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.