Xylanase production by a recombinant strain of Saccharomyces cerevisiae: the effect of carbon catabolite repression and ethanol concentration in batch and continuous cultures

English: Endo-β-I,4-xylanase production by a recombinant strain of Saccharomyces cerevisiae Y294, transformed with a Trichoderma reesei xylanase gene (XYN2), was evaluated in batch and continuous cultures. Expression of the heterologous xylanase gene by this yeast was under control of a promoter-terminator expression cassette derived from the alcohol dehydrogenase II gene (ADH2) of S. cerevisiae, which is mainly regulated through carbon catabolite repression. The 3,5-dinitrosalicylic acid and Sornogyi-Nelson assays for endo-β-I,4-xylanase activity (hereafter referred to as xylanase) in supernatants from cultures of the recombinant S. cerevisiae strain and in a commercial xylanase preparation were compared. These two assays gave widely differing activity values, even more so than anticipated. These huge discrepancies in activity values obtained indicated that these assays did not allow a reliable comparison of the activities of xylanolytic enzyme mixtures and preparations containing a single xylanase without accessory enzymes. In aerobic batch culture, the highest specific rate of xylanase production (20.8 nkat.mg biomassi-1.h-1) and activity (1 590 nkat.ml-1) were recorded with glucose as carbon source. The inclusion of ethanol as carbon source, which is a standard procedure for the derepression of ADH2, resulted in poor xylanase production. The influence of glucose flux, glucose concentration and growth rate on xylanase production by this recombinant strain of S. cerevisiae Y294 was investigated in carbon-limited continuous culture and compared to results obtained with recombinant xylanase-producing S. cerevisiae strains HI58 and CEN.PK Il 0-6C. The latter two strains harboured the same expression cassette and xylanase gene (XYN2) as the recombinant S. cerevisiae Y294 strain. Xylanase production by strains Y294, HI58 and CEN.PK110-6C was markedly reduced at glucose flux values greater than 0.98, l.44 and l.68 mmol glucose.g biomass-1.h-1, respectively. Despite the obvious strain differences, with all three yeast strains the glucose flux appeared to contribute to a greater extent towards the regulation of xylanase production than did the glucose concentration in the culture. Xylanase production by recombinant S. cerevisiae strains Y294 and CEN.PKII0-6C was found to be transcriptionally repressed by ethanol at and above 5.5 g ethanol.I". This finding was surprising, since the xylanase gene was under the regulation of the ADH2 promoter and alcohol dehydrogenase II is associated with the assimilation of ethanol by effecting the catalysis of ethanol to acetaldehyde. This finding adds a new perspective to the regulation of alchohol dehydrogenase II. In a fed-batch culture of recombinant S. cerevisiae Y294, the specific rate of xylanase production was increased 2.5-fold as compared to batch cultures, through control of the growth rate at 0.1 h-I by using an exponentially increasing feed rate. These observations also indicate the important role of the growth rate, and thus possibly the carbon flux, in regulating ADH2-mediated xylanase production.