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
2000-02-24
2003-02-11
Yucel, Remy (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...
C435S471000, C435S254110, C435S254300, C435S254700, C536S023100
Reexamination Certificate
active
06518042
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
A process for making DNA libraries in filamentous fungal cells using a novel cloned gene involved in the mismatch repair system of filamentous fungal cells.
2. Description of the Related Art
The mismatch repair system is a system within cells which recognizes mismatches in newly synthesized duplex DNA sequences.
The mismatch repair system then either corrects the mismatches which are seen as errors by e.g. using the methylated “old” strain as template or alternatively it may mediate degradation of the duplex DNA sequences which comprise the mismatches.
Independently on the precise mechanism the end result will be that the “mismatch repair system” will limit the “diversity” within a cell, diversity being represented as duplex DNA sequences which comprise mismatches.
For example a duplex DNA sequence which comprises a single mismatch represents a diversity of two different DNA sequences within the cell. If the mismatch repair system corrects the mismatch there will only be a diversity of one within the cell.
Alternatively, if the mismatch repair system mediates the degradation of such a duplex DNA sequence the diversity will be lost. See
FIG. 1
for a graphic illustration on how the mismatch repair system may work within a cell.
Consequently, if duplex DNA sequences comprising mismatches represent a DNA library of interest, then the diversity of this library may be limited when transformed (placed) into cells with an active mismatch repair system.
The art provides a solution to this problem by making cells wherein the mismatch system is inactive.
EP 449923 describes bacterial cells wherein the mismatch system is inactivated.
WO 97/37011 describes yeast cells wherein the mismatch system is inactivated. See the working examples of this document.
WO 97/05268 describes mice cells wherein the mismatch system is inactivated. See the working examples of this document.
SUMMARY OF THE INVENTION
The problem to be solved by the present invention is to provide an improved strategy for making DNA libraries in filamentous fungal cells. A filamentous fungal cell population comprising such a DNA library may then be used to select a polypeptide of interest. Also polynucleotide sequences with particular properties might be selected, such as promoters, terminators and other regulatory elements with changed/improved properties.
The solution is based on that the present inventors have cloned a novel gene involved in the mismatch repair system of a filamentous fungal cell. Further, this gene is the first gene cloned which is involved in the mismatch repair system of a filamentous fungal cell.
By inactivating this gene in a filamentous cell it is possible to obtain a filamentous cell which is deficient in its mismatch repair system and which is highly useful for preparing DNA libraries in filamentous fungal cells.
The gene comprises a very characterizing DNA sequence encoding the polypeptide sequence shown in positions 683-758 of SEQ ID NO:2.
This DNA has been used to clone the full length gene encoding the polypeptide sequence shown in positions 1-940 of SEQ ID NO:2. See working examples herein (vide infra).
The gene cloned as described in working examples herein is a gene cloned from an
Aspergillus oryzae
filamentous fungal cell.
However, based on the novel sequence information provided herein it is routine work for the skilled person to clone similar homologous genes from other filamentous fungal cells by, e.g., standard hybridization or PCR technology, preferably by using the DNA sequence encoding the polypeptide sequence shown in positions 683-758 of SEQ ID NO:2 as a basis for making a hybridization probe or PCR primers.
Accordingly, in a first aspect the present invention relates to a filamentous fungal cell, wherein a gene involved in the mismatch repair system has been inactivated and in which the gene involved in the mismatch repair system comprises:
(a) a DNA sequence encoding the polypeptide sequence shown in positions 683-758 of SEQ ID NO:2; or
(b) a DNA sequence encoding a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 683-758 of SEQ ID NO:2; and
in a second aspect the present invention relates to a filamentous fungal cell, wherein a gene involved in the mismatch repair system has been inactivated and in which the gene involved in the mismatch repair system comprises:
(a) a DNA sequence encoding the polypeptide sequence shown in positions 1-940 of SEQ ID NO:2; or
(b) a DNA sequence encoding a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 1-940 of SEQ ID NO:2.
As stated above a filamentous fungal cell of the first or second aspect of the invention is very suitable for making a DNA library of interest in filamentous fungal cells.
Accordingly, in a third aspect the present invention relates to a process for preparing a filamentous fungal cell population wherein individual cells in the population comprise individually different DNA sequences of interest representing a DNA library of interest comprising the following steps:
(a) placing individually different DNA sequences of interest in a filamentous fungal cell population comprising a filamentous fungal cell of the first or second aspect of the invention; and
(b) growing the population of (a) for a period of time allowing an individual DNA sequence of interest in the population to be duplicated at least once under conditions wherein the mismatch repair system gene of the first or second aspect of the invention has been inactivated.
One of the advantages of allowing an individual mismatch repair inactivated filamentous fungal cell duplicated DNA of interest at least once as described under step (b) of the third aspect is illustrated in FIG.
1
. As can be seen in
FIG. 1
the process of the third aspect using a filamentous fungal mismatch repair inactivated cell as described herein allows preparation of a DNA library wherein eventual hetero duplex DNA mismatches are not corrected. This gives a DNA library with a higher diversity as compared to a DNA library made in a filamentous fungal cell NOT having an inactivated mismatch repair system (see FIG.
1
). Duplication of DNA sequence of interest means that the two strands are replicated such that two separate sets of double stranded DNA are generated, each being based on a separate one of the two original strands.
A filamentous fungal cell population wherein individual cells in the population comprise a DNA library of interest as described above may be used to select a polypeptide of interest.
Accordingly, in a fourth aspect the present invention relates to a process for production of a polypeptide of interest comprising the steps of the third aspect and wherein the DNA sequences of interest encode a polypeptide of interest and which further comprises the following step:
(c) selecting from the resultant population of filamentous fungal cells of step (b) of the third aspect a desired polypeptide of interest.
An advantage of the process of the fourth aspect may be that the polypeptide of interest is selected from a filamentous fungal cell expressing the polypeptide. Consequently, it is directly known that the polypeptide can be expressed from a filamentous fungal cell, which is useful if it is subsequently required to produce the polypeptide in large scale in a filamentous fungal cell. This may be of particular interest when the DNA library encodes polypeptides of interest which are derived from filamentous fungal cells, since it is known that filamentous fungal polypeptides preferably are produced in industrial relevant high yields in filamentous fungal cells.
This is contrary to a similar selection process using e.g. a yeast cell. Here the only thing known is that the selected polypeptide is capable of being expressed in yeast and later expression a filamentous fungal cell might give problems, especially if high yields are required.
Definitions
The following section provides definitions of technical features in above-mentioned aspect
Borchert Torben Vedel
Christiansen Lars
Pedersen Dennis Holm
Vind Jesper
Davis Katharine F
Garbell Jason
Lambiris Elias
Novozymes A/S
Pedersen Dennis Holm
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