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S. cerevisiae has many properties which have made it the preferred host for the expression and production of a number of recombinant proteins. Xylose is the second most abundant sugar in nature and S. cerevisiae has been engineered to grow on this abundant sugar. Therefore, identifying S. cerevisiae promoters that are strongly induced during growth on xylose will be important in the production of recombinant proteins for the biofuel and other industries. Since xylose is not a native substrate for S. cerevisiae, it is not known how S. cerevisiae promoters will react during growth on xylose. The objective of the study was to evaluate the expression of a reporter gene, the Trichoderma reesei xylanase 2 (XYN2), under the control of five commonly used expression promoters (GPD3, ENO2, PGK1, ADH2 and YG100). Five episomal expression vectors were constructed for this purpose. These vectors were transformed to a recombinant xylose utilizing S. cerevisiae. Xylanase activity assays were used to determine the expression level from each of these promoters. The PGK1 promoter was observed to be the strongest promoter with average activity/OD of 130 nkat/ml/OD on both xylose and glucose. The GPD3 promoter showed the highest average activity/OD of 150 nkat/ml, but xylanase was only produced during growth on glucose. The data presented show that xylose is not a better carbon source than glucose for recombinant protein production in terms of the S. cerevisie promoters evaluated. Further research is required to obtain a yeast strain that grows well on xylose and promoters that show higher level on protein production. Keywords: xylose, promoter, expression, recombinant, S. cerevisiae

Differences between the recombinant xylose-utilizing Saccharomyces cerevisiae strain TMB 3399 and the mutant strain TMB 3400, derived from TMB 3399 and displaying improved ability to utilize xylose, were investigated by using genome-wide expression analysis, physiological characterization, and biochemical assays. Samples for analysis were withdrawn from chemostat cultures. The characteristics of S. cerevisiae TMB 3399 and TMB 3400 grown on glucose and on a mixture of glucose and xylose, as well as of S. cerevisiae TMB 3400 grown on only xylose, were investigated. The strains were cultivated under chemostat conditions at a dilution rate of 0.1 h-1, with feeds consisting of a defined mineral medium supplemented with 10 g of glucose liter-1, 10 g of glucose plus 10 g of xylose liter-1 or, for S. cerevisiae TMB 3400, 20 g of xylose liter-1. S. cerevisiae TMB 3400 consumed 31% more xylose of a feed containing both glucose and xylose than S. cerevisiae TMB 3399. The biomass yields for S. cerevisiae TMB 3400 were 0.46 g of biomass g of consumed carbohydrate-1 on glucose and 0.43 g of biomass g of consumed carbohydrate-1 on xylose. A Ks value of 33 mM for xylose was obtained for S. cerevisiae TMB 3400. In general, the percentage error was <20% between duplicate microarray experiments originating from independent fermentation experiments. Microarray analysis showed higher expression in S. cerevisiae TMB 3400 than in S. cerevisiae TMB 3399 for (i) HXT5, encoding a hexose transporter; (ii) XKS1, encoding xylulokinase, an enzyme involved in one of the initial steps of xylose utilization; and (iii) SOL3, GND1, TAL1, and TKL1, encoding enzymes in the pentose phosphate pathway. In addition, the transcriptional regulators encoded by YCR020C, YBR083W, and YPR199C were expressed differently in the two strains. Xylose utilization was, however, not affected in strains in which YCR020C was overexpressed or deleted. The higher expression of XKS1 in S. cerevisiae TMB 3400 than in TMB 3399