Chromosomal mutagenesis in Pichia methanolica
AssigneeZymoGenetics, Inc.
InventorChristopher K. Raymond
Life of this patent
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Abstract
Methods for altering a selected chromosomal locus in P. methanolica cells and cells comprising such altered loci are disclosed. A linear DNA construct comprising (i) a segment comprising a portion of the target locus in which at least one nucleotide pair is altered and (ii) a selectable marker that complements adenine auxotrophy is introduced into cells auxotrophic for adenine. The cells are cultured under selective conditions, and cells in which the linear DNA construct has been chromosomally integrated by homologous recombination are identified. The cells are then cultured under conditions whereby cells auxotrophic for adenine can be identified, and a subset of such cells in which the altered locus has been chromosomally integrated are identified.
Claims11
A method for altering a chromosomal locus of Pichia methanolica cells, comprising:
- (a) selecting a target chromosomal locus of said cells;
- (b) providing a population of P. methanolica cells each comprising a chromosomal copy of said locus, wherein said cells are auxotrophic for adenine;
- (c) introducing into said provided cells a linear DNA construct comprising (i) a segment comprising a portion of said target chromosomal locus in which at least one nucleotide pair is altered, and (ii) a selectable marker that complements adenine auxotrophy;
- (d) culturing said cells from step (c) under conditions that are selective for the presence in said cells of said selectable marker;
- (e) identifying a subset of said cultured cells in which said segment of said DNA construct and said selectable marker have been chromosomally integrated by homologous recombination, said recombination resulting in tandem duplication of said target chromosomal locus;
- (f) culturing said identified subset of cells under conditions wherein cells prototrophic for adenine grow and exhibit a first phenotype, and cells auxotrophic for adenine grow and exhibit a second phenotype;
- (g) recovering cells that are auxotrophic for adenine;
- and
- (h) identifying a subset of said auxotrophic cells in which said segment of said DNA construct has been chromosomally integrated, whereby the target chromosomal locus is altered.
Dependencies are inferred from the claim text.