Selection systems based on dominant-negative transcription factors for precise genetic engineering

Abstract

Diverse tools are available for performing genetic modifications of microorganisms. However, new methods still need to be developed for performing precise genomic engineering without introducing any undesirable side-alteration. Indeed for functional analyses of genomic elements, as well as for some industrial applications, only the desired mutation should be introduced at the locus considered. This article describes a new approach fulfilling these requirements, based on the use of selection systems consisting in truncated genes encoding dominant-negative transcription factors. We have demonstrated dominant-negative effects mediated by truncated Gal4p and Arg81p proteins in Saccharomyces cerevisiae, interfering with galactose and arginine metabolic pathways, respectively. These genes can be used as positive and negative markers, since they provoke both growth inhibition on substrates and resistance to specific drugs. These selection markers have been successfully used for precisely deleting HO and URA3 in wild yeasts. This genetic engineering approach could be extended to other microorganisms.

Figure 1.

Phenotype determination of GAL4Δ and ARG81Δ expressing S. cerevisiae strains. BY4709-derived strains carrying the various integrated vectors were streaked on diverse media for identifying phenotypes. P0int: negative control vector; pGint: GAL4Δ expression vector; pGVint: GAL4Δ:V5 expression vector; pAVint: ARG81Δ:V5 expression vector; pAint: ARG81Δ expression vector. First column: minimal medium (YNB) with 20 mg/ml glucose as carbon source (no significant effect resulting from expression of GAL4Δ or ARG81Δ). GAL4Δ-associated phenotypes were observed on YNB with 20 mg/ml galactose (second column) and on 2DG selection medium (YNB with 30 mg/ml glycerol, 20 mg/ml 2DG, 5 mg/ml casaminoacids, 1 mg/ml galactose and 0.2 mg/ml glucose, third column). ARG81Δ-related phenotypes were highlighted on YNB with 1 mg/ml ornithine as sole nitrogen source (fourth column) and on canavanine selection medium (YNB with 80 mM ammonium, 20 mg/ml glucose, 200 µg/ml ornithine and 8 µg/ml canavanine, (fifth column).

Figure 2.

Subcellular localization of truncated Gal4p-V5 fused to GFP.

Figure 3.

(A) Transcriptional analysis of GAL1, GAL2 and GAL10 expression in wild-type (carrying empty CYC1 integrative plasmid p0int) and truncated Gal4p overproducing S. cerevisiae strains (harboring CYC1 integrative pGint and pGVint vectors, enabling the expression of GAL4Δ and GAL4Δ:V5 respectively). Strains were cultivated on YNB with 20 mg/ml raffinose (glucose repression relieved, no galactose induction) and, for inducing GAL gene expression, 2 mg/ml galactose was added for one hour. mRNA of GAL1, GAL2 and GAL10 were quantified by qRT–PCR and the values were normalized with TBP1 mRNA. (B) Transcriptional analysis of ARG3 and CAR1 expression in wild-type (carrying empty integrative plasmid p0int) and truncated Arg81p overproducing S. cerevisiae strains (harboring CYC1 integrative pAint and pAVint vectors, enabling the expression of ARG81Δ and ARG81Δ:V5 respectively). Strains were cultivated either on YNB with 80 mM ammonium, 20 mg/ml glucose (expression of arginine biosynthesis genes, repression of arginine catabolic pathway genes) or on YNB with 80 mM ammonium, 20 mg/ml glucose and 1 mg/ml arginine (repression of arginine anabolic pathway genes, induction of arginine degradation genes). By qRT–PCR, mRNA of ARG3 and CAR1 were quantified and the values were normalized with TBP1 mRNA.

Figure 4.

Scheme of integrative cassettes for the deletion of HO and URA3. All cassettes are excised from pUC19 plasmid by a BamHI double digest. Up sequences are identical to a sequence upstream of targeted ORF and down sequences are identical to a sequence downstream of targeted ORF. Dashed yellow and blue boxes are small 30 repeats of upstream HO and URA3 sequences, respectively.

Figure 5.

Scheme of gene deletion by homologous recombination between linearized integrative vectors and targeted genomic sequences, and subsequent excision by homologous recombination between the designed short (30 bp) directly repeated genomic sequences.

Figure 6.

Plasmid maps of pCSC129, 130 and 131 containing GAL4Δ, ARG81Δ and GAL4Δ/KanMX4 selection markers respectively. These vectors are derived from pUC19 and contain two convenient polylinkers, BglII-AvrII-NotI and MluI-KpnI-BamHI, to introduce homologous sequences in view of genetic modification.