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ACS Synth Biol. 2015 Jul 17;4(7):842-52. doi: 10.1021/sb500363y. Epub 2015 Mar 17.

A tetO Toolkit To Alter Expression of Genes in Saccharomyces cerevisiae.

Author information

1
†Howard Hughes Medical Institute, ‡Department of Genome Sciences, §Department of Medicine, University of Washington, Seattle, Washington 98195, United States.

Abstract

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.

KEYWORDS:

genome engineering; homologous recombination; lycopene; metabolic engineering; tetO element; yeast

PMID:
25742460
PMCID:
PMC4506738
DOI:
10.1021/sb500363y
[Indexed for MEDLINE]
Free PMC Article

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