title: One-step assembly and targeted integration of multigene constructs assisted by the I-SceI meganuclease in Saccharomyces cerevisiae.
abstract: In vivo assembly of overlapping fragments by homologous recombination in Saccharomyces cerevisiae is a powerful method to engineer large DNA constructs. Whereas most in vivo assembly methods reported to date result in circular vectors, stable integrated constructs are often preferred for metabolic engineering as they are required for large-scale industrial application. The present study explores the potential of combining in vivo assembly of large, multigene expression constructs with their targeted chromosomal integration in S. cerevisiae. Combined assembly and targeted integration of a ten-fragment 22-kb construct to a single chromosomal locus was successfully achieved in a single transformation process, but with low efficiency (5% of the analyzed transformants contained the correctly assembled construct). The meganuclease I-SceI was therefore used to introduce a double-strand break at the targeted chromosomal locus, thus to facilitate integration of the assembled construct. I-SceI-assisted integration dramatically increased the efficiency of assembly and integration of the same construct to 95%. This study paves the way for the fast, efficient, and stable integration of large DNA constructs in S. cerevisiae chromosomes.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands; Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands.
authors.forename: Niels G A
authors.initials: NG
authors.lastname: Kuijpers
authors.forename: Soultana
authors.initials: S
authors.lastname: Chroumpi
authors.forename: Tim
authors.initials: T
authors.lastname: Vos
authors.forename: Daniel
authors.initials: D
authors.lastname: Solis-Escalante
authors.forename: Lizanne
authors.initials: L
authors.lastname: Bosman
authors.forename: Jack T
authors.initials: JT
authors.lastname: Pronk
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Daran
authors.forename: Pascale
authors.initials: P
authors.lastname: Daran-Lapujade
mesh_terms: D026923:Amino Acid Transport Systems, Basic / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics; D023061:Gene Order; D018390:Gene Targeting; D006019:Glycolysis / Q000235:genetics; D059765:Homologous Recombination; D016254:Mutagenesis, Insertional; D040641:Quantitative Trait Loci; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D026923:Amino Acid Transport Systems, Basic; C502212:CAN1 protein, S cerevisiae; D018797:Cell Cycle Proteins; C096553:SPR3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific; D020558:GTP Phosphohydrolases
keywords: homologous recombination; in vivo assembly; pathway engineering; synthetic biology
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