Construction of a Trp- commercial baker's yeast strain by using food-safe-grade dominant drug resistance cassettes.
We have designed a food-safe-grade module for gene disruptions in commercial baker's yeast strains, which contains the G418 resistance cassette, KanMX4, flanked by direct repeats from the MEL1 gene of Saccharomyces cerevisiae. This module was used to obtain a Trp(-) auxotrophic mutant of the polyploid HY strain by successive targeting to the TRP1 locus and later in vivo excision of the kan(r) marker. Southern blot analysis indicated that HY contains five copies of the TRP1 gene. However, after four disruption rounds, a strain named HYtrpM(4), unable to grow in the absence of tryptophan, was selected. Southern and Northern analysis of HYtrpM(4) cells showed that a remaining functional wild-type copy was still present, suggesting that the level of phosphoribosylanthranylate isomerase activity, resulting from a single copy of TRP1, is too low to sustain growth. Accordingly, a high reversion frequency of the Trp(-) phenotype, through gene conversion, was found in cells of the mutant strain. Nevertheless, this was not a drawback for its use as a recipient strain of heterologous genes. Indeed, YEpACT-X24 transformants were stable after 25 generations and expressed and secreted high levels of active recombinant xylanase. These data indicate that the new Trp(-) strain can be used to generate a stable recombinant yeast that fulfils all the requirements and market criteria for commercial utilisation.
Estruch
Francisco
F
Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), P.O. Box 73, 46100, Valencia, Burjassot, Spain.
Prieto
José Antonio
JA
D019747:Aldose-Ketose Isomerases; D001709:Biotechnology; D004352:Drug Resistance, Microbial / Q000235:genetics*; D005524:Food Technology; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D019747:Aldose-Ketose Isomerases; C490353:TRP1 protein, S cerevisiae