Two glucose-phosphorylating enzymes, a hexokinase phosphorylating both glucose and fructose, and a glucose-specific glucokinase were electrophoretically separated in the methylotrophic yeast Hansenula polymorpha. Hexokinase-negative, glucokinase-negative and double kinase-negative mutants were isolated in H. polymorpha by using mutagenesis, selection and genetic crosses. Regulation of synthesis of the sugar-repressed alcohol oxidase, catalase and maltase was studied in different hexose kinase mutants. In the wild type and in mutants possessing either hexokinase or glucokinase, glucose repressed the synthesis of maltase, alcohol oxidase and catalase. Glucose repression of alcohol oxidase and catalase was abolished in mutants lacking both glucose-phosphorylating enzymes (i.e. in double kinase-negative mutants). Thus, glucose repression in H. polymorpha cells requires a glucose-phosphorylating enzyme, either hexokinase or glucokinase. The presence of fructose-phosphorylating hexokinase in the cell was specifically needed for fructose repression of alcohol oxidase, catalase and maltase. Hence, glucose or fructose has to be phosphorylated in order to cause repression of the synthesis of these enzymes in H. polymorpha suggesting that sugar repression in this yeast therefore relies on the catalytic activity of hexose kinases.

Glucose transport was studied in a methylotrophic yeast Hansenula polymorpha. Two kinetically different glucose transport systems were revealed in cells grown under different growth conditions. Glucose-repressed cells exhibited a low-affinity transport system (K, for glucose 1.75 mM) while glucose-derepressed and ethanol-grown cells had a high-affinity transport system (K, for glucose 0.054.06 mM). The high-and low-affinity transport systems differed in substrate specificity, sensitivity to pH, dinitrophenol and protonophore carbonyl cyanide-m-chlorophenyl-hydrazone. The kinetic rearrangement of the glucose transport system in response to altered growth conditions was dependent on de novo protein synthesis. 0 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

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We previously showed that, unlike other yeasts, Hansenula polymorpha possesses a glucokinase HPGLK1 that can mediate glucose repression in this yeast, although it cannot replace the regulatory function of hexokinase 2 in Saccharomyces cerevisiae. In the present study, the H. polymorpha hexokinase gene HPHXK1 was cloned by complementation of the glucose growth deficiency of the H. polymorpha double kinase-negative mutant A31-10 with a genomic library. The sequence of the 483-amino acid hexokinase protein deduced from the HPHXK1 gene showed the highest degree of identity (56%) with hexokinase from Schwanniomyces occidentalis, whereas the identity with hexokinase from Kluyveromyces lactis and both hexokinases from Sac. cerevisiae was 55%. The hexokinase protein was purified from crude extracts of H. polymorpha, using ion exchange chromatography and gel filtration. The K(m) values of the purified enzyme for glucose, fructose and ATP were 0.26 mM, 1.1 mM and 0.32 mM, respectively. H. polymorpha hexokinase was inhibited by trehalose-6-phosphate ( K(i)=12 microM) and ADP ( K(i)=1.6 mM), but not by glucose-6-phosphate. Transformation of a H. polymorpha hexokinase-negative mutant with a plasmid carrying the HPHXK1 gene restored the ability of the mutant to phosphorylate fructose and to repress the synthesis of alcohol oxidase and catalase by fructose. Therefore, hexokinase is specifically needed for the establishment of fructose repression in H. polymorpha.

Hansenula polymorpha is an exception among methylotrophic yeasts because it can grow on the disaccharides maltose and sucrose. We disrupted the maltase gene (HPMAL1) in H. polymorpha 201 using homologous recombination. Resulting disruptants HP201HPMAL1Delta failed to grow on maltose and sucrose, showing that maltase is essential for the growth of H. polymorpha on both disaccharides. Expression of HPMAL1 in HP201HPMAL1Delta from the truncated variants of the promoter enabled us to define the 5'-upstream region as sufficient for the induction of maltase by disaccharides and its repression by glucose. Expression of the Saccharomyces cerevisiae maltase gene MAL62 was induced by maltose and sucrose, and repressed by glucose if expressed in HP201HPMAL1Delta from its own promoter. Similarly, the HPMAL1 promoter was recognized and correctly regulated by the carbon source in a S. cerevisiae maltase-negative mutant 100-1B. Therefore we suggest that the transcriptional regulators of S. cerevisiae MAL genes (MAL activator and Mig1 repressor) can affect the expression of the H. polymorpha maltase gene, and that homologues of these proteins may exist in H. polymorpha. Using the HPMAL1 gene as a reporter in a H. polymorpha maltase disruption mutant it was shown that the strength of the HPMAL1 promoter if induced by sucrose is quite comparable to the strength of the H. polymorpha alcohol oxidase promoter under conditions of methanol induction, revealing the biotechnological potential of the HPMAL1 promoter.