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DEVELOPMENT OF HOST VECTOR SYSTEMS IN DAIRY YEASTS

Objective

CONSTRUCTION OF EFFICIENT CLONING VECTORS ABLE TO TRANSFORM KLUYVEROMYCES AND OTHER YEASTS USED IN THE DAIRY INDUSTRY.
The genus Kluyveromyces is important in dairy industries and lately the pKD1 plasmid as well as the KARS based vectors have proved to be promising instruments in the physiological studies and genetic engineering of dairy yeasts.

Efficient expression vectors have been developed for the yeasts of industrial importance, in particular, the dairy yeasts.
Research has involved:
structural and functional characterization of the plasmid pKD1 and its derivative vectors;
analysis of the mechanism that gives autonomy and stability to the vectors derived from 2 and pKD1;
study of physiological regulation in Kluyveromyces lactis as a production organism.

The molecular structures of the plasmid pKD1 and of the autonomously replicating KARS sequence from K lactis chromosome were established. Plasmid and host functions important for the stable maintenance of the yeast plasmid were examined. Highly stable forms of pKD1 derived vectors were obtained. The host range of pKD1 was extended to several other species of Kluyveromyces. Shuttle deoxyribonucleic acid (DNA) banks were constructed for Kluyveromyces and Saccharomyces species. By combining the K lactis killer toxin gene sequence and pKD1 derived vectors, performing secretion vectors were obtained. Human interleukin-1 beta could be produced at high levels. To better control the gene expression of K lactis, several promoters were studied; in particular, new knowledge was obtained on the regulation of lactose metabolism and in the alcohol dehydrogenase system.
CONSTRUCTION OF NEW CLONING VECTORS FOR THE YEAST KLUYVEROMYCES.
STEPS :
1- DESCRIPTION AND SEQUENCING OF THREE NEW PLASMIDS RECENTLY DISCOVERED IN KLUYVEROMYCES SPECIES.
2- ISOLATION OF AUXOTROPHIC MUTATIONS IN KLUYVEROMYCES.
3- IDENTIFICATION OF REPLICATION ORIGINS ON THE NEW PLASMIDS.
4- IMPROVEMENT OF TRANSFORMATION EFFICIENCY USING CONSTRUCTIONS CONTAINING ORIGINS OF REPLICATIONS FROM THE NEW PLASMIDS AND THE S.CEREVISIAE MARKER URA3.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Institut Curie
Address
Université De Paris Xi (Université Paris-sud) 15 Rue Georges Clémenceau
91405 Orsay
France

Participants (2)

INSTITUT FÜR MIKROBIOLOGIE
Germany
Address

Düsseldorf
Università degli Studi di Roma
Italy
Address

Roma