Chemistry: molecular biology and microbiology – Vector – per se
Patent
1996-05-06
1998-07-28
Degen, Nancy
Chemistry: molecular biology and microbiology
Vector, per se
435 691, 435 911, 4351723, 4352528, 43525411, 536 231, 536 241, C12N 1581, C12N 1511, C12P 2102, C07H 2104
Patent
active
057862125
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to modified Yarrowia lipolytica LEU2 gene promoters and to modified Y. lipolytica LEU2 genes comprising such modified Y. lipolytica LEU2 gene promoters. This invention further relates to vectors comprising such modified Y. lipolytica LEU2 genes. Such vectors include vectors comprising a Y. lipolytica DNA sequence sufficient for integrative transformation of Y. lipolytica at a locus other that the LEU2 locus of Y. lipolytica; and vectors which comprise a nucleotide sequence coding for a polypeptide and a promoter functional in Y. lipolytica operably linked thereto. The latter vectors are also known as expression vectors. Further still, this invention relates to E. coli transformants comprising vectors of this invention.
This invention also relates to Y. lipolytica transformants which comprise an expression vector according to this invention; to methods of producing Y. lipolytica transformants comprising multiple integrated expression vectors; to strains of Y. lipolytica useful in the preparation of such transformants; and to methods of producing polypeptides with certain of the Y. lipolytica transformants. Further still, this invention relates to nucleotide sequences useful in the preparation of modified Y. lipolytica LEU2 gene promoters according to this invention and a method for producing modified Y. lipolytica LEU2 promoters.
BACKGROUND ART
Processes for transformation of Y. lipolytica as well as vectors useful therefor and transformants comprising such vectors, inter alia, are disclosed and claimed in U.S. Pat. Nos. 4,880,741 and 5,071,764, both of which are assigned to the assignee hereof. Vectors useful in transformation of Y. lipolytica for expression and secretion of heterologous proteins as well as transformants and processes for producing heterologous protein therewith, inter alia, are disclosed and claimed in U.S. Pat. No. 4,937,189 which also is assigned to the assignee hereof. U.S. Pat. No. 4,937,189 also discloses the nucleotide sequence of the LEU2 gene of Y. lipolytica (SEQUENCE I.D. NO: 1).
WO91/00920 (PCT/EP90/01138), published Jan. 24, 1991, discloses a process for preparing a homologous or heterologous protein by a yeast transformed by multicopy integration of an expression vector into the genome of the yeast. The expression vector used in that process contains both an "expressible gene" encoding the desired protein and a "deficient selection marker needed for the growth of the yeast in a specific medium" such as a defective LEU2 gene (leu2d). Also disclosed are such vectors which also contain DNA coding for ribosomal RNA.
WO92/01800 (PCT/US91/04899), published Feb. 6, 1992, discloses integrating plasmid vectors capable of inserting throughout the yeast genome with high copy number and a process to accomplish such integration. The vectors disclosed use "dispersed repetitive elements (DRE's)" such as the yeast DELTA sequences, Ty sequences and tRNA DNA sequences.
DISCLOSURE OF THE INVENTION
This invention provides modified Y. lipolytica LEU2 gene promoters useful in the preparation of modified Y. lipolytica LEU2 genes. More specifically, this invention provides modified Y. lipolytica LEU2 gene promoters selected from the group consisting of nucleotides 693 to 798 of SEQUENCE I.D. NO: 1, nucleotides 718 to 798 of SEQUENCE I.D. NO: 1, nucleotides 718 to 798 of SEQUENCE I.D. NO: 1 wherein nucleotide 724 is changed from A to G, nucleotides 718 to 798 of SEQUENCE I.D. NO: 1 wherein nucleotide 725 is changed from T to G, nucleotides 718 to 798 of SEQUENCE I.D. NO: 1 wherein nucleotide 722 is changed from A to G, nucleotides 745 to 798 of SEQUENCE I.D. NO: 1, and the functional equivalents thereof. This invention also provides modified Y. lipolytica LEU2 genes comprising a modified LEU2 gene promoter according to this invention functionally linked to a Y. lipolytica LEU2 structural gene coding sequence.
This invention further provides vectors comprising modified Y. lipolytica LEU2 genes as described hereinabove. Still further, this invention provide
REFERENCES:
Gaillardin et al. "Leu 2 Directed Expression of Beta-Galactosidase Activity and Phleomycin Resistance in Yarrowia lipolytica" Current Genetics 11(5) 369-375, 1987.
Matsuoka et al "Analysis of Regions Essential for the Function of Chromosomal Replicator Sequences from Yarrowia lipolytica" Mol. Gen Genet. 237 327-333, 1993.
James Larry C.
Strick Christine A.
Benson Gregg C.
Degen Nancy
Pfizer Inc.
Richardson Peter C.
Sheyka Robert F.
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