A tetO Toolkit To Alter Expression of Genes in Saccharomyces cerevisiae.
Strategies to optimize a metabolic pathway often involve building a large collection of strains, each containing different versions of sequences that regulate the expression of pathway genes. Here, we develop reagents and methods to carry out this process at high efficiency in the yeast Saccharomyces cerevisiae. We identify variants of the Escherichia coli tet operator (tetO) sequence that bind a TetR-VP16 activator with differential affinity and therefore result in different TetR-VP16 activator-driven expression. By recombining these variants upstream of the genes of a pathway, we generate unique combinations of expression levels. Here, we built a tetO toolkit, which includes the I-OnuI homing endonuclease to create double-strand breaks, which increases homologous recombination by 10(5); a plasmid carrying six variant tetO sequences flanked by I-OnuI sites, uncoupling transformation and recombination steps; an S. cerevisiae-optimized TetR-VP16 activator; and a vector to integrate constructs into the yeast genome. We introduce into the S. cerevisiae genome the three crt genes from Erwinia herbicola required for yeast to synthesize lycopene and carry out the recombination process to produce a population of cells with permutations of tetO variants regulating the three genes. We identify 0.7% of this population as making detectable lycopene, of which the vast majority have undergone recombination at all three crt genes. We estimate a rate of ∼20% recombination per targeted site, much higher than that obtained in other studies. Application of this toolkit to medically or industrially important end products could reduce the time and labor required to optimize the expression of a set of metabolic genes.
†Howard Hughes Medical Institute, ‡Department of Genome Sciences, §Department of Medicine, University of Washington, Seattle, Washington 98195, United States.
Josh T
JT
Cuperus
†Howard Hughes Medical Institute, ‡Department of Genome Sciences, §Department of Medicine, University of Washington, Seattle, Washington 98195, United States.
Russell S
RS
Lo
†Howard Hughes Medical Institute, ‡Department of Genome Sciences, §Department of Medicine, University of Washington, Seattle, Washington 98195, United States.
Lucia
L
Shumaker
†Howard Hughes Medical Institute, ‡Department of Genome Sciences, §Department of Medicine, University of Washington, Seattle, Washington 98195, United States.
Julia
J
Proctor
†Howard Hughes Medical Institute, ‡Department of Genome Sciences, §Department of Medicine, University of Washington, Seattle, Washington 98195, United States.
Stanley
S
Fields
D001426:Bacterial Proteins / Q000235:genetics*; D002338:Carotenoids / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics*; D004885:Erwinia / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D059765:Homologous Recombination; D000077276:Lycopene; D060847:Metabolic Engineering; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; C083139:Tet O resistance protein, Bacteria; D002338:Carotenoids; D000077276:Lycopene
genome engineering; homologous recombination; lycopene; metabolic engineering; tetO element; yeast