Transfer of genes for utilization of starch (sta2) and melibiose (mel) to industrial strains of Saccharomyces cerevisiae by single-chromosome transfer, using a kar1 mutant as vector.

A method has been developed for the transfer of genes from other yeast strains and species to industrial yeast strains, using a haploid, kar1-1 mutant strain of Saccharomyces cerevisiae as a vector. The sta2 gene, conferring the ability to metabolize starch was transferred from an auxotrophic haploid strain of S. cerevisiae (S. diastaticus) and the melibiose-metabolism (mel) gene(s), from S. kluyveri, to the kar1-1 mutant [K5-5A; (alpha ade2 his4 can1 gal) by normal mating and protoplast fusion. From this strain, the genes were transferred to baker's yeast and brewing yeast strains, which did not utilize starch, and to baker's yeast strains, which did not utilize melibiose, by protoplast fusion, spore-cell pairing, or rare-mating. Strains that utilized starch or melibiose were obtained by all three methods. Pulsed-field gel electrophoresis preparations showed little change in the mobility of the chromosomes of the hybrids. The most probable explanation for the results obtained is that single chromosomes were transferred, first, from the donor strains to the kar1-1 haploid mutant strain, and then from the kar1-1 vector to the recipient industrial strain of S. cerevisiae. The transfer of the genes is probably accomplished through formation of disomic strains and then, in the case of the hybrids that metabolize starch, by integration of the sta2 gene into the genome of the industrial yeast strains.

Actividad de beta-galactosidasa de cepas de Kluyveromyces spp. y produccíon de etanol a partir de lactosa / Beta-galactosidase activity of strains of Kluyveromyces spp. and production of ethanol from lactose

Se realizaron estudios sobre levaduras del género Kluyveromyces (K. fragilis 507, K. lactis 29 y K. lactis 10), que crecen en lactosa como única fuente de carbono, ya que poseen el sistema enzimático para la utilización de este azucar. Se determinó la actividad de ß-galactosidasa en medios con glucosa y con lactosa, en cultivos de estas tres cepas en fase log. Al agregar entre ) y 12% v/v de etanol a células tratadas con tolueno, no se observó inhibición enzimática para la cepa K. lactis 10 que tenía la mayor actividad enzimática (704,4 Unidades). Como existe la posibilidad de utilizar industrialmente suero concentrado 4 veces como sustrato que contenga lactosa, se realizaron ensayos de fermentación a 30 C las tres cepas, en medios que contenían inicialmente 16,5 y 24,5% de lactosa. Al cabo de 48 h la lactosa residual fue prácticamente cero, lográndose concentraciones de etanol directo entre 7,60 y 10,10% v/v. Es de esperar que la velocidad de fermentación de un disacárido como lactosa, esté relacionada con la velocidad de hidrólisis del mismo azúcar, por lo que cepas con una mayor velocidad enzimática hidrolítica debieran poseer mayor velocidad de fermentación. En este caso no se observó tal comportamiento, por cuanto cepas con actividad enzimática tan distinta como K. lactis 10 (704,4 U) y K lactis 29(189,7 U) no presentaron mayores diferencias en la producción de etanol a partir de lactosa

Actividad de beta-galactosidasa de cepas de Kluyveromyces spp. y produccíon de etanol a partir de lactosa / Beta-galactosidase activity of strains of Kluyveromyces spp. and production of ethanol from lactose

Se realizaron estudios sobre levaduras del género Kluyveromyces (K. fragilis 507, K. lactis 29 y K. lactis 10), que crecen en lactosa como única fuente de carbono, ya que poseen el sistema enzimático para la utilización de este azucar. Se determinó la actividad de ß-galactosidasa en medios con glucosa y con lactosa, en cultivos de estas tres cepas en fase log. Al agregar entre ) y 12% v/v de etanol a células tratadas con tolueno, no se observó inhibición enzimática para la cepa K. lactis 10 que tenía la mayor actividad enzimática (704,4 Unidades). Como existe la posibilidad de utilizar industrialmente suero concentrado 4 veces como sustrato que contenga lactosa, se realizaron ensayos de fermentación a 30 C las tres cepas, en medios que contenían inicialmente 16,5 y 24,5% de lactosa. Al cabo de 48 h la lactosa residual fue prácticamente cero, lográndose concentraciones de etanol directo entre 7,60 y 10,10% v/v. Es de esperar que la velocidad de fermentación de un disacárido como lactosa, esté relacionada con la velocidad de hidrólisis del mismo azúcar, por lo que cepas con una mayor velocidad enzimática hidrolítica debieran poseer mayor velocidad de fermentación. En este caso no se observó tal comportamiento, por cuanto cepas con actividad enzimática tan distinta como K. lactis 10 (704,4 U) y K lactis 29(189,7 U) no presentaron mayores diferencias en la producción de etanol a partir de lactosa (AU)

[Beta-galactosidase activity of strains of Kluyveromyces spp. and production of ethanol from lactose]. / Actividad de beta-galactosidasa de cepas de Kluyveromyces spp. y producción de etanol a partir de lactosa.

We investigated the behavior of yeast of the genus Kluyveromyces (K. fragilis 507, K. lactis 29 and K. lactis 10), which grow on lactose as sole carbon source, since they possess an enzyme system for the utilization of this sugar. We determined the beta-galactosidase activity of these strains, grown in the logarithmic phase in media containing glucose and lactose. On addition of 0 to 12% v/v ethanol to cells treated with toluene, we did not observe inhibition of the enzyme in strain 10 of Kluyveromyces lactis, which showed the greatest activity (704.4 Units). Since there exist the possibility of industrial utilization of concentrated whey (4 times), we performed fermentation tests of the three strains, at 30 C, in media containing initial lactose concentrations of 16.5 and 24.5%. After 48 h the residual lactose concentration was practically zero, and the ethanol concentrations had reached 7.60 and 10.10% v/v. It might be expected that the rate of fermentation of a disaccharide such as lactose would be related to the rate of hydrolysis of the sugar, so that strains having a higher rate of enzymatic hydrolysis should show a higher fermentation rate. However, we did not observe such behavior, as strains of Kluyveromyces having enzymatic activities as different as K. lactis 10 (704.4 U) and K. lactis 29 (189.7 U) did not show any great difference in the production of ethanol from lactose.

Actividad de beta-galactosidasa de cepas de Kluyveromyces spp. y producción de etanol a partir de lactosa. / [Beta-galactosidase activity of strains of Kluyveromyces spp. and production of ethanol from lactose]

We investigated the behavior of yeast of the genus Kluyveromyces (K. fragilis 507, K. lactis 29 and K. lactis 10), which grow on lactose as sole carbon source, since they possess an enzyme system for the utilization of this sugar. We determined the beta-galactosidase activity of these strains, grown in the logarithmic phase in media containing glucose and lactose. On addition of 0 to 12

v/v ethanol to cells treated with toluene, we did not observe inhibition of the enzyme in strain 10 of Kluyveromyces lactis, which showed the greatest activity (704.4 Units). Since there exist the possibility of industrial utilization of concentrated whey (4 times), we performed fermentation tests of the three strains, at 30 C, in media containing initial lactose concentrations of 16.5 and 24.5

. After 48 h the residual lactose concentration was practically zero, and the ethanol concentrations had reached 7.60 and 10.10

v/v. It might be expected that the rate of fermentation of a disaccharide such as lactose would be related to the rate of hydrolysis of the sugar, so that strains having a higher rate of enzymatic hydrolysis should show a higher fermentation rate. However, we did not observe such behavior, as strains of Kluyveromyces having enzymatic activities as different as K. lactis 10 (704.4 U) and K. lactis 29 (189.7 U) did not show any great difference in the production of ethanol from lactose.