Yeast strains with stable integration of heterologous genes
AssigneeCentre National De La Recherche Scientifique
InventorsDenis Pompon, Christophe Cullin, Gilles Truan, Philippe Urban, Piotr Slonimski
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Abstract
Yeast strains and a method of expression in yeast of the monooxygenase activity of heterologous cytochromes P450 are disclosed. Within the genome of the yeast strain, genes for NADPH-cytochrome P450 reductase and cytochrome b5 are stably integrated and coexpressed. The genome is transformed with a plasmid carrying a cassette for the expression of a heterologous P450 gene.
Claims16
A diploid yeast strain having a chromosomal genome comprising
- at least one heterologous gene stably integrated at a heterozygous locus,
- an inducible and regulable promoter controlling the heterologous gene such that said heterologous gene is expressed when said promoter is active,
- a mating-type locus, and
- isogenic alleles at the remainder of loci of the chromosomal genome,
- wherein the heterozygous locus lacks a wild-type allele.
A method of expressing monooxygenase activity of a heterologous cytochrome P450 gene comprising:
- integrating an NADPH-cytochrome P450 reductase gene and a cytochrome b5 gene into a locus of the chromosomal genome of a diploid yeast strain thereby forming a heterozygous diploid locus, said chromosomal genome including:
- a mating type locus;
- for each said gene, an inducible and regulable promoter controlling said gene;
- and isogenic alleles at the remainder of loci of the chromosomal genome, wherein the heterozygous diploid locus lacks a wild-type allele,
- transforming the diploid yeast strain with a plasmid carrying a cassette for expression of a heterologous cytochrome P450 gene, and
- expressing the monooxygenase activity of said heterologous cytochrome P450 gene.
A set of yeast strains, each yeast strain having:
- a chromosomal genome,
- an NADPH-cytochrome P450 reductase gene and a heterologous cytochrome b5 gene integrated into said chromosomal genome, the levels of expression of the NADPH-cytochrome P450 reductase and the cytochrome b5 genes being variable amongst the strains, and
- a plasmid carrying a cassette for the expression of a heterologous cytochrome P450 gene, said plasmid inserted into said chromosomal genome,
- wherein the strains of the set provide variable expression of the monooxygenase activity of the cytochrome P450 gene due to the variable levels of expression of the NADPH-cytochrome P450 reductase and the cytochrome b5 genes.
Dependencies are inferred from the claim text.