Yeasts selectivity

A variety of tiansformation techniques using E. co/ -ye st shuttle vectors and yeast selectable markets, as well as efficient yeast promoters and signal... 

Zuzuarregui, A. and del Olmo, M. (2004). Analyses of stress resistance under laboratory conditions constitute a suitable criterion for wine yeast selection. Antonie Van Leeuwenhoek 85, 271-280. 

As with other in vivo selections, it is critical to validate hits at the end of the selection experiment with a secondary screen. The most common secondary screen used with the Y2H assay is a lacZ screen. If either the DBD or AD fusion is under control of an inducible promoter, this screen can be carried out both under inducing and non-inducing conditions to ensure that transcription activation is protein dependent. Y3H systems, where transcription activation depends on a bridging RNA or small molecule, and reverse Y2H systems, where a third protein is disrupting the interaction, provide a built-in control. One can simply check that transcription activation is in fact dependent on the third component. As with any in vivo selection, the evolved plasmid should be isolated and retransformed into a fresh yeast selection strain to ensure that the phenotype is plasmid-dependent. Ultimately, the interaction will be confirmed with co-immunoprecipitation experiments or other in vitro binding assays [39]. 

To outline the process, the aqueous substrate for beer production is made by brewing cereals with hops, which provide the characteristic bitter flavor component. Barley is the chief cereal ingredient, which is first malted to give enzymes required to breakdown starches to simple sugars, and also to contribute an important flavor component. Barley is a fairly expensive source of starch, so it is usually supplemented by the addition of less expensive starch adjuncts such as corn (maize), oats, millet, wheat, or rice. The adjunct contributes to carbohydrates, but makes little contribution to the flavor. Various strains of yeasts selected for their efficient conversion of glucose to ethanol are refined by individual brewers to achieve desired product qualities. 

Two yeast selectable markers wild-type genes TRPl and URA3, which are located on opposite sides of the cloning sites. 

The reduction of racemic 5 is a nice example of product-selective biocatalytic kinetic resolution. This means that the enantiomer that is reduced affords only one of two possible diastereomers. Enzyme reductase from the cells of Saccharomyces cerevisiae (baker s yeast) selectively reduces the carbonyl group in the S-enantiomer of 5 into alcohol with an S configuration on the new stereogenic center, hence producing (IS, 2S)-6. Due to the C-H acidify of the a-C atom in 5, it easily tautomerizes to enols 5a and 5b in equilibrium (Scheme 5.36). 

---