No abstract

Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wildtype and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEMÒ-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k cat ) for D-xylose compared with the wild-type enzyme at 60°C, but they did not show any increase in catalytic eciency (k cat /K m ). For D-glucose, both the k cat and the k cat /K m values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K i ) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular¯exibility of the mutant enzymes at low temperatures.

The yeast Yarrowia lipolytica was shown to be sensitive to the aminoglycoside antibiotic hygromycin B. Spontaneous resistants appeared at a frequency of (2-5) x 10(-7) in media containing 100 mg/l drug. In order to develop a new selective marker for the transformation of this yeast, we constructed new plasmids expressing the Escherichia coli hygromycin-resistance gene (hph) under the control of the promoter and terminator sequences of the strongly expressed XPR2 gene of Y. lipolytica. Direct selection of hygromycin-B-resistant transformants on complete medium was very efficient and resulted in transformation frequencies comparable to those observed with conventional auxotrophic markers. This new marker can be used for integrating single copies of plasmid and for gene disruption and provides a convenient tag for genetic studies.

In the yeast Yarrowia lipolytica the levels of the alkaline extracellular protease (AEP) and acid extracellular protease (AXP) are controlled by the pH of the growth medium. When the pH of growth medium is kept close to 4.0, levels of AXP are high and those of AEP are low, whereas at pH above 6.0 the opposite is true. Mutations which mimic the effects on the protease system of growth at alkaline pH have been identified in two genes, RPH1 and RPH2, in Y. lipolytica. Detailed genetic studies showed that mutations in these two genes are dominant in heterozygous diploids, and that their effects are additive in haploid double mutants. These mutants show that pH regulates AEP expression independently from other metabolic signals. These mutants are not detectably affected in their growth rates, nor in internal pH homeostasis.

In the yeast Yarrowia lipolytica the levels of the alkaline extracellular protease (AEP) and acid extracellular protease (AXP) are controlled by the pH of the growth medium. When the pH of growth medium is kept close to 4.0, levels of AXP are high and those of AEP are low, whereas at pH above 6.0 the opposite is true. Mutations which mimic the effects on the protease system of growth at alkaline pH have been identified in two genes, RPH1 and RPH2, in Y. lipolytica. Detailed genetic studies showed that mutations in these two genes are dominant in heterozygous diploids, and that their effects are additive in haploid double mutants. These mutants show that pH regulates AEP expression independently from other metabolic signals. These mutants are not detectably affected in their growth rates, nor in internal pH homeostasis.