Saccharomyces pastorianus IMX1187
Saccharomyces pastorianus
To limit construct instability and facilitate successive genome editing events it was decided to integrate a copy of the Streptococcus pyogenes cas9 variant, cas9D147Y P411T [36] was integrated in the genome of S. pastorianus CBS1483. The S. cerevisiae SPR3/YGR059W locus involved in sporulation was chosen as integration site, a function which is impaired in S. pastorianus, therefore the impact on growth of deletion of SPR3 should be negligible. Additionally, SPR3 is located in the middle of the right arm of the S. cerevisiae CHRVII that counts only one copy in CBS1483, which should enable stable integration of a single cas9 copy [4]. To prevent off-target integration driven by homology of the promoter and terminator, cas9 was placed under the control of the TEF1 promoter from Arxula adeninivorans, which has been shown to be functional in Saccharomyces yeast. The nourseothricin acetyl transferase expression cassette natNT2 expressed from the TEF1 promoter from the yeast Ashbya gossypii was used as a marker to select for integration (Figure 2). To guide the chromosomal integration of the endonuclease construct, the cas9 containing fragment was flanked with an homology region of 480-bp targeting the SPR3 promoter region (HRL, Figure 1) and a 506-bp targeting the SPR3 terminator region (HRM) to complete the double cross over integration (Figure 1). These elements were assembled into a transformation cassette on pUD526 and the purified integration fragment was used to transform S. pastorianus CBS1483 yielding 14 transformants. In comparison the same transformation in the laboratory S. cerevisiae CEN.PK113-7D yielded 476 transformants. Both transformations were performed simultaneously and under identical experimental conditions, therefore the difference in obtained transformants reflected the strongest resilience of industrial S. pastorianus strains to transformation.
strain
IMX1187
host
CBS1483
collection_date
2017
geo_loc_name
Netherlands
sample_type
pure culture
BioSampleModel
Microbe, viral or environmental