From Genome to Life:

STASYK Oleh

Institute of Cell Biology Nat. Acad. Sci. of Ukraine, Molecular Genetics And Biotechnology, Drahomanov Str., 14/16, Lviv 79005, Ukraine

title: Glucose repression in methylotrophic yeast Hansenula polymorpha is controlled by hexose transporter homologue

Synthesis of peroxisomal alcohol oxidase (AOX) and peroxisome biogenesis in the methylotrophic yeast Hansenula polymorpha are strictly regulated by carbon sources. At optimal conditions in continuous culture, induced by methanol AOX can constitute 30% of soluble cellular protein, while in the glucose-grown cells AOX promoter is totally repressed. We have isolated glucose repression deficient mutants and cloned the H. polymorpha glucose catabolite repression gene (HpGCR1) by functional complementation. The HpGCR1 is first identified gene involved in repression in methylotrophic yeast and encodes for a homologue of hexose transporters. The predicted sequence of Gcr1 protein shares 44% identity and 62% similarity with a core region of Saccharomyces cerevisiae Snf3p, a putative high affinity glucose sensor, but lacks an elongated carboxyterminal extension. Deficiency in the GCR1 gene leads to multiple alterations in metabolism and pleiotropic phenotype that includes the constitutive synthesis of alcohol oxidase and presence of peroxisomes in glucose-grown cells. Moreover, gcr1 mutants appeared to exhibit even higher AOX activity in glucose medium relative to the methanol-grown cells. Glucose-transport and repression defects in UV-induced gcr1-2 mutant were found to result from a missense point mutation that substituted a serine residue (S85) with phenylalanine in the second predicted transmembrane segment of the deduced Gcr1 protein. In addition to glucose, mannose, trehalose and xylose fail to repress alcohol oxidase in gcr1-2. A mutant deleted for the GCR1 gene was additionally deficient in fructose repression. However, AOX is strongly repressed in the gcr1 mutants by sucrose or ethanol, thus remains selectively regulated. We suggest a regulatory role for Gcr1p in a repression pathway, either via glucose transport or glucose signalling. Sugar-specific phenotype of the gcr1 mutants provides an opportunity for their exploitation as the hosts for AOX production and AOX promoter-directed expression of heterologous proteins in glucose medium. Such expression system would combine advantages of the strong regulatable AOX promoter with utilization of the sugar carbon substrates for growth and induction of strains-producers, while excluding toxic and inflammable methanol from the production process. We demonstrated glucose-induced, and sucrose-repressed synthesis of several differently localized heterologous proteins expressed under AOX promoter in gcr1 mutants, namely cytosolic b-galactosidase, secreted glucose oxidase and peroxisome-targeted green fluorescent protein.