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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 4 (1988), S. 293-303 
    ISSN: 0749-503X
    Keywords: Hansenula polymorpha ; methylotrophic yeast ; genetic analysis ; methanol mutant ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Techniques are described for the induction, isolation, and characterization of mutants of Hansenula polymorpha. In addition, techniques for controlled passage through the life cycle and genetic analyses, including complementation, tetrad and random spore analysis, have been developed and used to assign mutants to 62 complementation groups. We report that organism conforms to the expected genetics of a homothallic yeast and displays a Mendelian segregation of genes through meiosis. Preliminary mapping data are presented indicating linkage of three genes on a single linkage fragment. Enymatic analysis of methanol-non-utilizing mutants identified one class which is totally deficient in the key assimilatroy enzyme, dihydroxyacetone synthase.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 6 (1990), S. 245-254 
    ISSN: 0749-503X
    Keywords: malate synthase gene ; nucleotide sequence ; topogenic signal ; yeast microbody ; Hansenula polymorpha ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have cloned the MAS gene, encoding the microbody matrix enzyme malate synthase (EC 4.1.3.2.) from the methylotrophic yeast Hensenula polymorphia. The gene was isolated by screening of a genomic library with a mixed-sequence probe, based on the partial amino acid sequence of the purified enzyme. The nucleotide sequence of a 2·4-kilobase stretch of DNA covering the MAS gene was determined. The gene contains an open reading frame 555 amino acids, amounting to a calculated molecular mass of 63 254 for the encoded protein. Comparison of the amino acid sequence with the malate synthase sequences of Escherichia coli, Brassica napus L. and Cucumis sativus L. Clearly establishes the homology of all four proteins. Compared to the soluble enzyme from E. coli, the malate synthases from H. polymorpha and both plant species, which are located in the microbodies, have a short carboxy-terminal extension. In the plant malate synthases, the extension is probably involved in routing to the microbodies, since it contains the potential peroxisomal targeting signal, Ser-Arg/Lys-Leu, at the carboxy terminus. The H. polymorpha enzyme terminates with similar amino acids, but their sequence, Ser-Leu-Lys, does not conform to any of the known peroxisomal targeting signals.
    Additional Material: 5 Ill.
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  • 3
    ISSN: 0749-503X
    Keywords: Peroxisomes ; oleic acid ; β-oxidation ; membrane proliferation ; Hansenula polymorpha ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We studied the physiological responses of Hansenula polymorpha during adaptation of cells to oleic acid-containing media. Growth experiments indicated that the organism was unable to use oleic acid as the sole source of carbon and energy. However, upon incubation of glucose-grown cells in mineral media containing oleic acid, activities of various enzymes of the β-oxidation pathway were induced. These enzymes were localized in microbodies together with alcohol oxidase. Furthermore, a drastic increase in phospholipid content of the cells was observed; this was due to a rapid proliferation of membranes. These consisted of a variable number of membranous layers which were continuous with the peroxisomal membrane. Upon continued incubation, the membrane proliferations extended and large compartments were formed. This process was dependent on the presence of peroxisomes in the cells since it was not observed in peroxisome-deficient mutant strains of H. polymorpha. The newly formed membranous compartments differed from peroxisomes since they did not contain peroxisomal matrix proteins; these were confined to the single enlarged organelle which was incorporated in the membranous structure and characterized by a large alcohol oxidase crystalloid. The membranous compartments are considered to be whole entities since they could not be separated from the peroxisomes by common cell fraction methods; also they were degraded entirely after a shift of cells to glucose-excess condition.Freeze fracturing reveled that the substructure of the membranes greatly resembled that of normal peroxisomal membranes. Since a distinct enhancement of different peroxisomal membrane proteins was observed during the initial hours after the shift, we assume that exposure of H. polymorpha to acid lead to a drastic overproduction of peroxisomal membranes.
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  • 4
    ISSN: 0749-503X
    Keywords: Hansenula polymorpha ; expression system ; S and L surface antigens ; hepatitis B vaccine ; multimeric ; non-homologous integration ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An expression system has been developed for the methylotrophic yeast Hansenula polymorpha and used to co-express both the L (preS1-S2-S) and S hepatitis B surface antigens (HBsAg) under the control of strong methanol-inducible promoters derived from the methanol oxidase and from the formate dehydrogenase genes. A unique feature of this H. polymorpha expression system is the possibility of integrating up to 100 copies of an expression cassette via a multimeric integration mechanism. Several multimeric integrants containing various numbers of L and S expression cassettes were constructed to give a spectrum of strains characterized by different L to S ratios. The expression level of S antigen was 5-8% of the total soluble cell protein. Analysis by sucrose and CsCl density gradient centrifugation and by particle-specific immunoassays demonstrated that the synthesized HBsAg spontaneously assembled into composite subviral particles containing both S and L proteins. Only a minor portion of the L protein was found to be glycosylated. These H. polymorpha-derived composite particles can be used for the production of a hepatitis B virus vaccine with the potential for improved immunogenicity due to the presence of a wider spectrum of epitopes and negligible glycosylation.
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  • 5
    ISSN: 0749-503X
    Keywords: Yeast ; Hansenula polymorpha ; microbodies ; biogenesis ; PER genes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In the course of our studies on the molecular mechanisms involved in peroxisome biogenesis, we have isolated several mutants of the methylotrophic yeast Hansenula polymorpha impaired in the import of peroximal matrix proteins. These mutants are characterized by the presence of small intact peroxisomes, while the bulk of the peroxisomal matrix protein is not imported and resides in the cytosol (Pim- phenotype). Genetic analysis of back-crossed mutants revealed five different complementation groups, which were designated PERI-PER5. Mapping studies to determine the linkage relationships indicated that the observed Pim- phenotypes were determined by single recessive nuclear mutations.The different mutants had comparable phenotypes: (i) they were impaired to utilize methanol as the sole source of carbon and energy but grew well on various other compounds, including nitrogen sources, the metabolism of which is known to be mediated by peroxisome-borne enzymes in wild-type cells; (ii) all peroxisomal enzymes tested were induced, assembled and activated as in wild-type cells although their activities varied between the different representative mutants; (iii) all peroxisomal proteins, whether constitutive or inducible, were found both in the cytosol and in the small peroxisomes. These results suggest that a general, major import mechanism is affected in all mutants.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 11 (1995), S. 1331-1344 
    ISSN: 0749-503X
    Keywords: Hansenula polymorpha ; Pichia pastoris ; heterologous gene expression ; peroxisomes ; peroxisome biogenesis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In this contribution we discuss the potential of methylotrophic yeasts as hosts for the high level production of valuable foreign proteins. Recent relevant achievements on the intracellular production or secretion of proteins are summarized. Special attention is paid to a specific advantage of the use of methylotrophic yeasts, namely the possibility of accumulating the foreign gene products inside peroxisomes. This approach may be of major advantage when the protein product is toxic for the host cell and, also, to protect these proteins these proteins from undesired side-effects such as proteolysis or aggregation.
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  • 7
    ISSN: 0749-503X
    Keywords: yeast ; PMR1 ; Hansenula polymorpha ; Ca2+-ATPase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha. The partial DNA fragment of the H. polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids. The putative gene product contains all ten of the conserved regions observed in P-type ATPases. The cloned gene product exhibits 60·3% amino acid identity to the S. cerevisiae PMR1 gene product and complemented the growth defect of a S. cerevisiae pmr1 null mutant in the EGTA-containing medium. The results demonstrate that the H. polymorpha gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca2+-ATPase. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession Number U92083. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 5 Ill.
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  • 8
    ISSN: 0749-503X
    Keywords: Hansenula polymorpha ; methanol ; dihydroxyacetone ; xylose ; mutants ; transketolase ; formaldehyde ; continuous culture ; peroxisome ; regulation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Contrary to expectation, a mutant of Hansenula polymorpha blocked in dihydroxyacetone (DHA) synthase was able to assimilate methanol-carbon when grown in chemostat culture on mixtures of xylose and methanol. Incubation of a DHA synthase- and DHA kinase-negative double mutant resulted in DHA accumulation, indicating that a DHA synthase-type of reaction was involved. Low residual DHA synthase activity subsequently was shown to be present when using an assay with improved sensitivity. This activity was not associated with the (mutated) DHA synthase protein, which was still present in the peroxisomes, but with the enzyme transketolase. Transketolase from methanol grown cells was purified (525-fold) to homogeneity in 9% yield. The native enzyme was dimeric, as has been reported fro other transketolases, with a subunit molecular weight of 74000. The affinity of the purified enzyme for formaldehyde was low (Km = 5 mM), but high for xylulose-5-phosphate (ca. 10 μM). The in vivo functioning of transketolase in formaldehyde assimilation, and the influence of the hydration state of formaldehyde is discussed.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 6 (1990), S. 87-97 
    ISSN: 0749-503X
    Keywords: Hansenula polymorpha ; methylotrophic yeast ; microbodies ; peroxisome-deficient mutants ; alcohol oxidase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: As a first step in a genetic approach towards understanding peroxisome biogenesis and function, we have sought to isolate mutants of the methylotrophic yeast Hansenula polymorpha which are deficient in peroxisomes. A collection of 260 methanol-utilization-defective strains was isolated and screened for the ability to utilize a second compound, ethanol, the metabolism of which involves peroxisomes. Electron microscopical investigations of ultrathin sections of selected pleiotropic mutants revealed two strains which were completely devoid of peroxisomes. In both, different peroxisomal matrix enzymes were active but located in the cytosol; these included catalase, alcohol oxidase, malate synthase and isocitrate lyase.Subsequent backcrossing experiments revealed that for all crosses involving both strains, the methanol- and ethanol utilizing-deficient phenotypes segregated independently of each other, indicating that different gene mutations were responsible for these phenotypes. The phenotype of the backcrossed peroxisome-deficient derivates was identical: defective in the ability to utilize methanol but capable of growth on other carbon sources, including ethanol.The mutations complemented and therefore were recessive mutations in different genes.
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  • 10
    ISSN: 0749-503X
    Keywords: Hansenula polymorpha ; methanol ; glycerol ; dihydroxyacetone ; xylose ; alcohol oxidase ; continuous culture ; regulation ; mutants ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The physiological responses of Hansenula polymorpha wild-type and mutant strains 17B (dihydroxyacetone kinase-negative) and 17BG51 (dihydroxyacetone kinase- and glycerol kinase-negative) to growth on mixtures of xylose and methanol in chemostats were investigated. Increasing methanol concentrations (0-110 mM) in the feed of the wild-type culture resulted in increasing cell densities and a gradual switch towards methanol metabolism. At the lower methanol feed concentrations the mutant cultures used methanol and xylose to completion and changes in enzyme patterns comparable to the wild type were observed. This was not reflected in significant changes in cell densities. Instead, formaldehyde assimilation resulted in dihydroxyacetone (DHA) production, which was proportional to the amount of methanol added. At intermediate methanol concentration the cultures showed a strong variation in DHA levels and cell densities. Further increased in the methanol feed concentrations resulted in a drop in DHA accumulation rates, repression of alcohol oxidase synthesis and accumulation of residual methanol. The phenomena were studied in more detail in transition experiments and with gradients of methanol. The results indicate that xylulose-5-phosphate (Xu5P) generated in xylose metabolism served as acceptor molecule for formaldehyde assimilation by the peroxisomal enzyme DHA synthase. Accumulation of DHA in the mutant cultures, however, further diminished the availability of carbon for growth. The data suggest that with increasing methanol concentrations Xu5P eventually became growth rate limiting. This resulted in an unstable situation but wash-out of the culture did not occur to a significant extent. Instead, DHA accumulation ceased and cell densities, and enzymes specifically involved in xylose metabolism increase, indicating that the organism resumed its xylose metabolism. The molecular mechanisms controlling the partitioning of Xu5P over xylose (pentose phosphate pathway) and methanol (peroxisome) metabolism under these conditions remain to be elucidated.
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