Acetamidase gene as a new plasmid marker for CRISPR modification of industrial and laboratory <em>Saccharomyces</em> yeasts

Valery Urakov; Vitaly Kushnirov

doi:10.21638/spbu03.2024.205

Authors

Valery Urakov A. N. Bach Institute of Biochemistry, Federal Research Centre "Fundamentals of Biotechnology" of the Russian Academy of Sciences, Leninskiy pr., 33, Moscow, 119071, Russian Federation https://orcid.org/0000-0002-0417-1822
Vitaly Kushnirov A. N. Bach Institute of Biochemistry, Federal Research Centre "Fundamentals of Biotechnology" of the Russian Academy of Sciences, Leninskiy pr., 33, Moscow, 119071, Russian Federation https://orcid.org/0000-0003-0316-0766

DOI:

https://doi.org/10.21638/spbu03.2024.205

Abstract

Genetic manipulation of laboratory yeast strains relies on the use of auxotrophic markers. However, such markers are usually missing and undesirable in industrial yeasts. The standard option is then to use antibiotic resistance markers such as KanMX. However, the required antibiotic concentration can vary significantly depending on the growth medium and yeast strain, often resulting in a high proportion of false-positive colonies. An alternative selection procedure could be based on the ability of yeast cells to utilize an uncommon source of nitrogen. Many yeasts, including Saccharomyces, are unable to utilize acetamide. This ability can be conferred by a single acetamidase gene. The CRISPR/Cas9 technology has an advantage over previous methods of yeast modification in that it allows genomic modifications to be introduced without leaving a selectable marker in the genome. Driven by these two motives, we constructed a CRISPR/Cas9 plasmid with an acetamidase gene from the fungus Aspergillus nidulans, which allows the selection of yeast transformants on medium with acetamide as sole nitrogen source, and tested this plasmid with a triploid wine strain and a haploid laboratory strain.

Keywords:

wine yeast, selective marker, acetamidase gene, CAR1 gene, CRISPR/Cas9, acetamide, arginine, nitrogen source

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