Rainer Roggenkamp

Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors

SummaryA high frequency transformation system for the methylotrophic yeast Hansenula polymorpha has been developed. This system depends on complementation of isolated uracil auxotrophs by the URA3 gene of Saccharomyces cerevisiae. Maintenance of the uracil prototrophy is based on integration of plasmid YIp5 at random sites within the H. polymorpha genome and on autonomously replicating plasmids containing ARS1 of S. cerevisiae or related sequences cloned from the host DNA. The sequence of one autonomously replicating sequence (HARS1) from H. polymorpha has been determined showing an AT-rich region of 9 bp with some similarity to the consensus sequence of known eukaryotic replication origins. Mitotic loss of autonomously replicating sequences is high; selection for stable uracil prototrophs yields multiple tandem arrangement of the transformed DNA with no detectable loss of the phenotype on non-selective medium. These features offer the possibility for extensive gene expression in H. polymorpha.

Biosynthesis and regulation of the peroxisomal methanol oxidase from the methylotrophic yeast Hansenula polymorpha

SummaryThe biosynthesis of methanol oxidase, a peroxisomal enzyme in the methanol-utilizing yeast Hansenula polymorpha, was studied in vitro. Translation of Hansenula mRNA in a rabbit reticulocyte lysate yields methanol oxidase protein in high amounts. The apparent molecular mass of the protein was found to be identical to the subunit of the functional multimeric enzyme, which indicates the absence of an N-terminal extension typical of most transported proteins. The regulation of methanol oxidase by glucose repression and derepression as well as by induction of methanol was shown to be controlled at the level of transcription. Two mutants of Hansenula polymorpha unable to grow on methanol as a carbon and energy source were shown to be affected in methanol oxidase synthesis.

Targeting signal of the peroxisomal catalase in the methylotrophic yeast Hansenula polymorpha

The methylotrophic yeast,Hansenula polymorpha, harbours a unique catalase (EC 1,11,1,6), which is essential for growth on methanol as a carbon source and is located in peroxisomes. Its corresponding gene has been cloned and the nucleotide sequence determined. The deduced amino acid sequence displayed the tripeptide serine‐lysine‐isoleucine at the extreme C‐terminus, which is similar to sequences of other peroxisomal targeting signals. Exchange of the ultimate amino acid, isoleucine, of catalase for serine revealed a cytosolic enzyme activity and a concomitant loss of peroxisome function. We concluded that the tripeptide is essential for targeting of catalase inH. polymorpha.

Targeting sequences of the two major peroxisomal proteins in the methylotrophic yeast Hansenula polymorpha

SummaryDihydroxyacetone synthase (DAS) and methanol oxidase (MOX) are the major enzyme constituents of the peroxisomal matrix in the methylotrophic yeast Hansenula polymorpha when grown on methanol as a sole carbon source. In order to characterize their topogenic signals the localization of truncated polypeptides and hybrid proteins was analysed in transformed yeast cells by subcellular fractionation and electron microscopy. The C-terminal part of DAS, when fused to the bacterial β-lactamase or mouse dihydrofolate reductase, directed these hybrid polypeptides to the peroxisome compartment. The targeting signal was further delimited to the extreme C-terminus, comprising the sequence N-K-L-COOH, similar to the recently identified and widely distributed peroxisomal targeting signal (PTS) S-K-L-COOH in firefly luciferase. By an identical approach, the extreme C-terminus of MOX, comprising the tripeptide A-R-F-COOH, was shown to be the PTS of this protein. Furthermore, on fusion of a C-terminal sequence from firefly luciferase including the PTS, β-lactamase was also imported into the peroxisomes of H. polymorpha. We conclude that, besides the conserved PTS (or described variants), other amino acid sequences with this function have evolved in nature.

Biosynthesis of the peroxisomal dihydroxyacetone synthase from Hansenula polymorpha in Saccharomyces cerevisiae induces growth but not proliferation of peroxisomes

SummaryThe DAS gene of Hansenula polymorpha was expressed in Saccharomyces cerevisiae under the control of different promoters. The heterologously synthesized dihydroxyacetone synthase (DHAS), a peroxisomal enzyme in H. polymorpha, shows enzymatic activity in bakers yeast. The enzyme was imported into the peroxisomes of S. cerevisiae not only under the appropriate physiological conditions for peroxisome proliferation (oleic acid media), but also in glucose-grown cells where it induced the enlargement of the few peroxisomes present. This growth process was not accompanied by an increase in the number of microbodies, which suggests a separate control mechanism for peroxisomal proliferation.

Constitutive appearance of peroxisomes in a regulatory mutant of the methylotrophic yeast Hansenula polymorpha

SummaryA selection by glucosamine for mutants of Hansenula polymorpha insensitive to glucose repression of methanol assimilation is described. Constitutive synthesis of enzymes is established in standard batch cultures of glucosegrown cells. Upon prolonged glucose metabolism the phenotype is masked by catabolite inactivation and degradation of enzymes. Addition of the substrate methanol remarkably improves constitutive synthesis by preventing catabolite inactivation and delaying degradation. Regular peroxisomes of reduced number are formed in mutant cells under repressed conditions. No constitutive synthesis is detectable using ethanol as a carbon source. In addition, this alcohol is detrimental to growth of the mutants, indicating that H. polymorpha is constrained to repress synthesis of enzymes involved in the C1-metabolism when ethanol is present as a substrate.

Deficiency of peroxisome assembly in a mutant of the methylotrophic yeast Hansenula polymorpha

SummaryWe have isolated a mutant of the metholotrophic yeast Hansenula polymorpha defective in peroxisomal biosynthesis. The mutant strain has been derived by a selection procedure from cells of a high-copy number transformant that overproduces the major peroxisomal enzyme methanol oxidase (MOX) and forms enlarged peroxisomes. In contrast to the parental strain the mutant lacks intact peroxisomes in thin sections, but exhibits electron-dense particles that are devoid of intact membranes and crystalloid cores. Consequently, peroxisomal enzymes show severe proteolytic degradation in crude cell lysates. Complementation of this, and analogous mutations, will offer the possibility to identify genes that are required for peroxisome assembly.

Identification and characterization of cytosolic Hansenula polymorpha proteins belonging to the Hsp70 protein family.

We have isolated two members of the Hsp70 protein family from the yeast Hansenula polymorpha using affinity chromatography. Both proteins were located in the cytoplasm. One of these, designated Hsp72, was inducible in nature (e.g. by heat shock). The second protein (designated Hsc74) was constitutively present. Peptides derived from both Hsp72 and Hsc74 showed sequence homology to the cytosolic Saccharomyces cerevisiae Hsp70s, Ssa1p and Ssa2p. The gene encoding Hsp72 (designated HSA1) was cloned, sequenced and used to construct HSA1 disruption and HSA1 overexpression strains. Comparison of the stress tolerances of these strains with those of wild‐type H. polymorpha revealed that HSA1 overexpression negatively affected the tolerance of the cells to killing effects of temperature or ethanol, but enhanced the tolerance to copper and cadmium. The tolerance for other chemicals (arsenite, arsenate, H2O2) or to high osmolarity was unaffected by either deletion or overexpression of HSA1. The nucleotide sequence of HSA1 was submitted to the EMBL data library and given the Accession Number Z29379.

Recombinational properties of the Saccharomyces cerevisiae FLP gene expressed in Escherichia coli

SummaryThe FLP gene from the 2-μm DNA of Saccharomyces cerevisiae is shown to be functionally expressed in Escherichia coli leading to site-specific intramolecular as well as intermolecular recombination between IR sequences. The expression was achieved under control of a low expression as well as a high expression E. coli promoter. The FLP gene was found to complement in trans a Flp− plasmid and promote its interconversion.By the use of a low Flp expression plasmid, it could be shown that the rate of interconversion of a Flp− plasmid by complementation in trans, was lower than that of a Flp+ plasmid, suggesting that in addition to the IR sequences another cis-acting function exists.Expression of the FLP gene fused to the lac promoter in an in vitro system yielded two polypeptides with apparent molecular weights of 44,000 and 37,000. The 37,000 dalton polypeptide can also be produced from Flp− plasmids and is generated from a translation start within the FLP gene. The 44,000 dalton polypeptide is considered to represent the FLP gene product.