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.

Development of Genetic Modification Tools for Hanseniasporauvarum

“…Although both methods yielded transformants, electroporation yielded more transformants. The specific electroporation protocol used, involved a sensitising step with lithium acetate [ 43 ]. We constructed selection cassettes containing the resistance markers for hygromycin ( hph ), geneticin ( neoR ), nourseothricin (clonNAT, natI ), and zeocin ( Sh Ble ), commonly used for yeasts.…”

“…For transformation of H . uvarum DSM2768, an adaption of the protocols described by Thompson et al [ 58 ] and Bernardi et al [ 43 ] was used. H .…”

“…The transformation of S. cerevisiae was done according to the lithium acetate/single strand DNA/polyethylene glycol 4000 protocol described by Gietz and Schiestl [57]. For transformation of H. uvarum DSM2768, an adaption of the protocols described by Thompson et al [58] and Bernardi et al [43] was used. H. uvarum DSM2768 was grown overnight in 5 mL YPD at 30 °C.…”

A Versatile Toolset for Genetic Manipulation of the Wine Yeast Hanseniaspora uvarum

“…In addition, the low efficiency of homologous recombination could be improved by diminishing the activity of the nonhomologous end joining repair, e.g., deleting the two copies of the HuKU80 gene, as proven valuable in K. lactis [54]. Alternatively, taking advantage of both the high expression levels and the mitotic instability of the HuARS vectors described here, ScRAD51, a component of the homologous repair system of S. cerevisiae, could be transiently over-expressed from the HuTEF1 promoter in co-transformations with deletion cassettes, as exercised in lager yeast [55].…”

Detection of Escherichia Coli Using PCR Analysis Without DNA Extraction

“…Polymerase Chain Reaction (PCR) analysis has been proven having high accuracy and using in various purposes, including for disease diagnosis (Cherpillod et al 2016), forensic (Widodo at al 2018), genetic engineering (Bernardi et al 2019) and others. Before the PCR analysis, DNA must be separated from cells through DNA extraction.…”