A tetO Toolkit To Alter Expression of Genes in Saccharomyces cerevisiae

ACS Synthetic Biology
Josh T CuperusStanley Fields

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 permutation...Continue Reading

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Citations

Aug 3, 2018·Proceedings of the National Academy of Sciences of the United States of America·Michael W DorrityChristine Queitsch
Aug 23, 2019·Trends in Biotechnology·Ronald P H de JonghDick de Ridder
May 12, 2018·ACS Synthetic Biology·Anssi RantasaloDominik Mojzita
Aug 6, 2021·Biotechnology Journal·Andrew P Cazier, John Blazeck
Jan 28, 2020·Journal of Agricultural and Food Chemistry·Yun DuMu Li
Jul 5, 2021·Current Opinion in Chemical Biology·William M MooreItay Budin

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