The methylotrophic yeast Hansenula polymorpha: a versatile cell factory

Dijk, Ralf van; Faber, Klaas Nico; Kiel, Jan A.K.W.; Veenhuis, Marten; Klei, Ida J. van der

doi:10.1016/s0141-0229(00)00173-3

Cited by 75 publications

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“…When cells are grown on glucose/ammonium sulphate, no formaldehyde dehydrogenase activity is measured, while a shift of cells to media containing methanol as carbon source or primary amines such as methylamine results in strong induction of the gene and high enzyme activities (Eggeling and Sahm, 1978;Zwart et al, 1980). So far, we have made use of the endogenous alcohol oxidase promoter for overexpression of endogenous or heterologous genes in H. polymorpha (van Dijk et al, 2000). In order to obtain a stable and high expression strain the constructed overexpression cassette is usually integrated into the genomic P AOX locus at a high copy number (Baerends et al, 1997;Faber et al, 1995).…”

Section: Resultsmentioning

confidence: 99%

“…Currently, the H. polymorpha system provides a remarkably versatile technology platform for heterologous gene expression (Gellisen and Veenhuis, 2001). The Hansenula toolbox includes a variety of host strains, various sites available for integration and several strong promoters (reviewed by van Dijk et al, 2000). These promoters originate from inducible genes encoding enzymes/proteins involved in: (a) methanol metabolism, e.g.…”

Section: Introductionmentioning

confidence: 99%

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Molecular characterization of the Hansenula polymorphaFLD1 gene encoding formaldehyde dehydrogenase

Baerends

Sulter

Jeffries

et al. 2001

Yeast

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Glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required for the catabolism of methanol as a carbon source and certain primary amines, such as methylamine as nitrogen sources in methylotrophic yeasts. Here we describe the molecular characterization of the FLD1 gene from the yeast Hansenula polymorpha. Unlike the recently described Pichia pastoris homologue, the H. polymorpha gene does not contain an intron. The predicted FLD1 product (Fld1p) is a protein of 380 amino acids (ca. 41 kDa) with 82% identity to P. pastoris Fld1p, 76% identity to the FLD protein sequence from n-alkane-assimilating yeast Candida maltosa and 63-64% identity to dehydrogenase class III enzymes from humans and other higher eukaryotes. The expression of FLD1 is strictly regulated and can be controlled at two expression levels by manipulation of the growth conditions. The gene is strongly induced under methylotrophic growth conditions; moderate expression is obtained under conditions in which a primary amine, e.g. methylamine, is used as nitrogen source. These properties render the FLD1 promoter of high interest for heterologous gene expression. The availability of the H. polymorpha FLD1 promoter provides an attractive alternative for expression of foreign genes besides the commonly used alcohol oxidase promoter. The sequence has been deposited in the GenBank/NCBI data library under Accession No. AF364077.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Molecular characterization of the Hansenula polymorphaFLD1 gene encoding formaldehyde dehydrogenase

Baerends

Sulter

Jeffries

et al. 2001

Yeast

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“…Pichia angusta) is an important organism for biotechnological use, e.g. heterologous gene expression, and for basic research on peroxisome biogenesis and degradation (7,10). There are several reports describing H. polymorpha mutants defective in glucose repression (11)(12)(13).…”

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confidence: 99%

A Hexose Transporter Homologue Controls Glucose Repression in the Methylotrophic Yeast Hansenula polymorpha

Stasyk

Komduur

et al. 2004

Journal of Biological Chemistry

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Peroxisome biogenesis and synthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha are under the strict control of glucose repression. We identified an H. polymorpha glucose catabolite repression gene (HpGCR1) that encodes a hexose transporter homologue. Deficiency in GCR1 leads to a pleiotropic phenotype that includes the constitutive presence of peroxisomes and peroxisomal enzymes in glucose-grown cells. Glucose transport and repression defects in a UV-induced gcr1-2 mutant were found to result from a missense point mutation that substitutes a serine residue (Ser 85 ) with a phenylalanine in the second predicted transmembrane segment of the Gcr1 protein. In addition to glucose, mannose and trehalose fail to repress the peroxisomal enzyme, alcohol oxidase in gcr1-2 cells. A mutant deleted for the GCR1 gene was additionally deficient in fructose repression. Ethanol, sucrose, and maltose continue to repress peroxisomes and peroxisomal enzymes normally and therefore, appear to have GCR1-independent repression mechanisms in H. polymorpha. Among proteins of the hexose transporter family of baker's yeast, Saccharomyces cerevisiae, the amino acid sequence of the H. polymorpha Gcr1 protein shares the highest similarity with a core region of Snf3p, a putative high affinity glucose sensor. Certain features of the phenotype exhibited by gcr1 mutants suggest a regulatory role for Gcr1p in a repression pathway, along with involvement in hexose transport.If provided with a mixture of carbon substrates, yeast preferentially utilizes the one that supports the fastest growth rate. This is achieved by several coordinated regulatory mechanisms of metabolic adaptation. They include: (i) the induction of enzymes involved in the metabolism of a preferred substrate and (ii) repression and/or inactivation of enzymes involved in the metabolism of less preferred carbon sources. Carbon sourcetriggered repression (or catabolite repression) generally affects expression of the corresponding target genes at the transcriptional level. Among co-repressor substrates in Saccharomyces cerevisiae, glucose is best known (for review see Refs. 1 and 2). The main targets of glucose repression in S. cerevisiae are enzymes of gluconeogenesis and the glyoxylate cycle, mitochondrial enzymes involved in oxidative phosphorylation, and enzymes involved in transport and metabolism of alternative carbon substrates, such as galactose, sucrose, and maltose. Despite extensive studies and a growing number of genes known to be involved in glucose repression in this and other species, its actual mechanism, especially in the early stages of sensing and signal transduction, is not fully understood.In methylotrophic yeasts, unique peroxisomal and cytosolic enzymes of methanol metabolism are under strict control of repression by various carbon substrates, e.g. glucose and ethanol (3, 4). Glucose and ethanol also trigger inactivation of peroxisomal enzymes by a process that involves the autophagic degradation of whole peroxisomes by vacuolar prot...

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“…In the presence of glucose, however, no methanol oxidase activity can be detected in crude extracts (Veenhuis et al, 1989). In view of this, the MOX promoter has been widely employed for heterologous protein expression, and H. polymorpha has been considered a "versatile protein factory" (Gellissen, 2000;van Dijk et al, 2000). Nevertheless, although it is a very suitable source of carbon for general industrial fermentation, glucose cannot be regularly employed for protein expression in H. polymorpha.…”

Section: Introductionmentioning

confidence: 99%

The transcription factor Snf1p is involved in a Tup1p-independent manner in the glucose regulation of the major methanol metabolism genes of Hansenula polymorpha

et al. 2003

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Hansenula polymorpha is a methylotrophic yeast widely employed in biotechnology as a "protein factory". Most promoters used for heterologous protein expression, like MOX (methanol oxidase) and DAS (di-hydroxy acetone synthase), are involved in the peroxisomal methanol metabolism (C 1 metabolism) and are under strong glucose repression. Interestingly, the MOX promoter is subjected to glucose regulation also in Saccharomyces cerevisiae, a non-methylotrophic yeast in which this phenomenon is well studied. In this species, the transcription factor Tup1p plays an essential role in glucose repression of several genes. This effect is counteracted by the activator Snf1p when glucose is exhausted from medium. Therefore, to test whether this regulatory circuit has been conserved in H. polymorpha, HpTUP1 and HpSNF1 were partially cloned and disrupted. Deletion of HpTUP1 did not affect glucose repression of the major C 1 metabolism genes (MOX, DAS). Thus, though conserved, HpTUP1 does not seem to take part in a general glucose repression in H. polymorpha. In contrast, the deletion of HpSNF1 led to significant decreases in the activation of these genes in the absence of glucose. Therefore, the effect of HpSNF1 in transcriptional activation may be through an HpTUP1-independent circuit.

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The methylotrophic yeast Hansenula polymorpha: a versatile cell factory

Cited by 75 publications

References 57 publications

Molecular characterization of the Hansenula polymorphaFLD1 gene encoding formaldehyde dehydrogenase

Molecular characterization of the Hansenula polymorphaFLD1 gene encoding formaldehyde dehydrogenase

A Hexose Transporter Homologue Controls Glucose Repression in the Methylotrophic Yeast Hansenula polymorpha

The transcription factor Snf1p is involved in a Tup1p-independent manner in the glucose regulation of the major methanol metabolism genes of Hansenula polymorpha

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