Overexpression of RAD51 Enables PCR-Based Gene Targeting in Lager Yeast

Abstract

Lager beer fermentations rely on specific polyploid hybrids between Saccharomyces cerevisiae and Saccharomyces eubayanus falling into the two groups of S. carlsbergensis/Saaz-type and S. pastorianus/Frohberg-type. These strains provide a terroir to lager beer as they have long traditional associations and local selection histories with specific breweries. Lager yeasts share, based on their common origin, several phenotypes. One of them is low transformability, hampering the gene function analyses required for proof-of-concept strain improvements. PCR-based gene targeting is a standard tool for manipulating S. cerevisiae and other ascomycetes. However, low transformability paired with the low efficiency of homologous recombination practically disable targeted gene function analyses in lager yeast strains. For genetic manipulations in lager yeasts, we employed a yeast transformation protocol based on lithium-acetate/PEG incubation combined with electroporation. We first introduced freely replicating CEN/ARS plasmids carrying ScRAD51 driven by a strong heterologous promoter into lager yeast. RAD51 overexpression in the Weihenstephan 34/70 lager yeast was necessary and sufficient in our hands for gene targeting using short-flanking homology regions of 50 bp added to a selection marker by PCR. We successfully targeted two independent loci, ScADE2/YOR128C and ScHSP104/YLL026W, and confirmed correct integration by diagnostic PCR. With these modifications, genetic alterations of lager yeasts can be achieved efficiently and the RAD51-containing episomal plasmid can be removed after successful strain construction.

Keywords: fermentation; gene function analysis; homologous recombination; hybrid yeast.

Figure 1

Generating heterologous synthetic marker and reporter genes. (A) Linear maps of pRS-AsTEF1p-LacZ and pRS-AsTEF1p-ScRAD51-HYG3 plasmids. (B) The AsTEF1p-LacZ reporter gene and the HYG3 resistance marker were tested in Saccharomyces cerevisiae BY4741. The presence of an active β-galactosidase was detected by adding X-Gal to the centre of the transformation plate (left panel, circle indicates the area of X-Gal application). The presence and function of the HYG3 marker was tested by transforming S. cerevisiae with pRS-AsTEF1p-ScRAD51-HYG3 and selecting the transformants in the presence of 100 µg/mL of hygromycin (right).

Figure 2

Comparison of two transformation methods in the Weihenstephan lager yeast strain WS 34/70. (A,C) A lithium-acetate/single strand DNA/polyethylene glycol (Li-Ac/ssDNA/PEG) and (B,D) a LiAc/PEG incubation combined with electroporation (Li-Ac/Ep) were used with two different antibiotic concentrations for the selection of transformants. The transformation of WS 34/70 with a freely replicative plasmid carrying a hygromycin resistance marker (pRS-AsTEF1p-ScRAD51-HYG3) yielded few transformants when using the Li-ac/ssDNA/PEG method (A,C). By contrast, the Li-Ac/Ep method together with antibiotic selection at 100 µg/mL final concentration (D), resulted in a higher transformation efficiency compared to the other combinations (A–C).

Figure 3

PCR-based gene targeting in lager yeast. The Weihenstephan strain WS34/70 carrying either an empty vector (pHYG3; A) or a RAD51 plasmid (pRAD51, C) was transformed with disruption cassettes containing the YES1 marker harboring 50 bp of flanking homology regions for targeting to the ScHSP104 locus. We used a Li-Ac/Ep transformation protocol and selected transformants on YPD plates supplemented with hygromycin and G418 (100 µg/mL final concentration each). Restreaking of putative transformant colonies on new selective plates indicated stable transformants were only obtained in the lager yeast strain overexpressing pRAD51 (B,D).

Figure 4

Diagnostic PCR for verification of correct marker integration. (A) Schematic presentation of PCR-based gene targeting of YES1 amplified with gene specific S1/S2-primers adding 50 bp of target homology region (orange bars) to ScHSP104 or ScADE2, respectively. Diagnostic G1/G2 and G3/G4 primers were used for verification of the correct insertion of YES1 and removal of the Sc HSP104 (B) and Sc ADE2 locus (C) ORFs.