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1

Classic techniques for improvement of industrial yeast strains. Part I? Construction of ethanol-resistant and osmophilic industrial strains of Saccharomyces cerevisiae by electrofusion

100%
Salek A. T.
Biotechnologia
|
2002
|
issue 1
153-174
EN
The effective production of biomass or ethanol in industrial media of high osmolality requires new yeast strains. The present work focused on the development of such strains. Genetic engineering methods using cytoplasmatically-marked yeast (electrofusion of protoplasts of heterothallic haploids; electrotransformation of killer dsRNA or VLPs into haploids; generation of rho-) were used. The characteristics of the hybrids were evaluated by conventional analytical and instrumental methods, followed by statistical interpretation. After screening for a minimum 10% increase in industrially-relevant parameters, 3 osmophilic hybrids of baker's yeast, as well as 8 improved strains of distillery yeasts were selected. The baker?s yeasts showed optimum growth in a relatively concentrated molasses wort (1:5 ratio of molasses to final volume). The alcohol-resistant yeasts (including killer) produced up to 14.5% (w/w) ethanol in a medium containing 34% dissolved solids (a mixed mash of sucrose and potato). The characteristics of the alcohol-resistant and osmophilic yeasts were stable over several years of their industrial applications.The results show that electrical techniques (fusion to obtain hybrids, with interpretation by computer-aided image analysis, and transformation to give marked strains) can be used effectively enough for the construction of some industrially-productive yeasts.
2

Intensification of lipase biosynthesis as a result of electrofusion of Rhizopus cohnii protoplasts

100%
Sawicka-Zukowska R., Juszczakiewicz D., Misiewicz A., Krakowiak A., Jedrychowska B.
Journal of Applied Genetics
|
2004
|
vol. 45
|
issue 1
37-48
EN
Our objective was to obtain products of fusion of the filamentous fungus Rhizopus cohnii Rh.c./1 with an increased capacity for lipase biosynthesis in comparison with the original strain. Protoplasts of auxotrophic mutants of the parent strain Rh.c./1 obtained after UV irradiation of the spores were subjected to electrofusion. We found that the largest number of electrofusion products could be obtained with the use of the following process parameters: 1 or 2 impulses immediately following one another with a field intensity of 200 V/cm and an exposition time of 1000 ms at the stage of dielectrophoresis, 1 impulse with a field intensity of 500 V/cm and an exposition time of 10 ms or 20 ms at the stage of fusion, regulated temperature of 4oC before and after the process, rounding time of ca 20 min. Electrofusion of protoplasts of auxotrophic mutants of the Rh.c./1 strain produced 19 fusion products whose lipase biosynthesis capacity in a liquid medium culture was higher than that of the parent strains. The fusion product labelled XIII-21 was selected as the best strain. Lipase activity obtained after its culture in the liquid medium was ca 3.5 times higher than that obtained after the culture of the original strain Rh.c./1.
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