ¶Department of Bioengineering, Berkeley , California 94720, United States.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Arkin
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, ‡Department of Chemical and Biological Engineering, §Department of Chemistry and Biochemistry, University of Colorado Boulder , Boulder, Colorado 80303, United States.|Lawrence Berkeley National Laboratory, Physical Bioscience Division, ¶Department of Bioengineering, Berkeley , California 94720, United States.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Gill
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D000440:Butanols / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D016680:Genome, Bacterial; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics; D009154:Mutation; D000094704:RNA, Guide, CRISPR-Cas Systems; D015203:Reproducibility of Results
chemical_list: D001426:Bacterial Proteins; D000440:Butanols; D029968:Escherichia coli Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D049452:Green Fluorescent Proteins; C040507:isobutyl alcohol; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli
keywords: CRISPR; genome editing; genome integration; metabolic pathways; synthetic biology