ABSTRACT
Methylotrophic yeasts such as Ogataea polymorpha and Komagataella phaffii (sin. Hansenula polymorpha and Pichia pastoris, respectively) are commonly used in basic research and biotechnological applications, frequently those requiring genome modifications. However, the CRISPR-Cas9 genome editing approaches reported for these species so far are relatively complex and laborious. In this work we present an improved plasmid vector set for CRISPR-Cas9 genome editing in methylotrophic yeasts. This includes a plasmid encoding Cas9 with a nuclear localization signal and plasmids with a scaffold for the single guide RNA (sgRNA). Construction of a sgRNA gene for a particular target sequence requires only the insertion of a 24 bp oligonucleotide duplex into the scaffold. Prior to yeast transformation, each plasmid is cleaved at two sites, one of which is located within the selectable marker, so that the functional marker can be restored only via recombination of the Cas9-containing fragment with the sgRNA gene-containing fragment. This recombination leads to the formation of an autonomously replicating plasmid, which can be lost from yeast clones after acquisition of the required genome modification. The vector set allows the use of G418-resistance and LEU2 auxotrophic selectable markers. The functionality of this setup has been demonstrated in O. polymorpha, O. parapolymorpha, O. haglerorum and Komagataella phaffii.
Subject(s)
CRISPR-Cas Systems , Gene Editing , CRISPR-Cas Systems/genetics , Plasmids/geneticsABSTRACT
We identified Saccharomyces cerevisiae mutants with 100% higher intracellular glutathione using 1-methyl-3-nitro-1-nitrosoguanidine mutagenesis. This method employs visual selection of the most pigmented colonies among met30 strains carrying ade1 and ade2 mutations. Since the method does not involve genetic engineering, the mutants are suitable for use in the food industry.
Subject(s)
Color , Glutathione/analysis , Mutation/genetics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/isolation & purification , Sulfur/analysis , Cell Separation/methods , Food Industry/methods , Genes, Fungal/genetics , Glutathione/metabolism , Pigmentation/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
Glutathione overproducers were detected by examining the pigmentation intensity of Saccharomyces cerevisiae met30 yeast carrying wild-type alleles for ADE1 and ADE2. Highly pigmented colonies, phenocopies of the ade2 or ade1 mutants, were observed among yeast grown in minimal biotin-free medium with a high methionine content.
