Scandinavian Technology Group Ab

Lisbeth Olsson

Rakesh Koppram

Eva Albers

Nicklas Bonander

Elia Tomas-Pejo

The present invention relates to a method of preparing a strain of sugar fermentingwith capability to ferment xylose, wherein said method comprises different procedural steps. The method comprises mating a first sporulatedstrain with a secondhaploid strain. Thereafter, screening for mated cells is performed, growing such mated cells, and verifying that mated cells exhibit basic morphology by microscopic inspection. Thereafter, creation of a mixture of the mated cells is performed, subjecting the mixture to continuous chemostat cultivation and obtaining the sugar fermentingcells with capability to ferment xylose is performed. The invention also comprises strains obtained by said method.

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A method for producing a strain ofwith introduced genes coding for xylose reductase, xylitol dehydrogenase and xylulokinase and with improved ethanol production, improved xylose conversion and reduced xylitol production is described. The method comprises culturing the cells in a repetitive batch series in a medium at a xylose concentration of about 15-25 g/l and at a temperature of about 28-32&#xb0; C., and thereafter lowering the xylose concentration in at least one step at a temperature of about 28-32&#xb0; C. to obtain an increased selection pressure for improved xylose fermentation, improved ethanol production and reduced xylitol production, and continuing the culturing of the cells in said repetitive batch series. Further, strains ofobtained by the method according to the invention are described.

A method for producing a strain ofwith introduced genes coding for xylose reductase, xylitol dehydrogenase and xylulokinase and with improved ethanol production, improved xylose conversion, reduced xylitol production and improved inhibitor tolerance is described. The method comprises culturing a strain ofat a continuous mode with a medium comprising essentially only xylose as carbon source at a temperature of 25-38&#xb0; C., preferably 30-35&#xb0; C., and an airflow of 0.040-0.055 vvm, and increasing the dilution rate to maintain a constant cell level, said cell level being in the range of 1.5-3.0 determined by optical density or equivalent analytical means, and adding at least one inhibitor to the cells and gradually increasing the addition of said inhibitor. Further, strains ofobtained by the method according to the invention are described.

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Göteborg, Sweden

Lisbeth Olsson