Vector YFRp1 allows transformant selection in Saccharomyces cerevisiae via resistance to formaldehyde

Abstract

Formaldehyde (FA), a chemical with low toxic potential, is used as sole selective agent for transformation in the yeast Saccharomyces cerevisiae. Neither stable auxotrophic markers in recipient cells nor defined synthetic media are needed when multicopy vector YFRp1, containing the yeast SFA gene, is employed for yeast transformation. The SFA gene gives stability to the vector and its yeast (and other) passenger genes when transformants are propagated in complex media supplemented with 3-5 mM-FA. Use of inexpensive FA and non-synthetic, undefined media will lower the cost of yeast transformant propagation considerably and thus make feasible large-volume industrial application of transformants containing YFRp1 derivatives.

Trifluoroleucine resistance as a dominant molecular marker in transformation of strains of Saccharomyces cerevisiae isolated from wine

“…To overcome this obstacle several dominant resistance markers have been developed. They include resistance to methylglyoxal [1], copper [2], methotrexate [3], sulfometuron methyl [4], G418 [5], cycloheximide [6], formaldehyde [7,8], o-£uoro-DL-phenylalanine [9] and £uoroacetate [8]. Regardless of their price, not one of these markers is without pitfalls, i.e.…”

Genetic engineering of baker's and wine yeasts using formaldehyde hyperresistance-mediating plasmids

“…In the present study we show the applicability of another selection system that seems to be advantageous for large-scale fermentations, because unlike most of the above-mentioned selection procedures, it requires an inexpensive chemical that is subject to degradation during the fermentation process of FA-hyperresistant yeast cultures. Selection based on FA hyperresistance allows growth of yeast transformants in the usual industrial media to which they are optimally adapted, with the selective agent being metabolized mainly to CO 2 and to traces of methanol (10,20). Thus, the use of FA reduces and sometimes practically eliminates the problem of removal (detoxification) of the employed selective chemical FA (poison, weak mutagen; 21).…”

“…This hyperresistance phenotype is achieved by overexpression of the S. cerevisiae gene SFA, coding for a glu-tathione-dependent formaldehyde dehydrogenase (9). Consequently, this led to the development of the formaldehyde selectable yeast vector YFRp1 (10). We demonstrate here the applicability of this vector and derivatives in genetic engineering of baker's and brewer's yeasts which paves the way for the development of simple to apply and inexpensive to use expression vectors for these hosts.…”

“…For transformation of yeast a lithium acetate protocol (13) was modified as suggested by Wehner and Brendel (10). Cultures were harvested in the exponential growth phase and concentrated to 2 x 10 9 cells/ml in 0.…”

2‐μm vectors containing the Saccharomyces cerevisiae metallothionein gene as a selectable marker: Excellent stability in complex media, and high‐level expression of a recombinant protein from a CUP1‐promoter‐controlled expression cassette in cis

“…An alternative to autoselection systems consists in the use of (semi)dominant selectable marker genes. Quite a few such markers, most of them heterologous to yeast, have been described (Webster and Dickson, 1983;Zhu et al, 1986;Hadfield et al, 1986;Kunze et al, 1389;Wehner and Brendel, 1993). Unfortunately, these systems most often require the application of antibiotics.…”