TY - CHAP
T1 - Coupling yeast golden gate and VEGAS for efficient assembly of the violacein pathway in saccharomyces cerevisiae
AU - Chuang, James
AU - Boeke, Jef D.
AU - Mitchell, Leslie A.
N1 - Funding Information:
This work was supported by the National Science Foundation grant MCB-0718846 and Defense Advanced Research Projects Agency grant N66001-12-C-4020.
Publisher Copyright:
© 2018, Springer Science+Business Media, LLC.
PY - 2018
Y1 - 2018
N2 - The ability to express non-native pathways in genetically tractable model systems is important for fields such as synthetic biology, genetics, and metabolic engineering. Here we describe a modular and hierarchical strategy to assemble multigene pathways for expression in S. cerevisiae. First, discrete promoter, coding sequence, and terminator parts are assembled in vitro into Transcription Units (TUs) flanked by adapter sequences using “yeast Golden Gate” (yGG), a type IIS restriction enzyme-dependent cloning strategy. Next, harnessing the natural capacity of S. cerevisiae for homologous recombination, TUs are assembled into pathways and expressed using the “Versatile Genetic Assembly System” (VEGAS) in yeast. Coupling transcription units constructed by yGG with VEGAS assembly is a generic and flexible workflow to achieve pathway expression in S. cerevisiae. This protocol describes assembly of a five TU pathway for yeast production of violacein, a pigment derived from Chromobacterium violaceum.
AB - The ability to express non-native pathways in genetically tractable model systems is important for fields such as synthetic biology, genetics, and metabolic engineering. Here we describe a modular and hierarchical strategy to assemble multigene pathways for expression in S. cerevisiae. First, discrete promoter, coding sequence, and terminator parts are assembled in vitro into Transcription Units (TUs) flanked by adapter sequences using “yeast Golden Gate” (yGG), a type IIS restriction enzyme-dependent cloning strategy. Next, harnessing the natural capacity of S. cerevisiae for homologous recombination, TUs are assembled into pathways and expressed using the “Versatile Genetic Assembly System” (VEGAS) in yeast. Coupling transcription units constructed by yGG with VEGAS assembly is a generic and flexible workflow to achieve pathway expression in S. cerevisiae. This protocol describes assembly of a five TU pathway for yeast production of violacein, a pigment derived from Chromobacterium violaceum.
KW - Hierarchical DNA assembly
KW - Metabolic engineering
KW - Saccharomyces cerevisiae
KW - Versatile Genetic Assembly System (VEGAS)
KW - Violacein
KW - Yeast Golden Gate (yGG)
KW - Metabolic Engineering
KW - Cloning, Molecular/methods
KW - Escherichia coli/genetics
KW - Saccharomyces cerevisiae/genetics
KW - Metabolic Networks and Pathways
KW - Genetic Vectors/genetics
KW - Polymerase Chain Reaction
KW - Indoles/metabolism
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U2 - 10.1007/978-1-4939-7295-1_14
DO - 10.1007/978-1-4939-7295-1_14
M3 - Chapter
C2 - 29170962
AN - SCOPUS:85035151257
VL - 1671
T3 - Methods in molecular biology (Clifton, N.J.)
SP - 211
EP - 225
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -