Characterization of genetically transformed Saccharomyces cerevisiae baker's yeasts able to metabolize melibiose

Abstract

Three transformant (Mel+) Saccharomyces cerevisiae baker's yeast strains, CT-Mel, VS-Mel, and DADI-Mel, have been characterized. The strains, which originally lacked α-galactosidase activity (Mel-), had been transformed with a DNA fragment which possessed an ILV2-SMR1 allele of the ILV2 gene and a MEL1 gene. The three transformed strains showed growth rates similar to those of the untransformed controls in both minimal and semi-industrial (molasses) media. The α-galactosidase specific activity of strain CT-Mel was twice that of VS-Mel and DADI-Mel. The yield, Y(X/S) (milligrams of protein per milligram of substrate), in minimal medium with raffinose as the carbon source was 2.5 times higher in the transformed strains than in the controls and was 1.5 times higher in CT-Mel than in VS-Mel and DADI-Mel. When molasses was used, Y(X/S) (milligrams of protein per milliliter of culture) increased 8% when the transformed strains CT-Mel and DADI-Mel were used instead of the controls. Whereas no viable spores were recovered from either DADI-Mel or VS- Mel tetrads, genetic analysis carried out with CT-Mel indicated that the MEL1 gene has been integrated in two of three homologous loci. Analysis of the DNA content by flow cytometry indicated that strain CT-Mel was 3n, whereas VS- Mel was 2n and DADI-Mel was 1.5n. Electrophoretic karyotype and Southern blot analyses of the transformed strains showed that the MEL1 gene has been integrated in the same chromosomic band, probably chromosome XIII, in the three strains. Tetrads of CT-Mel strain segregated 4+:0-, 3+:1- and 2+:2-, for both positive MEL1 Southern blots and α-galactosidase activity, indicating, as expected, integration of the MEL1 gene in two of three homologous chromosomes.