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Contenuto archiviato il 2022-12-23

Genetic dissection of different pathways of H. polymorpha and functional screening of genes involved in methanol metabolism, peroxisome homeostasis, protein glycosylation, cell wall integrity and secretion

Obiettivo

Opportunities provided by methylotrophic yeasts for studies of different pathways in eukaryotic cell attract the attention of many scientists. Several laboratories in Europe, Ukraine, Russia and Korea are involved in studies of one of the methylotrophic yeasts, Hansenula polymorpha. However genetic studies of this organism are hampered by absence of sets of mutations, genetically dissecting particular pathways, absence of a complete genome sequence and low frequency of homologous integration of transforming DNA in this organism. Approaches for homologous integration and the use of high frequency of non-homologous integration for generation of mutation and rapid identification of corresponding genes have been developed previously by participants of the consortium. Rhein Biotech GmbH will complete the genome sequence in the near future. Experience and skills of different laboratories working with Hansenula polymorpha are combined in this project to generate mutations and identify corresponding genes of this yeast for facilitating studies of eukaryotic secretion, regulation of methanol metabolism, cell wall biogenesis and protein glycosylation.

During the work on this project, sets of mutations genetically dissecting H. polymorpha pathways involved in control of cell wall integrity, protein secretion and glycosylation will be obtained by random integration of linear DNA fragments. Mutations altering cell wall integrity and protein glycosylation will be identified by inability of integrants to grow in the presence of a detergent or aminoglycoside antibiotics.

The glycosylation pattern of chitinase and invertase will be analysed to determine whether N- or O- glycosylation is altered. Mutants hypersensitive to detergents or aminoglycoside antibiotics and showing no alterations of protein glycosylation will be analysed for cell wall content. Mutations perturbing the secretion pathways will be identified by alteration of the secretion rate of a reporter protein. The use of different reporter genes at the same time will allow rapid distinction between mutations affecting secretion or expression of reporter protein and will get rid of mutations destroying expression cassettes of the reporter protein. Genes defined by these mutations will be identified. The same approach will be used for identification of genes, mutations of which increase secretion efficiency of heterologous proteins. Obtaining such mutations is important for engineering H. polymorpha strains efficiently producing a wide range of recombinant proteins. Approaches utilizing novel reporter proteins, carboxymethyl cellulase and fungal b-galactosidase will be designed to identify genes responsible for regulation of expression of the MOX gene and genes regulated by methanol. Also genes involved in glutathione biosynthesis and catabolism will be cloned, sequenced and disrupted. Their role in maintaining glutathione intracellular pool and in providing resistance to methanol, formaldehyde and cadmium ions will be elucidated.

Invito a presentare proposte

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Meccanismo di finanziamento

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Coordinatore

Rhein Biotech GmbH
Contributo UE
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Indirizzo
Eichsfelder Strasse 11
40595 Düsseldorf
Germania

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Costo totale
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Partecipanti (5)