title: Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae.
abstract: We have developed a method for assembling genetic pathways for expression in Saccharomyces cerevisiae. Our pathway assembly method, called VEGAS (Versatile genetic assembly system), exploits the native capacity of S. cerevisiae to perform homologous recombination and efficiently join sequences with terminal homology. In the VEGAS workflow, terminal homology between adjacent pathway genes and the assembly vector is encoded by 'VEGAS adapter' (VA) sequences, which are orthogonal in sequence with respect to the yeast genome. Prior to pathway assembly by VEGAS in S. cerevisiae, each gene is assigned an appropriate pair of VAs and assembled using a previously described technique called yeast Golden Gate (yGG). Here we describe the application of yGG specifically to building transcription units for VEGAS assembly as well as the VEGAS methodology. We demonstrate the assembly of four-, five- and six-gene pathways by VEGAS to generate S. cerevisiae cells synthesizing β-carotene and violacein. Moreover, we demonstrate the capacity of yGG coupled to VEGAS for combinatorial assembly.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University Langone School of Medicine, New York City, NY 10016, USA Institute for Systems Genetics, New York University Langone School of Medicine, New York City, NY 10016, USA High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Mitchell
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA Department of Biomedical Engineering and Institute of Genetic Medicine, Whiting School of Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: James
authors.initials: J
authors.lastname: Chuang
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University Langone School of Medicine, New York City, NY 10016, USA Institute for Systems Genetics, New York University Langone School of Medicine, New York City, NY 10016, USA High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Neta
authors.initials: N
authors.lastname: Agmon
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA Department of Biomedical Engineering and Institute of Genetic Medicine, Whiting School of Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Chachrit
authors.initials: C
authors.lastname: Khunsriraksakul
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA Department of Biomedical Engineering and Institute of Genetic Medicine, Whiting School of Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Nick A
authors.initials: NA
authors.lastname: Phillips
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Yizhi
authors.initials: Y
authors.lastname: Cai
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University Langone School of Medicine, New York City, NY 10016, USA Institute for Systems Genetics, New York University Langone School of Medicine, New York City, NY 10016, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Truong
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Ashan
authors.initials: A
authors.lastname: Veerakumar
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Yuxuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: DSM Nutritional Products, Lexington, MA 02421, USA.
authors.forename: María
authors.initials: M
authors.lastname: Mayorga
authors.affiliation: DSM Nutritional Products, Lexington, MA 02421, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Blomquist
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Praneeth
authors.initials: P
authors.lastname: Sadda
authors.affiliation: DSM Nutritional Products, Lexington, MA 02421, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: Trueheart
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University Langone School of Medicine, New York City, NY 10016, USA Institute for Systems Genetics, New York University Langone School of Medicine, New York City, NY 10016, USA High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA jef.boeke@nyumc.org.
authors.forename: Jef D
authors.initials: JD
authors.lastname: Boeke
mesh_terms: D053898:Biosynthetic Pathways / Q000235:genetics*; D005800:Genes, Fungal; D005822:Genetic Vectors; D059765:Homologous Recombination; D007211:Indoles / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D058615:Synthetic Biology / Q000379:methods; D014158:Transcription, Genetic; D019207:beta Carotene / Q000096:biosynthesis
chemical_list: D007211:Indoles; D019207:beta Carotene; C063155:violacein