Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-05-20
2001-01-30
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S468000
Reexamination Certificate
active
06180366
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods for producing heterologous polypeptides in trichothecene-deficient filamentous fungal mutant cells. The present invention also relates to such mutant cells of filamentous fungal cells and methods for obtaining the mutant cells. The present invention also relates to isolated trichodiene synthases and isolated nucleic acid sequences encoding the trichodiene synthases. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the trichodiene synthases. The present invention further relates to mutants cells comprising a marker-free modification of a gene, and methods for obtaining and using such mutant cells.
2. Description of the Related Art
Trichothecenes are sesquiterpene epoxides which are named after the fungus
Trichothecium oseum
from which the first trichothecene was isolated. The trichothecenes T-2 toxin, diacetoxyscirpenol, and deoxynivalenol are most commonly found in agricultural commodities infected with Fusarium species. Interest in these compounds is due primarily to the discovery that trichothecene contamination of foods and feeds may be detrimental in humans and animals.
Trichothecenes are produced by a sequence of oxygenations, isomerizations, cyclizations, and esterifications leading from trichodiene, which is produced from the cyclization of trans, trans-farnesyl pyrophosphate by the enzyme trichodiene synthase (Desjardins, Hohn, and McCormick, 1993
, Microbiological Reviews
57: 595-604).
The trichodiene synthase gene (tri5 or tox5) has been cloned from
Fusarium sporotrichioides
(Hohn and Beremand, 1989
, Gene
79: 131-138); Gibberella pulicaris (Hlohn and Desjardins, 1992
, Molecular Plant
-
Microbe Interactions
5: 249-256);
Gibberella zeae
(Proctor et al., 1995
, Molecular Plant
-
Microbe Interactions
4: 593-601);
Myrothecium roridin
(Trapp, et al., 1995
, Journal of Cellular Biochemistry Supplement
19B: 154); and
Fusarium poae
(Fekete et al, 1997
, Mycopathologia
138: 91-97).
Tri5- mutants of
Gibberella pulicaris
(Hohn and Desjardins, 1992, supra) and
Gibberella zeae
(Proctor et al., 1995, supra) have been generated which do not produce trichothecenes.
Other genes in the trichothecene biosynthetic pathway have been cloned including the tri3 gene, encoding a 15-O-acetyltransferase, from
Fusarium sporotrichioides
(McCormick et al., 1996
, Applied and Environmental Microbiology
62: 353-359); the tri4 gene, encoding a cytochrome P450 monooxygenase, from
Fusarium sporotrichioides
(Hohn et al., 1995
, Molecular and General Genetics
248: 95-102); the tri6 gene, encoding a zinc finger protein involved in the regulation of trichothecene biosynthesis, from
Fusarium sporotrichioides
(Proctor et al., 1995
, Applied and Environmental Microbiology
61: 1923-1930); the tri11 gene, encoding a cytochrome P450 monooxygenase required for C-15 hydroxylation, from
Fusarium sporoirichioides
(Alexander et al., 1997
, Applied and Environmental Microbiology
64: 221-225); the tri12 gene, which encodes an apparent transport protein involved in trichothecene production, from
Fusarium sporotrichioides
(Alexander et al., 1997
, Cereal Research Communications
25: 347-348); and the tri101 gene, encoding a 3-O-acetyltransferase, from
Fusarium graminearum
(Kimura et al., 1998
, Journal of Biological Chemistry
272: 1654-1661).
It is an object of the present invention to provide methods for producing polypeptides in mutant cells.
SUMMARY OF THE INVENTION
The present invention relates to methods for producing a heterologous polypeptide, comprising: (a) cultivating a mutant cell of a parent filamentous fungal cell under conditions conducive for the production of the heterologous polypeptide, wherein (i) the mutant cell comprises a first nucleic acid sequence encoding the heterologous polypeptide and a second nucleic acid sequence comprising a modification of at least one of the genes involved in the production of a trichothecene and (ii) the mutant cell produces less of the trichothecene than the parent filamentous fungal cell when cultured under the same conditions; and (b) isolating the heterologous polypeptide from the cultivation medium. The present invention also relates to mutant cells of filamentous fungal cells and methods for obtaining the mutant cells.
The present invention also relates to isolated trichodiene synthases and isolated nucleic acid sequences encoding the trichodiene synthases. The invention further relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the trichodiene synthases.
The present invention also relates to methods for obtaining a mutant cell, comprising:
(a) introducing into a parent cell, having a first nucleic acid sequence encoding a first polypeptide, a first nucleic acid construct comprising a nitrate reductase gene as a selectable marker and a modification of the first nucleic acid sequence, wherein the first construct incorporates into the genome of the parent cell replacing the endogenous first nucleic acid sequence with the modified first nucleic acid sequence resulting in reduced production of the first polypeptide compared to the parent cell when cultivated under the same conditions; and
(b) selecting a mutant cell from step (a) for the presence of the nitrate reductase gene and reduced production of the first polypeptide.
In a preferred embodiment, the methods for obtaining a mutant cell may further comprise (c) selecting a mutant cell from step (b) under culturing conditions in which the nitrate reductase gene is deleted. In another preferred embodiment, the methods for obtaining a mutant cell may further comprise (c) introducing into the mutant cell from step (b) a second nucleic acid construct comprising a second nucleic acid sequence comprising 5′ and 3′ regions of the modified first nucleic acid sequence, but lacking the nitrate reductase gene, wherein the second construct incorporates into the genome of the parent cell replacing the modified first nucleic acid sequence with the second nucleic acid sequence; and (d) selecting a mutant cell from step (c) under culturing conditions in which the nitrate reductase gene is deleted.
The present invention further relates to mutant cells obtained from such methods and methods for producing polypeptides with such mutant cells.
REFERENCES:
Trapp, SC, Hohn, TM., McCormick, S, Jervis, BB (1998); Mol. Gen. Genetics 257:421-432. Characterization of the Gene Cluster for Biosynthesis of Macrocyclic Trichothecenes in Myrothecium Roridium.
Desjardins, Hohn, and McCormick, 1993, Microbiological Reviews 57: 595-604.
Hohn and Beremand, 1989, Gene 79: 131-138.
Hohn and Desjardins, 1992, Molecular Plant-Microbe Interactions 5: 249-256.
Proctor et al., 1995, Molecular Plant-Microbe Interactions 4: 593-601.
Trapp et al., 1995, Journal of Cellular Biochemistry Supplement 19B: 154.
Fekete et al., 1997, Mycopathologia 138: 91-97.
McCormick et al., 1996, Applied and Environmental Microbiology 62: 353-359.
Hohn et al., 1995, Molecular and General Genetics 248: 95-102.
Proctor et al., 1995, Applied and Environmental Microbiology 61: 1923-1930.
Alexander et al., 1997, Applied and Environmental Microbiology 64: 221-225.
Kimura et al., 1998, Journal of Biological Chemistry 273: 1654-1661.
Brody Howard
Christianson Lynne M.
Gambetta Gregory A.
Otani Suzanne M.
Royer John C.
Lambiris Esq. Elias
Novo Nordisk Biotech Inc.
Ousley Andrea
Schwartzman Robert A.
Starnes Robert L.
LandOfFree
Methods for producing heterologous polypeptides in... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods for producing heterologous polypeptides in..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods for producing heterologous polypeptides in... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2447426