Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae

Abstract

The widely used pESC vector series (Stratagene, La Jolla, CA, USA) with the bidirectional GAL1 /GAL10 promoter provides the possibility of simultaneously expressing two different genes from a single vector in Saccharomyces cerevisiae. This system can be induced by galactose and is repressed by glucose. Since S. cerevisiae prefers glucose as a carbon source, and since its growth rate is higher in glucose than in galactose-containing media, we compared and evaluated seven different promoters expressed during growth on glucose (pTEF1, pADH1, pTPI1, pHXT7, pTDH3, pPGK1 and pPYK1 ) with two strong galactose-induced promoters (pGAL1 and pGAL10 ), using lacZ as a reporter gene and measuring LacZ activity in batch and continuous cultivation. TEF1 and PGK1 promoters showed the most constant activity pattern at different glucose concentrations. Based on these results, we designed and constructed two new expression vectors which contain the two constitutive promoters, TEF1 and PGK1, in opposite orientation to each other. These new vectors retain all the features from the pESC-URA plasmid except that gene expression is mediated by constitutive promoters.

Novel Method for Genomic Promoter Shuffling by Using Recyclable Cassettes

“…The PGK1 promoter is considered one of the most powerful constitutive promoters when cells are grown in glucose as the preferred carbon source (21,22). The GAL1 gene, along with GAL7 and GAL10, is induced in the presence of galactose and strongly repressed in the presence of glucose (22)(23)(24)(25), and the GAL1 promoter is commonly used for the induction of target genes. P tetO7 is known as a tetracyclineregulatable promoter and is used in the Tet-off gene expression system (26)(27)(28).…”

Recovery of Phenotypes Obtained by Adaptive Evolution through Inverse Metabolic Engineering

“…Other strains carrying plasmids were maintained on the same SM medium, except for the complete supplement mixture without uracil (0.77 g/liter). To avoid uracil supplement-related effects, an empty plasmid with the URA3 gene as a marker, pSP-GM2, was used for transformation into the CEN.PK113-5D strain and the site-directed mutants (22). To overexpress the PGM2 gene, pPGM2 was used, which is a high-copy-number plasmid containing the constitutive promoter of the PMA1 gene and URA3 as a marker (3).…”

Engineering yeast shikimate pathway towards production of aromatics: rational design of a chassis cell using systems and synthetic biology

“…The same principles were used for ARO7 deletion / ARO4 K229L integration with a few differences: After incorporating the point mutation in codon 229 of the original ARO4 (as already described in (Williams et al, 2015)), which changes the respective amino acid residue from Lys to Leu and renders the protein feedback inhibition resistant, it was cloned to pCV3. The plasmid pCV3- Plasmid vectors for expression of the genes for pABA formation from chorismate were constructed based on pSP-G1 (Partow et al, 2010). The ABZ1 genes were always cloned to the TEF1 promotor's multi-cloning site while the ABZ2 genes were cloned into the PGK1 promotor's polylinker.…”

“…-74 - (Williams et al, 2015) pSP-G1 Ura + selectable double expression vector, contains TEF1-PGK1 bidirectional promoter (Partow et al, 2010) pSP-G1-ABZ1 AWRI1631 - and pH, collecting supernatant for analysis every three hours during exponential growth and twice a day in stationary phase (after 9 h in case of GLC, after 24 h in case of GLY/ETH).…”