CN1341146A - Fungal cells with inactivated DNA mismatch repair system - Google Patents
Fungal cells with inactivated DNA mismatch repair system Download PDFInfo
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- CN1341146A CN1341146A CN00804283A CN00804283A CN1341146A CN 1341146 A CN1341146 A CN 1341146A CN 00804283 A CN00804283 A CN 00804283A CN 00804283 A CN00804283 A CN 00804283A CN 1341146 A CN1341146 A CN 1341146A
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Abstract
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.
Description
Invention field
A kind of clone gene of novelty that utilizes prepares the method in filamentous fungal cells DNA library, and this gene participates in the mismatch repair system of filamentous fungal cells.
Background of invention
Mismatch repair system is the system of the mispairing in the new synthetic double chain DNA sequence of identification in the cell.
Mismatch repair system can utilize methylated " old " strain system to correct as template then and perceived as wrong mispairing, perhaps can mediate the degraded of the double chain DNA sequence that comprises mispairing.
Be independent of outside the accurate mechanism, net result will be " mismatch repair system " with " diversity " in the restrictive cell, diversity shows as the double chain DNA sequence that comprises mispairing.
For example, the double chain DNA sequence that comprises single mispairing is represented the diversity of two kinds of different dna sequence dnas in the cell.If mismatch repair system is corrected mispairing, in cell, will have only a kind of dna sequence dna so.
Perhaps, if the degraded of a kind of like this double chain DNA sequence of mismatch repair system mediation, diversity will disappear so.Referring to Fig. 1, it is schematically set forth mismatch repair system and how to work in cell.
So, represent DNA library of interest if comprise the double chain DNA sequence of mispairing, when conversion (placement) was in the cell with active mismatch repair system, the diversity in this library can be restricted so.
This area provides a kind of solution of this problem, promptly prepares the wherein mispairing system cell of inactivation.
EP449923 has described the bacterial cell that wherein mispairing system is inactivated.
WO97/37011 has described the yeast cell that wherein mispairing system is inactivated.Work example referring to the document.
WO97/05268 has described the mouse cell that wherein mispairing system is inactivated.Work example referring to the document.
Summary of the invention
Problem to be solved by this invention provides the improvement strategy in preparation filamentous fungal cells DNA library.Comprise the filamentous fungal cells colony in a kind of like this DNA library thereby can be used to select desired polypeptides.Can also select to have the polynucleotide sequence of special properties, for example have promotor, terminator and other regulatory element of change/improvement character.
The basis of solution is that the contriver has cloned the new gene that participates in the filamentous fungal cells mismatch repair system.In addition, this gene is the gene of first kind of quilt participation filamentous fungal cells mismatch repair system of cloning.
This gene by among the deactivation filiform cell might obtain such filiform cell, and it is defectiveness in its mismatch repair system, and is very useful the DNA library in the preparation filamentous fungal cells.
This gene comprises the dna sequence dna of very individual coding peptide sequence shown in the 683-758 position of SEQ ID NO 2.
This DNA has been used for the full-length gene of clones coding peptide sequence shown in the 1-940 position of SEQ ID NO 2.Work example (as follows) referring to this paper.
As being from aspergillus oryzae filamentous fungal cells cloned genes as described in this paper work example by cloned genes.
But, based on the new sequence information that this paper provided, be regular works from the similar homologous gene of other filamentous fungal cells clone for a person skilled in the art, for example use the hybridization or the round pcr of standard, preferably the dna sequence dna of peptide sequence shown in the 683-758 position of use coding SEQ ID NO 2 is as the basis of preparation hybridization probe or PCR primer.
Therefore, the present invention relates to filamentous fungal cells in first aspect, and the gene that wherein participates in mismatch repair system is inactivated, and the gene of this participation mismatch repair system comprises:
(a) dna sequence dna of peptide sequence shown in the 683-758 position of coding SEQ ID NO 2; Or
(b) coding at least 70% is equal to the dna sequence dna of peptide sequence of the 683-758 position of SEQ ID NO 2;
The present invention relates to filamentous fungal cells in second aspect, and the gene that wherein participates in mismatch repair system is inactivated, and the gene of this participation mismatch repair system comprises:
(a) dna sequence dna of peptide sequence shown in the 1-940 position of coding SEQ ID NO 2; Or
(b) coding at least 70% is equal to the dna sequence dna of peptide sequence of the 1-940 position of SEQ ID NO 2.
As mentioned above, the filamentous fungal cells of the present invention first or second aspect is very suitable for preparing DNA library of interest in the filamentous fungal cells.
Therefore, the present invention relates to the method for preparing filamentous fungal cells colony in the third aspect, and wherein the individual cells in the colony comprises the various objectives dna sequence dna of representing DNA library of interest respectively, and this method comprises the following steps:
(a) respectively different target DNA sequences is placed in the filamentous fungal cells colony that comprises the present invention first or second aspect filamentous fungal cells;
(b), single target DNA sequence in the colony is duplicated at least once under the condition that the present invention first or second aspect mismatch repair system gene have been inactivated with (a) population growth for some time.
Fig. 1 has set forth as described in third aspect step (b) and has made single mispairing reparation deactivation type filamentous fungal cells duplicate one of target DNA advantage at least once.Fig. 1 can see, the third aspect method that as killed cells is repaired in use filamentous fungus as described herein mispairing makes the preparation in the DNA library that wherein final heteroduplex dna mismatch is not repaired become feasible.Gained DNA library has more highly diverse (referring to Fig. 1) than the DNA library that makes in the filamentous fungal cells of the mismatch repair system that does not have deactivation.Duplicating of target DNA sequence means that two strands all are replicated, the two cover double-stranded DNAs that make generation respectively, and every cover is all based on one of two original stocks.
Wherein the filamentous fungal cells colony that comprises above-mentioned purpose DNA library of the individual cells in the colony can be used to select desired polypeptides.
Therefore, the present invention relates to the method for producing desired polypeptides in fourth aspect, and it comprises the step of the third aspect, and target DNA sequence encoding desired polypeptides wherein, and this method further comprises the following steps:
(c) from third aspect step (b) gained filamentous fungal cells colony, select required desired polypeptides.
The advantage of fourth aspect method is that desired polypeptides is to select from the filamentous fungal cells of expressing this polypeptide.So, can know directly that described polypeptide can express from filamentous fungal cells, if subsequently need be in filamentous fungal cells the mass preparation polypeptide, this is useful so.When the DNA library was encoded from filamentous fungal cells deutero-desired polypeptides, this point merited attention especially, because known to industrial, the filamentous fungus polypeptide preferably prepares in filamentous fungal cells, and the output height.
This is with similarly system of selection is opposite, and described similar approach is for example used the method for yeast cell.Here uniquely be known that selected polypeptide can be expressed in yeast, the expression in filamentous fungal cells later on may have problems, especially when the needs high yield.
Definition:
The definition of the invention described above each side technical characterictic is provided below.
Term " gene " represents that herein gene (dna sequence dna) can be expressed as polypeptide in described cell.Therefore, described gene order will be defined as starting from initiator codon (being generally " ATG ", " GTG " or " TTG "), end at the exploitation reading frame of terminator codon (being generally " TAA ", " TAG " or " TGA ").
As known in the art, for expressing said gene, the element that links to each other with this gene must be arranged, they are that this gene is necessary at cell inner expression.Such standard component can comprise promotor, ribosome bind site, end sequence, can also be other elements known in the art.
According to this area, term " mismatch repair system " is appreciated that the system of identification double chain DNA sequence mispairing in the cell herein.For example capable referring to WO97/37011 page 1 21-28.Mismatch repair system or correction are found to be wrong mispairing, for example methylate " old " chain as template by use, or it can mediate the degraded of the double chain DNA sequence that comprises mispairing.
Be independent of outside the accurate mechanism, net result will be " mismatch repair system " with " diversity " in the restrictive cell, diversity shows as the double chain DNA sequence that comprises mispairing.
For example, the double chain DNA sequence that comprises single mispairing is represented the diversity of two kinds of different dna sequence dnas in the cell.If mismatch repair system is corrected this mispairing, in cell, will have only a kind of dna sequence dna so.Perhaps, if the degraded of a kind of like this double chain DNA sequence of mismatch repair system mediation, diversity will disappear so.
The polypeptide of the genes encoding of quilt participation mismatch repair system is by discerning this mispairing with the mechanism of mispairing purpose.
Therefore, whether the mismatch repair system of the checking filamentous fungal cells as described herein assay method of deactivation adopts " gel moves (shift) test ", or claims " gel is postponed test ".This is the used a kind of standard test in this area.Referring to the 16th, 17 and 25 pages of WO97/05268.
The principle of this class test is that cell extract is made from following: (a) filamentous fungal cells, and the gene of participation mismatch repair system wherein as described herein is deactivation; (b) corresponding filamentous fungal cells, gene wherein is not deactivation.These extracts combine/mix with oligonucleotide and the superhelix TG dinucleotides of the G that contains base-pair mismatch: T, G: A, G: G, A: C then, measure on native gel.
If gel move the contained gene of (shift) evidence not the contrast filamentous fungal cells of deactivation comprise and any above-mentioned oligonucleotide bonded arbitrary protein matter, and do not see in the filamentous fungal cells that these conjugated protein genes at contained participation mismatch repair system as described herein are deactivations, confirm that so latter's mismatch repair system is deactivation.
This paper work example 1 provides the gel that is fit to move the detailed description of (shift) test.
The sequence identity that relates to term " coding at least 70% is equal to the dna sequence dna of the peptide sequence of peptide sequence shown in the 683-758 position of SEQ ID NO 2 " and " coding at least 70% is equal to the dna sequence dna of the peptide sequence of peptide sequence shown in the 1-940 position of SEQ ID NO 2 " is confirmed as the degree that is equal between two kinds of sequences, and it illustrates that first kind of sequence is from second kind of derivation.Identity can suitably be measured by computer program known in the art, for example the GAP in the GCG routine package (Program Manual for the Wisconsin Package, Version 8, August 1994, GeneticsComputer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, C.D., (1970) " molecular biology magazine " 48,443453).GAP adopts following peptide sequence relatively to be provided with: GAP generates point penalty 3.0, GAP prolongs point penalty 0.1, the result, first aspect according to invention, at aminoacid sequence shown in the 683-758 position of SEQ ID NO 2, perhaps according to the first aspect of inventing, at aminoacid sequence shown in the 1-940 position of SEQ ID NO 2, the degree that is equal to that polypeptide showed by the similar dna sequence encoding of the present invention is preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, especially be at least 97%.
The library of at least two kinds of different dna sequence dnas represented herein in term " DNA library ".For most practical purposes, the library must be bigger.Therefore, the DNA library preferably comprises at least 1000 kinds of different dna sequence dnas, at least 10000 kinds of different dna sequence dnas more preferably, and then at least 100000 kinds of different dna sequence dnas more preferably.
The term that relates to third aspect present invention method steps (a) " is placed on different target DNA sequences in the filamentous fungal cells colony respectively " herein and should extensively understands on " it is not to be equal to the target DNA sequence that is placed in the filamentous fungal cells colony " this meaning.In the context of this article, when relating to the method for using mispairing rectification of defects type cell preparation DNA library, a kind of like this situation should preferably be represented in this term, wherein the intragroup cell of filamentous fungal cells comprises at least two kinds of different target DNA sequences, they are homeologous, so that they can hybridization/reorganization each other in cell.Measure this class sequence how portion homologous with general knowledge that to obtain described intracellular reorganization be those skilled in the art.
Example can be to be placed in the cell with the homeologous sub-thread oligonucleotide sequence of chromosomal DNA sequence, and the double chain DNA sequence that perhaps will comprise mispairing (for example being included in the carrier) is placed in the cell.Further specifying about these examples as follows.
The concrete experimental technique that these dna sequence dnas are placed in the filiform cell can carry out according in the multiple proper technology any, for example transformation technology.
According to third aspect present invention step (b), term " with (a) population growth for some time; single target DNA sequence is duplicated at least once under the condition that the mismatch repair system gene has been inactivated " expression, after at least one time of individual cells self-replacation, mismatch repair system can be activated again, and does not lose the advantage of this method.Fig. 1 has set forth technical reason wherein.In this example, the double chain DNA sequence that will comprise single mispairing is placed among the filiform cell.After cell duplicated once under the condition that the mismatch repair system gene has been inactivated, two kinds of different single-stranded dna sequences were replicated respectively in the double-stranded DNA, obtain two kinds of different double-stranded sequences, contain one in each replicating cell, without any mispairing.Therefore, because such cell does not comprise the purpose double chain DNA sequence with mispairing, therefore there is not the Technology Need of keeping the mismatch repair system deactivation.
The preferred embodiment of the invention is described below, only supplies illustration.
Description of drawings
Fig. 1:
This figure sets forth the double chain DNA sequence will comprise single mispairing and is placed on example among the filiform cell.After cell duplicated once under the condition that the mismatch repair system gene has been inactivated, two kinds of different single-stranded dna sequences were replicated respectively in the double-stranded DNA, obtain two kinds of different double-stranded sequences, contain one in each replicating cell, without any mispairing.On the contrary, be in the active cell at mismatch repair system, two intrachain mispairing have been repaired.
Fig. 2:
This figure shows the three kinds of incomplete aspergillus oryzae peptide sequences of PCR fragment deutero-from being cloned: ' msh2 ' Ao-co110/13/15.With these three kinds of incomplete peptide sequences and other two kinds of known mismatch repair proteins: people's mismatch repair protein, i.e. msh2-human.p; With the yeast saccharomyces cerevisiae mismatch repair protein, i.e. the incomplete peptide sequence contrast of S.c.msh2 is arranged.The sequence of band underscore is derived from the segmental structure of PCR among the figure.
Fig. 3:
What this figure showed generally acknowledged (putative) aspergillus oryzae mismatch repair protein (Ao.MSH2) infers (proposed) peptide sequence and three kinds of known mismatch repair proteins: the originate arrangement of (S.c.msh2) peptide sequence of human origin (msh2-human.p), muroid source (msh2-mus.p) and yeast.
Detailed description of the invention
Filamentous fungal cells as described herein, the wherein base of participation mismatch repair system as described herein Because being inactivated.
Participate in the deactivation of the gene of mismatch repair system:
The novel gene of participation mismatch repair system as described herein can be by known to the skilled many The in addition deactivation of one of kind of known technology.
Embodiment of the present invention relate to filamentous fungal cells as described herein, wherein participate in mispairing and repair Multiple gene is defective.
When dna sequence dna was known, the method that makes in a large number gene defect all was known to the skilled. These methods comprise a part of dna sequence dna of deleting gene; Move by deletion or the introducing of insertion nucleotides The code sudden change; Introduce terminator codon etc.
The preferred embodiment of the invention relates to filamentous fungal cells as described herein, wherein participates in wrong Join the gene of reparation by temporary transient deactivation.
Similar to the above, the big metering method that can accomplish this point all is known to the skilled, is included in Upstream region of gene inserts adjustable promoter, perhaps for example on the permanent delet chromosome one one of this gene Divide, then carrier (for example plasmid) is inserted in the cell that comprises this gene. Plasmid can be on gene Trip comprises adjustable promoter, and perhaps when mismatch repair system needed by temporary transient deactivation, plasmid could From cell, remove, when mismatch repair system need be reactivated, can reinsert carefully then Among the born of the same parents.
Selecting suitable strategy for concrete technical purpose is technical staff's general knowledge.
The preferred side who prepares the filamentous fungal cells of temporarily deactivation mismatch repair system as described herein Method is that the mismatch repair gene on the permanent as killed cells chromosome at first as described herein is then with matter Grain is inserted in the cell that comprises this gene, and wherein this plasmid is characterised in that it comprises suitable copying Homing sequence and the selected marker that is fit to.
Preferably, the replication initiation sequence that is fit to be AMA1 (Gems, D. etc. (1991) " gene " 98: 61-67).
The more detailed theory of the selected marker that suitable replication initiation sequence is provided below immediately and is fit to Bright, this paper work example 4 provides the example of this strategy, uses the plasmid that comprises AMA1 as multiple Homing sequence processed uses AmdS as selected marker.
The replication initiation sequence
Term used herein " the replication initiation sequence of fungi " is defined as so a kind of nucleotide sequence, It can support the oneself of extrachromosomal molecule, for example plasmid or dna vector in the fungal host cells Copy, the structural rearrangement of plasmid does not usually take place or to the integration of host cell gene group. Replication initiation Sequence can be to originate arbitrarily, as long as it can mediate the initiating activity that copies among the fungal cell.
Preferably, the replication initiation sequence is from filamentous fungal cells, more preferably aspergillus, Fusarium Or the bacterial strain of Alternaria, and then more preferably aspergillus nidulans, aspergillus oryzae, aspergillus niger, fusarium oxysporum Or Alternaria alternata.
The replication initiation sequence can be identified by method well known in the art. For example, can be from choosing Identify the sequence of lasting self-replacation that can be in yeast in the fixed biologically-derived genomic fragment (Ballance and Turner " gene " 36 (1985), 321-331), in its indication filamentous fungal cells The of self-replication capacity. The fungi of specified sequence is copied initiating activity and can also measure like this, uses selected matter The grain replicon transforms fungi, the bacterium colony that selection has irregular form, and the forfeiture of its indication segmentation plasmid, Growth when this segmentation plasmid can cause the gene of finding at selective medium screening plasmid lacks (Gems etc. " gene " 98 (1991) 61-67). AMA1 analyzes by this way. Analyze multiple The another kind of mode of homing sequence processed be analyze naturally occurring plasmid (disclosed such as Tusge etc., " science of heredity " 146 (1997) 111-120 are about Alternaria alternata).
The example of replication initiation sequence includes but not limited to ANS1 and the AMA1 sequence of aspergillus nidulans, For example be described in respectively Cullen, D. etc. (1987 " nucleic acids research " 15:9163-9175) and Gems, D. Deng (1991 " gene " 98:61-67).
Term " copies initiating activity " uses its conventional sense in this article, just represents the sequence energy Enough support the self-replacation of extrachromosomal molecule, for example plasmid or dna vector among the fungal cell.
Term " structural rearrangement of plasmid does not take place " and be used in this article expression had not both deleted plasmid A part or insert another part of this plasmid also is inserted into plasmid without any host genome DNA In.
The filamentous fungi selected marker
Term " selection pressure " is defined in the suitable selection that is with or without effective dose in this article Under the existence of agent, cultivate the filamentous fungal cells that contains dna vector, this carrier contains can handle connection Fungi selected marker on the polynucleotide of interest sequence. The selective agent of effective dose in this article Be defined as being enough to never containing and select the cell that contains selected marker in the cell of selected marker Amount.
In preferred embodiments, the fungi selected marker is selected from, and coding can provide the thing of killing livestock The resistance of agent or viral toxicity, resistance or the prototrophy of heavy metal toxicity is become auxotrophic product The gene of thing.
In a more preferred embodiment, prototrophy is to obtain from the enzyme that is selected from such one group of metabolic pathway , these metabolic pathways be synthetic by nucleotides, co-factor synthetic, amino acid synthetic, acetamide generation Thank, Proline Metabolism, sulfuric acid metabolism and nitric acid metabolism form.
So preferred embodiment in, in the method for the invention, the fungi selected marker is So a kind of gene, it is selected from argB (ornithine transcarbamylase), amdS (acetamidase), bar (phosphine Silk rhzomorph acetyltransferase), hemA (5-aminolevulinate synthetase), (porphobilinogen closes hemB Become enzyme), hygB (hygromix phosphotransferase), niaD (nitrate reductase), prn (proline permease), PyrG (Orotidine-5 '-'-phosphate decarboxylase), pyroA, riboB, sC (sulfuric acid adenylate transferase) And trpC (anthranilate synthase).
With the fungal cell in the culture medium that is fit to, be fit to screening or select to carry the purpose of variation many Cultivate under the condition of the transformant of nucleotide sequence, this sequence has or the required feature of encoding. Cultivate Can carry out according to the method for screening polynucleotides variant library well known in the art.
Filamentous fungal cells
Filamentous fungal cells as described herein comprises Eumycotina (Eumycota) and oomycetes subphylum (Oomycota) the thread form of all. The feature of filamentous fungi be the mycelia wall by chitin, cellulose, Glucan, chitosan, mannosan and other complicated composition of Salvia polysaccharides. Nourish and grow by In the elongation of mycelia, the catabolism of carbon is the obligate aerobic-type. On the contrary, the battalion of the yeast such as saccharomyces cerevisiae Health length is by means of unicellular thalloid budding, and the metabolism of carbon can be anaerobic fermentation. Preferably Embodiment in, filamentous fungal cells is the cell of following species, Acremonium, aspergillus, sickle Spore genus, Humicola, mucor, myceliophthora, neurospora, Penicillium, the mould genus of pillar spore (Scytalidium), careless rhizopus, Tolypocladium and trichoderma, but be not limited thereto.
Filamentous fungal cells example used in this invention comprise aspergillus cell, Acremonium cell, Fusarium cell, Humicola cell, mucor cell, myceliophthora cell, neurospora cell, Penicillium cell, careless rhizopus cell, Tolypocladium cell and trichoderma cell.
More specifically, filamentous fungal cells be aspergillus awamori, smelly aspergillus, aspergillus japonicus, aspergillus nidulans, Aspergillus niger or aspergillus oryzae cell; Bar spore shape sickle spore, Fusarium cerealis, Fusarium Crookwellense, machete sickle spore, F.graminearum schw, the red sickle spore of standing grain, different spore sickle spore, albizzia sickle spore, Fusarium oxysporum, racemosus sickle spore, rose-colored sickle spore, elder sickle spore, colour of skin sickle spore, plan branch spore sickle Spore, sulphur look sickle spore, Fusarium torulosum, Fusarium trichothecioides, Fusarium The cell of venenatum or Fusarium venenatum cell (Nirenberg sp.nov); Humicola Insolens cell or Humicola lanuginosa cell; The conspicuous Mucor cell of rice; The thermophilic fungus destroyed wire cell; The Neurospora crassa cell; The penicillium purpurogenum cell; Thielavia terrestris cell; Trichoderma Harzianum, Kang Ningshi wood mould, Trichoderma longibrachiatum, Trichoderma reesei Or Trichoderma viride cell.
Preparation comprises the method for the filamentous fungal cells colony in DNA library according to third aspect present invention:
Step (a) according to the third aspect present invention method is placed on different target DNA sequences in the filamentous fungal cells colony respectively:
As mentioned above, these dna sequence dnas are placed on concrete experimental technique in the filamentous fungal cells Can carry out according to one of multiple proper technology, for example transformation technology. Referring to common fungi textbook " genetic of fungi " (1996, ISBN 0-8247-9544-X) further specifying about these standard techniques.
Example can be to place with the homeologous sub-thread oligonucleotide sequence of chromosomal DNA sequence In cell. Referring to " genetic of fungi communication (Fungal genetic newsletter) " 43 such as Calissano: 15-16 (1995) further specifying about this point.
Another kind of example can be the double chain DNA sequence that will comprise mispairing (for example shown in Figure 1, comprise In carrier) be placed in the cell.
In preferred embodiments, different target DNA sequences is included in the plasmid, wherein This plasmid is characterised in that, it comprise aforesaid suitable replication initiation sequence and be fit to selective Mark.
Preferably, the replication initiation sequence that is fit to be AMA1 (Gems, D. etc. (1991) " gene " 98: 61-67).
According to the step (b) of third aspect present invention, population growth a period of time of step (a) is made colony In single target DNA sequence under the mismatch repair system gene condition that is inactivated as described herein Copy at least one times.
The growth of colony can multiple suitable becoming known for grow filamentous fungal cells culture medium it Carry out in arbitrary. The general knowledge that to select so a kind of suitable culture medium be the technical staff.
Above explained, the individual cells in the colony must can the mismatch repair system gene as Copy at least one times under the condition that is inactivated described herein.
Described cell certainly can under the condition that the mismatch repair system gene has been inactivated, copy once with On.
Because the deactivation of mismatch repair system will cause the tired of the upper sudden change of endocellular chromosome DNA usually Long-pending, may become thus the lethal mutation of cell, therefore actual preferred above-mentioned replicative cycle number of times will Depend on that these potential lethal mutations occur to such an extent that how soon have.
Determine how much preferred replicative cycle is technical staff's general knowledge if having.
Because these potential lethal mutations, the mismatch repair system under the preferred steps (b) is by temporarily Deactivation.
After the replicative cycle that is fit to according to third aspect step (b), activate the mismatch repair system of temporary transient deactivation again, purpose is these lethal mutations of avoiding in the filamentous fungal cells itself.The strategy of this temporary transient deactivation can be any above-mentioned strategy.
The strategy that another kind of restriction sudden change is incorporated on the karyomit(e) is temporarily to stop THE REPLICATION OF CHROMOSOME under the condition of mispairing rectification of defects, and the duplicated chromosome external component.This can be by only being realized for introducing sudden change in the necessary element of chromosome duplication.
Preferred strategy is to use such filamentous fungal cells, the permanent deactivation of the gene of participation mismatch repair system wherein as described herein quilt on cell chromosome, insert plasmid then in the cell that comprises this gene, wherein this plasmid is characterised in that the selected marker that it comprises suitable replication initiation sequence and is fit to.See the further explanation of going up about this strategy.
Preferably, the replication initiation sequence of Shi Heing is AMA1 (Gems, D. etc. (1991) " gene " 98:61-67).
Further embodiment relates to the method for third aspect present invention, and wherein the mismatch repair system under the step (b) is defective.
In further embodiment, the present invention relates to method as described herein, wherein under the step (b) of third aspect present invention, exist between the vivo gene of homeologous target DNA sequence and recombinate.
Because general conception of the present invention provides the method that relates to the deactivation of mispairing system, certainly preferred described homeologous dna sequence dna can form the double chain DNA sequence that comprises mispairing in the body.
Prepare the method for desired polypeptides according to a fourth aspect of the present invention, it comprises the step of third aspect present invention and target DNA sequence encoding desired polypeptides wherein:
According to the step (c) of fourth aspect, from third aspect step (b) gained filamentous fungal cells colony, select required desired polypeptides.
Required desired polypeptides can be any polypeptide that comprises required technical characterictic, the stability that described technical characterictic has for example improved; Required activity specific; Required best pH; Improved scourability in washing composition; Deng.
Being used to select the special strategy of this required desired polypeptides can be the arbitrary of a large amount of selection strategy known to the skilled, for example plate screening test, based on the test of droplet plate etc.
Embodiment of the present invention relate to the method for fourth aspect present invention, further comprise the following steps:
(d) optional step, it comprises the aminoacid sequence of modifying the desired polypeptides of selecting on demand according to further specific needs;
(e) will the encode dna sequence dna of modification type desired polypeptides of the desired polypeptides of fourth aspect step (c) or step (d) is placed in the filamentous fungal cells that is suitable for this desired polypeptides of mass preparation;
(f) under the condition that allows desired polypeptides to express, at 10000m at least
3Fermentor tank in the filamentous fungal cells of culturing step (e);
(g) analysis purposes polypeptide.
This embodiment relates to and the closely-related method of industrialization, and wherein selected desired polypeptides is a mass preparation.
Optionally step (d) relates to a kind of like this situation, and wherein selecting for example required desired polypeptides, purpose for example is to identify according to polypeptide third aspect present invention step (c), have the scourability of improvement in washing composition.Although this peptide species has improved scourability in washing composition, may stablize inadequately as commerical prod.Therefore, may need to carry out some further aminoacid replacement in this selected polypeptide, for example the proline(Pro) that is fit to replaces, and purpose is to obtain enough stability, so that this peptide species commercialization.
Further embodiment relates to the method for embodiment just now, and the filamentous fungal cells that wherein is suitable for the desired polypeptides of the described embodiment step of mass preparation (e) is the another kind of filamentous fungal cells that is different from the filamentous fungal cells of third aspect present invention step (a).
This embodiment relates to a kind of like this situation, wherein is used to select the filamentous fungal cells of desired polypeptides to be different from the filamentous fungal cells that is used for mass preparation.
Further embodiment relates to method as described herein, and wherein desired polypeptides is from the filamentous fungal cells polypeptides derived.
The term amino acid sequence information that is appreciated that desired polypeptides of " deriving from filamentous fungal cells " is from deriving from the polypeptide deutero-of filamentous fungal cells.
So it can be wild-type filamentous fungus variant polypeptides and/or can be the reorganization/reorganization product of two or more different filamentous fungus polypeptide.
So further in the embodiment, the present invention relates to method as described herein, wherein desired polypeptides is a kind of enzyme, for example amylase, proteolytic enzyme, cellulase, lipase, zytase, Phospholipid hydrolase.
Embodiment:
Material
Chemical as buffer reagent and substrate is the commerical prod that is at least SILVER REAGENT.
Embodiment 1:
Be suitable for measuring the gel whether filamentous fungal cells as described herein is inactivated in mismatch repair system:
The principle that this gel moves (shift) test is that cell extract is made from following: (a) filamentous fungal cells, and the gene of participation mismatch repair system wherein as described herein is deactivation; (b) corresponding filamentous fungal cells, gene wherein is not deactivation.
These extracts combine/mix with the oligonucleotide of the G that contains base-pair mismatch: T, G: A, G: G, A: C and the dinucleotides of supercoiled TG then, measure on native gel.
If gel move the contained gene of (shift) evidence not the contrast filamentous fungal cells of deactivation comprise and any above-mentioned oligonucleotide bonded arbitrary protein matter, and these conjugated protein genes at contained participation mismatch repair system as described herein are not seen in the filamentous fungal cells of deactivation, confirm that so latter's mismatch repair system is deactivation.
Experimental program:
The preparation of cell extract is carried out (Mol.Gen Genet (1993) 237:251-260 as described in Nagata etc.; See material and method).
Basically as carrying out annealing, the cell extract of oligonucleotide as described in the document and containing the combining and native polyacrylamide gel electrophoresis (Stephenson and Karran of double chain oligonucleotide of mispairing; From human cell's the protein and the selective binding of DNA base-pair mismatch; " journal of biological chemistry " 264:2177-21182 (1989)).
But, gel electrophoresis is carried out in TAE damping fluid rather than tbe buffer liquid.In order to obtain double chain oligonucleotide, make oligonucleotide U by radio-labeling, and anneal with any unlabelled oligonucleotide L-G.T, L-G.A, L-G.C, L-A.C, L-T.G or L-HOM.Oligonucleotide sequence obtains from " institute of NAS newspaper " 91:8905-8909 (1994) such as Aquilina.
U：5′-GGGAAGCTGCCAGGCCCCAGTGTCAGCCTCCTATGCTC-3′(SEQ?ID?NO?3)；
L-G.T:5 '-GAGCATAGGAGGCTGACATTGGGGCCTGGCAGCTTCCC-3 ' (SEQ ID NO 4) (causing the G.T mispairing);
L-G.A.:5 '-GAGCATAGGAGGCTGACAATGGGGCCTGGCAGCTTCCC-3 ' (SEQ ID NO 5) (causing the G.A mispairing);
L-G.G.:5 '-AGCATAGGAGGCTGACAGTGGGGCCTGGCAGCTTCCC-3 ' (SEQ ID NO 6) (causing the G.G mispairing);
L-A.C.:5 '-GAGCATAGGAGGCTGACACCGGGGCCTGGCAGCTTCCC-3 ' (SEQ ID NO 7) (causing the A.C mispairing);
L-TG:5 '-GAGCATAGGAGGCTGACACTGTGGGGCCTGGCAGCTTCCC-3 ' (SEQ ID NO 8) (causing supercoiled TG dinucleotides);
L-HOM:5 '-GAGCATAGGAGGCTGACACCGGGGCCTGGCAGCTTCCC-3 ' (SEQ ID NO 9) (causing homoduplex).
All experiments include the double-stranded competitive oligonucleotide of the excessive unlabelled homotype of twice.
Embodiment 2:
Participate in the clone of the gene of aspergillus oryzae cell mismatch repair system
As described in this example, be presented among SEQ ID NO 1 (dna sequence dna) and the SEQ IDNO 2 (aminoacid sequence of translation) by cloned genes.
Some sequences from the biological mismatch repair protein of difference are known, only use wherein three kinds below: yeast saccharomyces cerevisiae (M84170), H.sapiens (L47580) and mouse (U21011).
Shown in numeral be the inquiry number of public's GenBank database that can obtain.
Based on the end of the C-between known mismatch repair protein homology, designed one group of different primer of letter, purpose is the incomplete sequence of amplification aspergillus oryzae homologue:
Pr117858(SEQ?ID?NO?10)：P-GGCNCARATHGGNTGYTTYGTNCC
Pr117859(SEQ?ID?NO?11)：P-GCCCANGCNARNCCRAANCC
Be used to chromosomal DNA from aspergillus oryzae strain JaL142 (WO96/29391) as template and above-mentioned primer, at eight kinds of MgSO
4(0.5mM to 4.0mM is according to manufacturer's recommendation for concentration; Boehringer M.) carries out following PCR reaction under based on 50 μ l PWO polysaccharases.Find 1mMMgSO
4Being optimum, obtaining the single band of about 230bp, is the result who estimates not contain in the sequence intron.
The PCR circulation is: [96 ℃ of 2min-(94 ℃ of 15s; 50 ℃ of 15s; 72 ℃ of 30s) circulation 30 times-72 ℃ 7min-4 ℃ keep].
230bp PCR fragment is connected the BamH1 site of pUC19 through blunt end.PUC19 through the BamH1 cracking, mends flat sticky end by klenow polysaccharase and dNTP then in the presence of calf intestinal alkaline phosphatase.From the intestinal bacteria XL1 transformant of above-mentioned ligation, analyze to carry and insert segmental three plasmids and order-checking.The polypeptide of clone's PCR fragment translation arranged disclosed it and known mismatch repair protein sequence has higher homology (see figure 2).
The sequence of band underscore is from above-mentioned total PCR primer deutero-sequence among Fig. 2.
Three kinds of Aspergillus sequences of Fig. 2 equal sequence shown in the 683-758 position of SEQ ID NO 2, but wherein 685 be Thr (T) in final sequence of cloning, rather than shown Ile (I).This is to be caused by the sequence in the above-mentioned total primer.
Arrangement shown in Figure 2 proves that clearly the fragment of being cloned is from the aspergillus oryzae homologue of mismatch repair protein.
In order to clone complete gene, generate the radiolabeled probe of institute's cloned sequence by PCR, promptly, use 0.5mg pUC19 ' msh2 '-13 (on seeing) as template, in 100ml, react with Taq polysaccharase, 30pmol pUC forward and reverse primer, 0.2mM dG-, dC-, dTTP and 0.2mM dATP+32P-dATP.The radiolabeled probe who is generated discharges from the pUC19 sequence by the EcoR1-Hind3 digestion, carries out the segmental gel-purified of gained 293bp then.
Make probe with from (WO96/29391) film lattice point (gridded) the cosmid library hybridization of genomic dna of aspergillus oryzae strain A1560 (parent of JaL142).Logical phosphoric acid imaging is only analyzed in (Phosphoimager) and is identified that the positive colony on the filter membrane is λ 31A2.
Breeding λ 31A2 cosmid DNA uses above-mentioned identical radio-labeling primer to carry out Southern and analyzes.Identified about 9Kb Pst fragment, it is split into 5.8 and the 3.2kb fragment by BstX (finding in the past) in the PCR fragment of being cloned, all send signal, this 9kb fragment cloning to the pUC19 that has cut with Pst, is obtained the plasmid of pUC19msh2P by name because of carrying probe.The primer of the sequence of measuring before show begins in order below to be based on last and to take turns the primer that institute checks order and is listed as inserting sequencing fragment:
130740(SEQ?ID?NO?12)：GCTCGAAACATCCAACATCC
130741(SEQ?ID?NO?13)：GCTGTGAATCACTTGCACC
131928(SEQ?ID?NO?14)：CTTCATAAACTGCGACAAATCATGC
131929(SEQ?ID?NO?15)：GGAGGAGCATCTTCGC
131930(SEQ?ID?NO?16)：GGAACTTGAAGACTTTACTTCATCC
134608(SEQ?ID?NO?17)CCAGAAACTCGCTAACACC
134609(SEQ?ID?NO?18)：GTGCTTTGCGGACGC
134610(SEQ?ID?NO?19)：CAGGACAGTAGGACGC
135320(SEQ?ID?NO?20)：CGAGCGATGAACTCTGC
135321(SEQ?ID?NO?21)：GCGTTGGTGGATTATCC
136105(SEQ?ID?NO?22)：CGTTGCATCTATCATATACC
136106(SEQ?ID?NO?23)：GGTATATGATAGATGCAACGC
Translate in the former framework of in the PCR fragment, measuring of the 3825bp sequence of Ce Dinging (SEQ ID NO 1) in view of the above.Gained protein (SEQ ID NO 2) is called as Ao.MSH2, and the protein sequence of known mismatch repair protein among itself and Fig. 3 is arranged.From arrangement shown in Figure 3 as can be seen, the DNA that clones and check order has clearly been contained the encoding sequence of the homologue of yeast, people and mouse mismatch repair protein, and it has an intron in the N-end parts.Montage rule according to standard is inferred the position of intron, and constitutes unique possibility.
Embodiment 3:
Destroy 1 cloned genes of embodiment on the aspergillus oryzae cell chromosome:
About breaking test, from pUC19msh2P (seeing embodiment 2), delete msh2CDS by PCR, introduce the NotI site with for it.The primer that carries out this reaction is:
137208(SEQ?ID?NO?24)：5′P-CCGCGTCTCCAACAAGATGAATGG
137207(SEQ?ID?NO?25)：5′P-CCGCTTTCTCGGGGTCATAGC
With 2.5mM MgSO
4The PCR based on the Pwo polysaccharase that is carried out with 50pg pUC19msh2P reacts in (according to the condition of manufacturers's suggestion):
The PCR circulation is: [96 ℃ of 2min-(94 ℃ of 30s; 52 ℃ of 30s; 72 ℃ of 3min) 4 times-(94 ℃ of 30s of circulation; 59 ℃ of 30s; 72 ℃ of 3min) 25-72 ℃ of 10min of circulation].
Analyze the PCR product of the about 8.9Kb of gained, be connected to pUC19, then transformed into escherichia coli XL1.Analyze the pMsh2 Δ from the gained transformant, by order-checking [primer 138149 (SEQ ID NO26): CCTTTCCACTTTAATCCTAAGC] (X11/pMsh2 Δ: Lac3073) new joint of checking and exactness on every side thereof.
Aspergillus oryzae pyrG (WO96/29391) is inserted the NotI site of this construct.
By utilizing this construct, can be by known in the art about in the aspergillus oryzae cell, deleting described chromogene (Miller in the standard technique that makes this class deletion gene intersection (crossing) on the karyomit(e) by homologous recombination, B.L. etc., 1985 " molecule and cytobiology " 5:1714-1721).
Embodiment 4:
The structure that comprises the plasmid of mismatch repair gene, AMA1 replication initiation sequence and AmdS selected marker shown in SEQ ID NO 1:
The plasmid of structure as described below is very suitable for preparing the filamentous fungal cells of contained mismatch repair system deactivation temporarily, wherein when mismatch repair system should be activated, this plasmid can be inserted in the destructive of the mispairing bacterial strain of embodiment 3, when mismatch repair system should be inactivated, this plasmid can be deleted from bacterial strain.
The destruction of mismatch repair gene can cause accumulating of new chromosome mutation, and therefore such bacterial strain may be that heredity is gone up unsettled.So decision is carried out karyomit(e) and destroyed in following bacterial strain, mismatch repair gene is expressed by extra-chromosomal element in the described bacterial strain, and this element can be lost easily when needs mispairing repair-deficient phenotype.
Here, extra-chromosomal element is to comprise AMA1 as replication initiation sequence and the AmdS plasmid as selected marker.
For this reason, mismatch repair gene (SEQ ID NO 1) is cloned in the self-replacation type construct that carries an AMA1 tumor-necrosis factor glycoproteins.
From pMT1505 (embodiment 5 is about the explanation of pMT1505 as follows), separate following fragment, and they linked together:
5.16kb?NotI-[Hind3]
*＋3.515kb[Sal]
*－BamH1＋757bp?BamH1-NotI
[]
*Represent that this site is flat with klenow-polysaccharase and dNTP benefit
Analyze pMT1505DHS (LaC 3212) by this ligation, the corresponding site that expression cassette is introduced pMT1505DHS as the BamH1-Mun1 fragment is repaired in mispairing, obtain plasmid pAma-msh2 (LaC3216).
Aspergillus oryzae JaL250 (seeing embodiment 5) is converted into AmdS with pAma-msh2
+, check that non-selected transformant (transformant 50%) loses the ability of amdS proterties, illustrate that this plasmid keeps as the karyomit(e) element.(LaC3244 remains on ethanamide+uridine).
Embodiment 5:
The structure of embodiment 4 used plasmid pMT1505:
Plasmid
PMT1505: following embodiment 5 described structures
PHelpl: contain aspergillus oryzae pyrG gene as selected marker, also contain can self-replacation in Aspergillus nidulans the AMA1 sequence, as Gems, D. etc. (1991. " gene " 98:61-67) are described
PToC68: as described in EP0 531 372 (Novo Nordisk A/S)
Bacterial strain
JaL250: the derivative of aspergillus oryzae A1560, wherein pyrG gene deactivation is as described in WO98/01470
DH5a: e. coli host cell, available from GIBCO BRL (Life Technologies, Inc., Rockville MD)
PMT1466 makes up by being inserted among the pToC68 from the SphI/NarI fragment of pHelpl.PMT1489 is by with SphI and StuI digestion pMT1466, reconnects then and makes up.PMT1500 is by with AatII and NarI digestion pMT1489, connects a joint again and makes up.PMT1504 is by digesting pMT1500 with NheI, reconnects then and makes up.
The 2.7kbXbaI fragment (Corrick, C.M. etc. 1987 " gene " 53:63-71) of pMT1505 by will containing amdS encoding gene in the Aspergillus nidulans genomic dna inserted with among the pMT1504 of NheI cutting and make up.
Embodiment 6:
The deletion of last minute mshII gene of karyomit(e)
Plasmid p418MsHII (from lac3159) with SalI and XhoI cutting, is handled with the calf intestinal Phosphoric acid esterase again.Cut the part of msHII gene by this way.From 1% sepharose, analyze the big band (6400bp) that contains carrier and most of msHII gene.
Plasmid pJal554 is connected with the Asp718/NheI cutting fragment (316bp) of pS02 by the SpeI/SspBI cutting fragment (5330bp) with pS02 and makes up.PJal554 cuts with SalI with plasmid, analyzes the 2350bp band that contains the pyrG gene on 1% sepharose.The 2350bp band that will contain pyrG is connected with the p418MsHII plasmid of cutting, is transformed in the intestinal bacteria.Identify correct intestinal bacteria transformant by restriction analysis, prepare the plasmid preparation from this transformant.
Prepared plasmid is cut with EcoRI, and purpose is to make plasmid linearizing before for example being transformed into aspergillus oryzae Jal250.On basic flat board, select transformant.
The transformant that two heredity exchanges take place is to identify like this, and preparation Aspergillus chromosomal DNA preparation utilizes suitable primer that total length mshII gene is carried out the PCR screening then.Utilization is carried out the Southern engram analysis by the chromosomal DNA of suitable enzyme random fragmentation and suitable probe and positive control probe at deleted msHII fragment (not existed).
For the mutation frequency that is increased in the bacterial strain of measuring msHII gene with deactivation, the sudden change in the screening niaD gene.This point realizes by cultivation parent bacterial strain aspergillus Jal250 and msHII deactivation type bacterial strain on flat board.
The preparation spore suspension, the spore sample is seeded on the flat board that contains oxymuriate, as (S.E.Unkles as described in Unkles etc., E.C.Campbell.Y.M.J.T.de Ruite Jacobs, M.Broekhuisen, J.A.Macro, D.Carrez, R.Contreras, C.A.M.J.J.van den Hondel J.R.Kinghorn. is based on the exploitation of the aspergillus oryzae autogenic transformation system of nitrate assimilation approach: be used for convenience that filamentous fungus transforms and general selective system, " molecule and General Genetics " V:218 p 99-104 (1989)).
There is not the bacterial strain of MsHII protein expression will compare the niaD sudden change (more oxymuriate resistance clone) that has higher frequency according to bacterial strain.
Embodiment 7:
Thereby use the interior antisense msHII RNA of body that preparation suppresses msHII mRNA translation that transcribes of TAKA promoters driven antisense mRNA
It is the mode (antisense and nucleic acid drug exploitation be (1997) V.7P.439-444 for the design of sense-rna, Georg Sczakiel) of any genetic expression of downward modulation in the known body that sense-rna is expressed.
Use following oligo:000120j2 (SEQ ID NO 27): TCTGCGAATCGCTTGGATCCCGAACGCGACAACAC, 000120j4 (SEQ ID NO 28): GAGCTCAGATCTCTTAGGTTCTGGACGAGAAGA also prepares the PCR fragment with pUC19msh2P as template.This PCR fragment contains 5 ' end of msHII gene, comprises the supposition part of 5 ' msHII mRNA.Use following oligo:000120j3 (SEQ ID NO 29): GTTGTCGCGTTCGGGATCCAAGCGATTCGCAGAAG, 1298-TAKA (SEQ ID NO 30): GCAAGCGCGCGCAATACATGGTGTTTTGATCAT and with pENI1298 as the another kind of PCR fragment of template (PCTDK99/00552) preparation.Twice PCR reaction is all according to manufacturers's handbook (Boehringer-Mannheim) and use the PWO polysaccharase to carry out.
The PCR fragment is with Qiagen PCR purification kit (Qiagen) purifying in addition.Two kinds of PCR fragments are mixed, utilize primer 1298-TAKA and 000120j4 to carry out PCR reaction for the third time.Assemble described two kinds of PCR fragments by this way.
PCR fragment after the assembling is with BssHII and BglII cutting, and purifying from 1.5% sepharose is connected with pENI1298 again, and the latter is with BssHII and BglII cutting (purifying from 1% sepharose).To connect mixture is transformed in the intestinal bacteria.The DNA preparation of every kind of intestinal bacteria transformant of preparation gained.PCR fragment after the assembling is checked order, to confirm not introduce during operation unwanted sudden change.Correct construct contains the TAKA promotor, and it drives transcribing of msHII antisense mRNA.
With pENI1298 in contrast, the gained plasmid is transformed into for example aspergillus oryzae Jal250, on basic flat board, selects transformant.On basic flat board, analyze the gained transformant, cultivate down, form spore up to them at 37 ℃.
In order to measure the mutation frequency that is increased in the bacterial strain (wherein the translation of msHII mRNA is subjected to impedance because of the expression of msHII sense-rna), carry out the screening that suddenlys change in the niaD gene.The spore suspension of preparation contrast transformant (pENil298) and msHII sense-rna transformant, the equivalent spore inoculating is being contained on the flat board of oxymuriate, as (S.E.Unkles as described in Unkles etc., E.C.Campbell.Y.M.J.T.de Ruite Jacobs, M.Broekhuisen, J.A.Macro, D.Carrez, R.Contreras, C.A.M.J.J.van den Hondel J.R.Kinghorn. is based on the exploitation of the aspergillus oryzae autogenic transformation system of nitrate assimilation approach: be used for convenience that filamentous fungus transforms and general selective system, " molecule and General Genetics " V:218 p 99-104 (1989)).
The bacterial strain of nothing or low MsHII protein expression will be compared the niaD sudden change (more oxymuriate resistance clone) that has higher frequency according to bacterial strain.
Claims (15)
1, filamentous fungal cells, the gene that wherein participates in mismatch repair system is inactivated, and the gene of this participation mismatch repair system comprises:
(a) dna sequence dna of peptide sequence shown in the 683-758 position of coding SEQ ID NO 2; Or
(b) coding at least 70% is equal to the dna sequence dna of peptide sequence of the 683-758 position of SEQ ID NO 2.
2, filamentous fungal cells, the gene that wherein participates in mismatch repair system is inactivated, and the gene of this participation mismatch repair system comprises:
(a) dna sequence dna of peptide sequence shown in the 1-940 position of coding SEQ ID NO 2; Or
(b) coding at least 70% is equal to the dna sequence dna of peptide sequence of the 1-940 position of SEQ ID NO 2.
3, claim 1 or 2 filamentous fungal cells, wherein the gene of this participation mispairing reparation is defective.
4, claim 1 or 2 filamentous fungal cells wherein should participate in gene that mispairing repairs by temporary transient deactivation.
5, each filamentous fungal cells in the claim formerly, wherein this filamentous fungal cells is the fusarium bacterial strain, perhaps Aspergillus bacterial strain more preferably.
6, the method for preparing filamentous fungal cells colony, wherein the individual cells in the colony comprises the various objectives dna sequence dna of representing DNA library of interest respectively, and this method comprises the following steps:
(a) respectively different target DNA sequences is placed in the filamentous fungal cells colony of the filamentous fungal cells that comprises claim 1 or 2;
(b), single target DNA sequence in the colony is duplicated at least once under the condition that 1 or 2 mismatch repair system gene has been inactivated with (a) population growth for some time.
7, the method for claim 6, wherein this mismatch repair system under the step (b) is defective.
8, the method for claim 6, wherein this mismatch repair system under the step (b) is by temporary transient deactivation.
9, each method among the claim 6-8, wherein this filamentous fungal cells is the fusarium bacterial strain, perhaps Aspergillus bacterial strain more preferably.
10, each method among the claim 6-9 wherein under the step (b) of claim 6, exists between the vivo gene of homeologous target DNA sequence and recombinates.
11, produce the method for desired polypeptides, it comprises the step of claim 6, and wherein said target DNA sequence encoding desired polypeptides, and this method further comprises the following steps:
(c) Accessory Right requires to select in 6 steps (b) the gained filamentous fungal cells colony required desired polypeptides.
12, the method for claim 11, it further comprises the following steps:
(d) optional step comprises the aminoacid sequence of modifying required selected desired polypeptides according to further specific needs;
(e) will the encode dna sequence dna of modification type desired polypeptides of the desired polypeptides of claim 11 step (c) or step (d) is placed in the filamentous fungal cells that is suitable for the mass preparation desired polypeptides;
(f) under the condition that allows desired polypeptides to express, at 10000m at least
3Fermentor tank in the filamentous fungal cells of culturing step (e);
(g) separate desired polypeptides.
13, the method for claim 12, the filamentous fungal cells that wherein is suitable for the desired polypeptides of mass preparation claim 12 step (e) are the another kind of filamentous fungal cells that is different from the filamentous fungal cells of claim 6 step (a).
14, each method among the claim 11-13, wherein said desired polypeptides is from the filamentous fungal cells polypeptides derived.
15, each method among the claim 11-14, wherein said desired polypeptides is a kind of enzyme, for example amylase, proteolytic enzyme, cellulase, lipase, zytase, Phospholipid hydrolase.
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| EP (1) | EP1157095A1 (en) |
| JP (1) | JP2002536990A (en) |
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| WO (1) | WO2000050567A1 (en) |
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| US7883872B2 (en) | 1996-10-10 | 2011-02-08 | Dyadic International (Usa), Inc. | Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose |
| KR20020026456A (en) | 2000-04-13 | 2002-04-10 | 에말파브 마크 아론 | High-throughput screening of expressed dna libraries in filamentous fungi |
| EP1341911A1 (en) * | 2000-12-06 | 2003-09-10 | Novozymes A/S | Method for producing a polynucleotide library |
| US6783941B2 (en) | 2000-12-06 | 2004-08-31 | Novozymes A/S | Method for producing a polynucleotide library in vitro by mismatch repair of heteroduplexes |
| WO2002059331A1 (en) * | 2001-01-24 | 2002-08-01 | Novozymes A/S | In vivo mutation and recombination in filamentous fungi |
| US8680252B2 (en) | 2006-12-10 | 2014-03-25 | Dyadic International (Usa), Inc. | Expression and high-throughput screening of complex expressed DNA libraries in filamentous fungi |
| WO2009033071A2 (en) | 2007-09-07 | 2009-03-12 | Dyadic International, Inc. | Novel fungal enzymes |
| EP2527432A1 (en) | 2011-05-23 | 2012-11-28 | Novozymes A/S | Bi-directional cytosine deaminase-encoding selection marker |
| EP2527448A1 (en) | 2011-05-23 | 2012-11-28 | Novozymes A/S | Simultaneous site-specific integrations of multiple gene-copies in filamentous fungi |
| CN109072209A (en) | 2016-03-24 | 2018-12-21 | 诺维信公司 | Cellobiohydrolase variant and the polynucleotides for encoding it |
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| FR2641793B1 (en) * | 1988-12-26 | 1993-10-01 | Setratech | METHOD OF IN VIVO RECOMBINATION OF DNA SEQUENCES HAVING BASIC MATCHING |
| ES2230554T3 (en) * | 1995-07-26 | 2005-05-01 | Mixis France S.A. | HOLOGA RECOMBINATION OF EUCHARIOT CELLS WITH THE REPAIR SYSTEM OF EVIL MATCHING. |
| WO1997037011A1 (en) * | 1996-04-01 | 1997-10-09 | Setratech S.A.R.L. | Meiotic recombination in vivo of partially homologous dna sequences |
| DK1717322T3 (en) * | 1997-01-17 | 2012-10-22 | Codexis Mayflower Holdings Llc | Development of whole cells and organisms by recursive sequence recombination |
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