Objective
General objectives are:
to bring non-conventional yeast species to be genetically manipulatable organisms;
to develop expertise for the control of their physiology;
to create new combinations of genetic elements suitable for the production of medical and agricultural interest.
From a newly discovered plasmid pKD1, stable, high copy number vectors have been constructed. The system allows cloning and expression of genes in Kluyveromyces lactis and related species. In a model system, secreted production of a human protein has been made. This is the first stable vector system available for the biotechnological manipulation of the lactic yeast Kluyveromyces.
Research is progressing on the line defined objectives. Mapping of genes K lactis has progressed by construction and use of multiply labelled strains and centromere linked markers. Data on physical mapping on chromosomes of cloned genes are accumulating in parallel, confirming genetic data. To support mapping work, many new mutations were isolated and several genes were cloned;
4 genes encoding alcohol dehydrogenase isozymes have been identified by hybridiztion to ADH2 probes of S cerevisiae. The genes have been isolated and sequenced;
Chromosomal deoxyribonucleic acid (DNA) fragments from K lactis have been tested for the ability of efficiently sustaining autonomous replication of plasmids in K lactis or in S cerevisiae. Some of these fragments have ARS function only in K lactis while others support autonomous replication in both yeasts;
Studies on the strong promoter of the methanol oxidase gene in Hansenula polymorpha have revealed a complex mode of regulation;
In Kluyveromyces lactis, the detailed knowledge on the structure and mode of regulation of the beta-galactosidase (LAC4) promoter is being exploited to establish expression systems for heterologous genes;
The resistance of different strains of Yarrowia lipolytica was analyzed and copper sensitive mutants have been obtained. Using an integrative bank of Yarrowia lipolytica it has been possible to complement one of the mutations;
4 clones of stress inducible genes isolated from Saccharomyces cerevisiae (HSP12, HSP26, ESP30 and ESP35) were used as hybridization probes for a Southern blot of analysis of K lactis genomic DNA. Clones hybridizing with these probes (except HSP26) could be isolated. It has not been necessary to construct complementary deoxyribonucleic acid (DNA) libraries for their isolation;
Many new mutants of Kluyveromyces lactis affected in carbohydrate metabolism were isolated. The genetic analysis demonstrated that the isolated mutants define several complementation groups;
Ka ryotyping of wild type Y lipolytica isolates revealed a pronounced diversity of patterns and individual gene probes showed that gross chromosomal rearrangements are associated with chromosomal polymorphism;
Work has started on the construction of recombinant Kluyveromyces lactis strains harboring expression cassettes either integrated at a defined chromosomal locus (RAG2) or contained on multi-copy vectors derived from the nature Kluyveromyces plasmid pKD1.
The fermentation characteristics of Kluyveromyces marxianus have been studied. The fermentation process is aiming at a very high cell density and high production/secretion of the homologous enzyme inulinase.
The proposal involves 7 academic institutions and 3 companies. The existing 400 species of yeast, represented by a large number of strains in the worldwide collection centres, are a great resource for scientific and industrial exploitation. Yeast can be used in two kinds of production:(i) primary metabolites and derivatives, (ii) homologous and heterologous proteins. In the first category, non-conventional species have already been used mostly on the basis of classical technology of physiological regulation. In the second category where the products are of high added value, recombinant DNA techniques are the essential tool. Therefore, recent attempts for the latter type of production have been made almost exclusively with Saccharomyces in which gene transfer systems are well established, while other species are only at the beginning of such molecular approaches. The experience with Saccharomyces and recent results with several non-conventional species have shown the limitations of the Saccharomyces system and the new possibilities offered by other species, in particular, Kluyveromyces, Hansenula/pichia and Yarrowia, for protein production. The major objectives of the proposal are:(i) to bring these species to be genetically manipulatable organisms, (ii) to develop expertise for the control of their physiology, and (iii) to create new combinations of genetic elements suitable for the production of proteins of medical and agricultural interest.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences genetics DNA
- natural sciences biological sciences genetics mutation
- natural sciences biological sciences biochemistry biomolecules carbohydrates
- natural sciences chemical sciences organic chemistry alcohols
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
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Coordinator
91405 Orsay
France
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