JP5170397B2 - Method for producing mutant strains of filamentous fungi using ion beam irradiation - Google Patents
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Description
本発明は、イオンビーム照射により、糸状菌ゲノム中の目的遺伝子の機能が破壊された糸状菌の変異株を作出する方法に関する。更に詳細には、目的遺伝子の機能が破壊され、かつ、目的遺伝子の機能を破壊するために用いたマーカー遺伝子全体が欠損した、産業上利用する上で不都合な形質を示さない有用な変異株の作出方法に関する。 The present invention relates to a method for producing a mutant strain of a filamentous fungus in which the function of a target gene in the filamentous fungus genome is disrupted by ion beam irradiation. In more detail, a useful mutant that does not show an inconvenient trait for industrial use, in which the function of the target gene is disrupted and the entire marker gene used for disrupting the function of the target gene is deleted. It relates to the production method.
麹菌などに代表される糸状菌は、醤油、味噌、清酒などの食品の生産はもとより、酵素、医薬品、化成品等の生産・開発において欠くことのできない有用な微生物である。これら糸状菌の有用性をさらに高めるため、紫外線や薬剤などを用いた突然変異誘発法による有用菌株の育種が古くから精力的に行われてきた。しかしながら、突然変異誘発法による育種は、変異率自体が低く、かつ特定遺伝子のみを変異させることができないなどの理由から、目的とする有用菌株を得るまでに非常に多くの労力と時間を必要とした。
ゲノム中の特定遺伝子の機能を確認する方法としては、遺伝子破壊法が既に知られている。遺伝子破壊法の１つの手法では、選抜に用いるためのマーカー遺伝子が目的とする遺伝子配列中に挿入された形のコンストラクトを作成し、このコンストラクトを相同組換えによって当該糸状菌のゲノム中に組み込むことで当該遺伝子の機能を破壊する。遺伝子破壊法を用いれば、糸状菌を産業利用する上で不都合な形質に関わる遺伝子の機能を喪失させ、有用な表現型を示す糸状菌変異株を得ることができる。しかし、遺伝子破壊法では、遺伝子組換えによって外来の生物由来のマーカー遺伝子が糸状菌のゲノムに組み込まれるため、パブリックアクセプタンス等の観点から当該糸状菌を食品産業等に利用できないという問題があった。
Filamentous fungi typified by koji molds are useful microorganisms that are indispensable in the production and development of enzymes, pharmaceuticals, chemical products and the like as well as the production of foods such as soy sauce, miso, and sake. In order to further increase the usefulness of these filamentous fungi, breeding of useful strains by mutagenesis using ultraviolet rays and drugs has been vigorously performed for a long time. However, breeding by mutagenesis requires a great deal of labor and time to obtain the desired useful strain because the mutation rate itself is low and only specific genes cannot be mutated. did.
As a method for confirming the function of a specific gene in the genome, a gene disruption method is already known. In one method of gene disruption, a construct in which a marker gene to be used for selection is inserted into a target gene sequence is prepared, and this construct is incorporated into the genome of the filamentous fungus by homologous recombination. To destroy the function of the gene. By using the gene disruption method, it is possible to obtain a mutant of a filamentous fungus exhibiting a useful phenotype by losing the function of a gene related to a trait that is inconvenient for industrial use of the filamentous fungus. However, the gene disruption method has a problem that a marker gene derived from a foreign organism is incorporated into the genome of the filamentous fungus by genetic recombination, so that the filamentous fungus cannot be used in the food industry or the like from the viewpoint of public acceptance.
一方、イオンビームは、サイクロトロンやシンクロトロンなどの加速器を使って様々なイオンを高速に加速したものである。このイオンビームを生物に照射した場合、イオンの飛跡に沿って高いエネルギーが放出されることから、ＤＮＡの２本鎖切断などが局所的に起こるといわれている。
イオンビームを生物の育種に用いた例としては、植物の例が数多く報告されていたが、近年、糸状菌を材料にしたものも知られている（特許文献１、非特許文献１〜３）。すなわち、特許文献１には、紅麹菌などの糸状菌の分生子に重イオンビームを照射し、これを培養して形成した糸状菌の中から変異の生じた糸状菌を選抜することを特徴とする、突然変異糸状菌の作出方法が開示されている。また、非特許文献１には、アスペルギルス・ソーヤ（Aspergillus sojae）及びアスペルギルス・オリゼ（A.oryzae）にイオンビームを照射することで、プロテアーゼの生産量が多く、かつ増殖率や酵素活性の低下等を示さない変異株を得たことが述べられている。さらに、非特許文献２には、アスペルギルス・アワモリ（A.awamori）にイオンビームを照射することでα−アミラーゼ活性の強い変異株を得たことが記載されている。
また、非特許文献３には、アスペルギルス・オリゼに炭素イオンビームを照射した後にセレン酸耐性を示す変異株を選抜したこと、当該耐性株の中から、セレン酸耐性の原因遺伝子の候補であるｓＢ、ｓＣ遺伝子について欠損などの大規模な変異が生じた株をさらにスクリーニングしたことが記載されている。
Many examples of plants have been reported as examples of using ion beams for biological breeding, but in recent years, those using filamentous fungi as a material are also known (Patent Document 1, Non-Patent Documents 1 to 3). . That is, Patent Document 1 is characterized in that a filamentous fungus having a mutation is selected from filamentous fungi formed by irradiating a conidia of a filamentous fungus such as red yeast with a heavy ion beam and culturing it. A method for producing mutant filamentous fungi is disclosed. Non-Patent Document 1 discloses that aspergillus sojae and Aspergillus oryzae are irradiated with an ion beam, thereby producing a large amount of protease and reducing the growth rate and enzyme activity. It has been stated that a mutant strain that does not show is obtained. Further, Non-Patent Document 2 describes that a mutant strain having a strong α-amylase activity was obtained by irradiating an Aspergillus awamori with an ion beam.
In Non-Patent Document 3, a mutant strain exhibiting selenate resistance was selected after irradiating Aspergillus oryzae with a carbon ion beam, and sB, which is a candidate gene responsible for selenate resistance, was selected from the resistant strains. Furthermore, it is described that a strain in which a large-scale mutation such as a deletion has occurred in the sC gene was further screened.
従来の遺伝子破壊法は、上述したように、様々な欠点を克服し、実用に耐え得る方法ではないことから、糸状菌中の特定遺伝子を欠損させる実用的な方法の確立が切望されていた。
本発明者らは、糸状菌中の特定の目的遺伝子を効率的に欠損させる手段として、イオンビームによる照射を利用できるのではないかと考えた。しかし、上記イオンビームに関する文献のいずれにおいても、イオンビーム照射の後の変異株は酵素活性の強さや有用物質の生産性という表現型の指標のみを用いて選抜されるものであり、糸状菌ゲノム中の特定の目的遺伝子の機能が破壊されたことを指標として選抜を行う方法はこれまで知られていなかった。
As described above, since the conventional gene disruption method is not a method that can overcome various disadvantages and can be used practically, establishment of a practical method for deleting a specific gene in a filamentous fungus has been desired.
The present inventors thought that irradiation with an ion beam could be used as a means for efficiently deleting a specific target gene in a filamentous fungus. However, in any of the above documents related to ion beams, mutant strains after ion beam irradiation are selected using only phenotypic indicators such as the strength of enzyme activity and the productivity of useful substances. Until now, no method has been known for selection based on the destruction of the function of a specific target gene.
本発明者らは、糸状菌中の特定遺伝子を欠損させ、かつ外来生物由来のマーカー遺伝子を当該糸状菌のゲノム中に存在させない実用的な方法を確立すべく鋭意検討を行った結果、遺伝子組換えによって糸状菌のゲノム中の機能を破壊しようとする任意の遺伝子の配列内にマーカー遺伝子を挿入し、当該マーカー遺伝子をイオンビーム照射によって欠損させることで、従来の遺伝子破壊法と同様に任意の遺伝子の機能が破壊されていながら、しかもゲノム中に外来のマーカー遺伝子を全く含まない遺伝子突然変異株を取得できることを見出し、本発明を完成するに至った。 As a result of intensive studies to establish a practical method in which a specific gene in a filamentous fungus is deleted and a marker gene derived from a foreign organism is not present in the genome of the filamentous fungus, By inserting a marker gene into the sequence of an arbitrary gene whose function in the genome of the filamentous fungus is to be destroyed by replacement, and deleting the marker gene by ion beam irradiation, an arbitrary gene similar to the conventional gene disruption method is used. The inventors have found that a gene mutant strain in which the function of a gene is destroyed and which does not contain any foreign marker gene in the genome can be obtained, and the present invention has been completed.
すなわち、本発明は、
[１] 糸状菌の変異株を作出する方法において、
（ａ）糸状菌ゲノム中の機能を破壊しようとする目的遺伝子内にマーカー遺伝子を挿入させる工程、
（ｂ）マーカー遺伝子を挿入した当該糸状菌にイオンビームを照射し、変異を誘発する工程、
（ｃ）イオンビーム照射後の糸状菌の中から、変異によってマーカー遺伝子の機能が欠損した株をスクリーニングする工程、
（ｄ）スクリーニングされた株において、マーカー遺伝子を含む外来遺伝子全体がイオンビーム照射によって欠損し、かつ目的遺伝子の機能が破壊されていることを確認する工程、
を含む、糸状菌ゲノム中の目的遺伝子の機能を破壊することで有用な変異株を作出する方法；
[２] 糸状菌が麹菌（Aspergillus 属）である上記[１]に記載の方法；
[３] 工程（ａ）において、遺伝子置換破壊法または遺伝子挿入破壊法により、糸状菌ゲノム中の機能を破壊しようとする目的遺伝子内にマーカー遺伝子を挿入させる上記[１]または[２]に記載の方法；
[４] 工程（ｂ）において、炭素イオンビームを２００〜６００Ｇｒａｙの照射量で照射する上記[１]から[３]のいずれかに記載の方法；
[５] 糸状菌ゲノム中の機能を破壊しようとする目的遺伝子が、ａｍｙＲ遺伝子、ａｆｌＲ遺伝子またはｘｌｎＲ遺伝子である上記[１]から[４]のいずれかに記載の方法；および
[６] マーカー遺伝子が、ｓＣ遺伝子、ｎｉａＤ遺伝子、ｐｙｒＧ遺伝子またはｐｔｒＡ遺伝子である上記[１]から[５]のいずれかに記載の方法
に関するものである。
That is, the present invention
[1] In a method for producing a mutant of a filamentous fungus,
(A) a step of inserting a marker gene into a target gene whose function in the genome of the filamentous fungus is to be disrupted;
(B) irradiating the filamentous fungus into which the marker gene has been inserted with an ion beam to induce mutation;
(C) screening a strain deficient in the function of the marker gene due to mutation from among filamentous fungi after ion beam irradiation;
(D) in the screened strain, confirming that the entire foreign gene including the marker gene is deleted by ion beam irradiation and that the function of the target gene is destroyed;
A method for producing a useful mutant strain by disrupting the function of a target gene in a filamentous fungus genome,
[2] The method according to [1] above, wherein the filamentous fungus is Aspergillus (genus Aspergillus);
[3] The method according to [1] or [2] above, wherein in the step (a), a marker gene is inserted into a target gene whose function in the genome of the filamentous fungus is to be disrupted by a gene replacement disruption method or a gene insertion disruption method. the method of;
[4] The method according to any one of [1] to [3] above, wherein in step (b), the carbon ion beam is irradiated at a dose of 200 to 600 Gray;
[5] The method according to any one of [1] to [4] above, wherein the target gene whose function in the genome of the filamentous fungus is to be destroyed is an amyR gene, an aflR gene, or an xlnR gene;
[6] The method according to any one of [1] to [5], wherein the marker gene is an sC gene, a niaD gene, a pyrG gene, or a ptrA gene.
本発明の方法によれば、特定の遺伝子の機能が破壊されたことによって、産業利用する上で不都合な形質を示さない糸状菌の有用菌株を得ることができる。しかも従来の遺伝子破壊法とは異なり、当該菌株のゲノムには外来生物由来のマーカー遺伝子等が含まれないため、得られた菌株を食品産業等に利用することも可能である。 According to the method of the present invention, it is possible to obtain a useful strain of a filamentous fungus that does not show an inconvenient trait for industrial use due to the destruction of the function of a specific gene. In addition, unlike the conventional gene disruption method, since the genome of the strain does not contain a marker gene or the like derived from a foreign organism, the obtained strain can be used in the food industry and the like.
本発明方法は、上記の（ａ）〜（ｄ）の工程を含む糸状菌ゲノム中の目的遺伝子の機能を破壊し、有用な変異株を作出する方法に関するものであり、以下に工程毎に詳細に説明する。 The method of the present invention relates to a method for destroying the function of a target gene in a filamentous fungus genome including the steps (a) to (d) described above and creating a useful mutant strain. Explained.
（ａ）糸状菌ゲノム中の機能を破壊しようとする目的遺伝子内にマーカー遺伝子を挿入させる工程：
本発明で用いる糸状菌は、菌糸を形成する生物であればいかなるものでもよい。中でも麹菌（Aspergillus属）が好ましく、さらには食品産業でよく用いられるアスペルギルス・オリゼ（A.oryzae）、アスペルギルス・ソーヤ（A.sojae）、アスペルギルス・アワモリ（A.awamori）などが特に好ましいが、これに限定されるものではない。
本発明では、まず、当該糸状菌のゲノム中の破壊したい遺伝子の領域中にマーカー遺伝子を挿入させる。ターゲットとする遺伝子は、糸状菌ゲノム中に含まれるものであれば、いかなるものでもよい。中でも、当該遺伝子の機能を破壊することによって糸状菌の産業上の有用性が向上するようなものが好ましい。例としては、毒性物質であるアフラトキシンの生合成酵素をコードするａｆｌＲ遺伝子や、α−アミラーゼの発現を正に制御する転写因子をコードしており、機能を破壊した変異株は淡色醤油の製造に有用となるａｍｙＲ遺伝子、キシラナーゼの発現を制御する転写因子をコードし、機能を破壊した株を用いることによって淡色醤油を効率的に生産できるｘｌｎＲ遺伝子などが挙げられる。
挿入するマーカー遺伝子は、遺伝子組換えのスクリーニングの指標として使用できるような遺伝子であれば、いかなるものでもよい。例としては、挿入後の株が亜硝酸非要求性株となるｎｉａＤ遺伝子、メチオニン非要求性株となるｓＣ遺伝子、ウラシル非要求性株となるｐｙｒＧ遺伝子、ピリチアミン耐性となるｐｔｒＡ遺伝子などが利用することができる。
また、使用する糸状菌とマーカー遺伝子の組み合わせは、マーカー遺伝子がゲノム中の適切な部位に組み込まれたこと、およびイオンビーム照射によって当該マーカー遺伝子が欠損したことの双方を、それぞれスクリーニングによって評価できるようなものであることが好ましい。例えばマーカー遺伝子が、特定の物質への耐性ないしは感受性を付与する遺伝子であって、かつ当該糸状菌が、マーカー遺伝子と同様の機能をもつ遺伝子を有していないもの、あるいは機能を喪失しているものなどが挙げられる。
(A) A step of inserting a marker gene into a target gene whose function in the genome of the filamentous fungus is to be destroyed :
The filamentous fungus used in the present invention may be any organism that forms a mycelium. Of these, Aspergillus is preferable, and Aspergillus oryzae (A.oryzae), Aspergillus sojae (A.sojae), and Aspergillus awamori (A.awamori), which are often used in the food industry, are particularly preferable. It is not limited to.
In the present invention, first, a marker gene is inserted into the region of the gene to be disrupted in the genome of the filamentous fungus. The target gene may be any gene as long as it is contained in the filamentous fungus genome. Among them, those that improve the industrial utility of the filamentous fungus by destroying the function of the gene are preferable. Examples include the aflR gene, which encodes the biosynthetic enzyme of aflatoxin, which is a toxic substance, and a transcription factor that positively regulates the expression of α-amylase. Examples include an amyR gene that is useful, and an xlnR gene that can efficiently produce light-colored soy sauce by using a strain that encodes a transcription factor that controls the expression of xylanase and disrupts its function.
The marker gene to be inserted may be any gene as long as it can be used as an index for screening for genetic recombination. Examples include the niaD gene in which the inserted strain is a nitrite non-requiring strain, the sC gene that is a methionine non-requiring strain, the pyrG gene that is a uracil non-requiring strain, the ptrA gene that is resistant to pyrithiamine, etc. be able to.
In addition, the combination of the filamentous fungus and the marker gene to be used can be evaluated by screening both of the fact that the marker gene has been incorporated into an appropriate site in the genome and that the marker gene has been deleted by ion beam irradiation. It is preferable that it is a thing. For example, a marker gene is a gene that confers resistance or sensitivity to a specific substance, and the filamentous fungus does not have a gene having the same function as the marker gene, or has lost its function Things.
目的遺伝子内にマーカー遺伝子を挿入する方法は、糸状菌の形質転換に利用できる方法であればいかなるものでもよい。遺伝子破壊法で通常用いられるような方法に準じればよく、例としては機能を破壊したい目的遺伝子の断片とマーカー遺伝子とを組み合わせたようなＤＮＡ断片を適当なベクターに組み込み、プロトプラスト−ＰＥＧ法やエレクトロポレーション法などによってベクターを糸状菌に取り込ませ、当該ＤＮＡ断片を糸状菌のゲノム中に導入する方法などを挙げることができるが、マーカー遺伝子が適切な位置に適切に導入される方法であればこれに限定されない。 Any method can be used for inserting the marker gene into the target gene as long as it can be used for transformation of filamentous fungi. For example, a DNA fragment such as a combination of a fragment of a target gene whose function is to be disrupted and a marker gene is incorporated into an appropriate vector, and the protoplast-PEG method or the like. Examples thereof include a method of incorporating a vector into a filamentous fungus by electroporation and the like, and introducing the DNA fragment into the genome of the filamentous fungus. It is not limited to this.
このような方法としては、より具体的には、遺伝子置換破壊法または遺伝子挿入破壊法が挙げられる。遺伝子置換破壊法については、図１の下段の左側に模式的に示されており、図２には、機能を破壊したい目的遺伝子としてａｍｙＲ遺伝子、マーカー遺伝子としてｓＣ遺伝子を用いた、遺伝子置換破壊法に使用するベクターの例が示されている。これらの図に示されているように、マーカー遺伝子の５’末端側および３’末端側のそれぞれに、機能を破壊したい目的遺伝子の少なくとも５’末端側および３’末端側の配列を含む遺伝子配列を結合させた領域を含むベクターを構築し、このベクターで糸状菌を形質転換して、相同組換えにより、糸状菌のゲノム中の機能を破壊したい目的遺伝子内にマーカー遺伝子を挿入することができる。
遺伝子挿入破壊法については、図３に、機能を破壊したい目的遺伝子としてａｍｙＲ遺伝子、マーカー遺伝子としてｓＣ遺伝子を用いた例が模式的に示されている。図３に示すように、目的遺伝子の一部分とマーカー遺伝子とを隣接して含むベクターを構築し、このベクターで糸状菌を形質転換することによって、開裂したベクターの全配列を糸状菌のゲノム中に導入させ、糸状菌のゲノム中の機能を破壊したい目的遺伝子内に、ベクター由来の遺伝子と共に、マーカー遺伝子を挿入することができる。この遺伝子挿入破壊法については、O.Yamada et.al.（J.Biosci.Bioeng.95(1),82-88,2003）等の文献が参考とされる。
More specifically, such a method includes a gene replacement disruption method or a gene insertion disruption method. The gene replacement disruption method is schematically shown on the left side of the lower part of FIG. 1, and FIG. 2 shows a gene replacement disruption method using an amyR gene as a target gene whose function is to be disrupted and an sC gene as a marker gene. Examples of vectors used are shown in FIG. As shown in these figures, a gene sequence comprising at least the 5 ′ end side and the 3 ′ end side sequence of the target gene whose function is desired to be destroyed on the 5 ′ end side and 3 ′ end side of the marker gene, respectively. By constructing a vector containing the region to which the ligase is bound, transforming a filamentous fungus with this vector, a marker gene can be inserted into the target gene whose function in the genome of the filamentous fungus is to be disrupted by homologous recombination .
Regarding the gene insertion disruption method, FIG. 3 schematically shows an example in which the amyR gene is used as a target gene whose function is to be disrupted and the sC gene is used as a marker gene. As shown in FIG. 3, by constructing a vector containing a part of the target gene and a marker gene adjacent to each other, and transforming the filamentous fungus with this vector, the entire sequence of the cleaved vector is put into the genome of the filamentous fungus. A marker gene can be inserted together with a vector-derived gene into a target gene to be introduced and to destroy the function in the genome of the filamentous fungus. For this gene insertion disruption method, references such as O. Yamada et.al. (J. Biosci. Bioeng. 95 (1), 82-88, 2003) are referred to.
（ｂ）マーカー遺伝子を挿入した当該糸状菌にイオンビームを照射し、変異を誘発する工程：
次に、上記の工程（ａ）で得られる、糸状菌のゲノム中の機能を破壊したい目的遺伝子内にマーカー遺伝子が挿入された形質転換体に対してイオンビーム照射を行う。
イオンビームの種類は、当該糸状菌に変異を与えられるようなものであればいかなるものでも用いることができる。例えば、炭素イオンビーム、アルゴンイオンビーム、窒素イオンビームなどを挙げることができる。
イオンビームの照射は、当該形質転換体の凍結乾燥分生子を作成し、これに対して行うのが好ましいが、イオンビーム照射による変異誘発が適切に行われる方法であれば、これに限定されない。
イオンビームの照射量は、当該糸状菌に過度の損傷を与えず、かつ変異を与えられるような範囲であれば特に限定されるものではない。例えばアスペルギルス・オリゼ（Aspergillus oryzae）に炭素イオンビームを照射する場合、好ましい照射量の範囲として２００〜６００Ｇｒａｙ、特に好ましい範囲として約３００〜５００Ｇｒａｙを挙げることができるが、これに限定されるものではない。
特に、本発明では、炭素イオンビームを２００〜６００Ｇｒａｙ、更には約３００〜５００Ｇｒａｙの照射量で照射する方法が好ましい。
(B) Irradiating the filamentous fungus into which the marker gene has been inserted with an ion beam to induce mutation :
Next, ion beam irradiation is performed on the transformant obtained by the above step (a) in which the marker gene is inserted into the target gene whose function in the genome of the filamentous fungus is desired to be destroyed.
Any kind of ion beam can be used as long as it can mutate the filamentous fungus. For example, a carbon ion beam, an argon ion beam, a nitrogen ion beam, etc. can be mentioned.
Irradiation with an ion beam is preferably carried out by producing freeze-dried conidia of the transformant, but is not limited to this as long as mutagenesis by ion beam irradiation is appropriately performed.
The irradiation amount of the ion beam is not particularly limited as long as it does not cause excessive damage to the filamentous fungus and can be mutated. For example, when irradiating a carbon ion beam to Aspergillus oryzae, a preferable irradiation range is 200 to 600 Gray, and a particularly preferable range is about 300 to 500 Gray, but is not limited thereto. .
In particular, in the present invention, a method of irradiating a carbon ion beam with a dose of 200 to 600 Gray, more preferably about 300 to 500 Gray is preferable.
（ｃ）イオンビーム照射後の糸状菌の中から、変異によってマーカー遺伝子の機能が欠損した株をスクリーニングする工程：
変異株のスクリーニング方法としては、導入したマーカー遺伝子の種類および用いた糸状菌株の特性に準じ、それぞれに適切な方法を用いればよい。
例えば、導入したマーカー遺伝子が薬剤耐性を付与するものである場合、イオンビーム照射後、当該分生子を培養し、寒天培地上などコロニーを形成させた後、適当な薬剤を含む培地上に菌株を移植し、感受性を示す株のみを選抜するなどの方法があるが、これに限定されるものではない。
(C) screening a strain deficient in the function of a marker gene from mutations among filamentous fungi after irradiation with an ion beam :
As a screening method for mutant strains, an appropriate method may be used for each according to the type of the marker gene introduced and the characteristics of the filamentous strain used.
For example, when the introduced marker gene confers drug resistance, the conidia is cultured after irradiation with an ion beam, colonies such as on an agar medium are formed, and then the strain is placed on a medium containing an appropriate drug. There are methods such as selecting only strains that are transplanted and exhibit sensitivity, but are not limited thereto.
（ｄ）スクリーニングされた株において、マーカー遺伝子を含む外来遺伝子全体がイオンビーム照射によって欠損し、かつ目的遺伝子の機能が破壊されていることを確認する工程：
本発明においては、食品産業等に使用する観点から、最終的に得られる変異株が、外来種由来のマーカー遺伝子全体が完全に欠損していることが必要である。
変異株のゲノムにおいて、マーカー遺伝子が完全に欠損していることを確認する手段は、分子生物学で通常用いられる方法であればどのようなものでもよい。例として、ＰＣＲ法、サザンブロッティング法、塩基配列のシーケンシングなどの方法を挙げることができる。また、本発明では、得られる変異株においては、目的遺伝子の機能が破壊されている必要があり、従って、目的遺伝子の機能が破壊されていれば、目的遺伝子の一部が残っていても構わない。目的遺伝子の機能が破壊されていることを確認する手段は、目的遺伝子の機能に応じて任意に採用することができる。例えば、機能を破壊したい目的遺伝子としてａｍｙＲ遺伝子を選択した場合には、変異株のデンプン分解能を解析すればよい。ａｆｌＲ遺伝子を選択した場合には、毒性物質であるアフラトキシンが生成されないことを確認すればよい。ｘｌｎＲ遺伝子を選択した場合には、例えば、変異株のキシラナーゼ活性を測定すればよい。
(D) A step of confirming that the entire foreign gene including the marker gene is deleted by ion beam irradiation in the screened strain and that the function of the target gene is destroyed :
In the present invention, from the viewpoint of use in the food industry and the like, it is necessary that the finally obtained mutant strain is completely deficient in the entire marker gene derived from the foreign species.
The means for confirming that the marker gene is completely deficient in the genome of the mutant strain may be any method that is usually used in molecular biology. Examples include PCR, Southern blotting, and nucleotide sequence sequencing. Further, in the present invention, in the obtained mutant strain, the function of the target gene needs to be disrupted. Therefore, if the function of the target gene is disrupted, a part of the target gene may remain. Absent. A means for confirming that the function of the target gene is destroyed can be arbitrarily adopted depending on the function of the target gene. For example, when the amyR gene is selected as the target gene whose function is to be disrupted, the starch resolution of the mutant strain may be analyzed. When the aflR gene is selected, it may be confirmed that aflatoxin, which is a toxic substance, is not produced. When the xlnR gene is selected, for example, the xylanase activity of the mutant strain may be measured.
以下、実施例によって本発明をさらに具体的に説明するが、これらの実施例は本発明を限定するものではない。 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but these examples do not limit the present invention.
実施例１
炭素イオンビーム照射を用いた、アスペルギスル・オリゼ（Aspergillus oryzae）におけるａｍｙＲ遺伝子欠損株の作出
図１に示すように、麹菌の機能を破壊したい目的遺伝子としてａｍｙＲ遺伝子を選択し、マーカー遺伝子としてｓＣ遺伝子を用いて、遺伝子置換破壊法により、ａｍｙＲ遺伝子内にｓＣ遺伝子を挿入して、炭素イオンビーム照射を行い、ｓＣ遺伝子全体が完全に欠損し、かつ、ａｍｙＲ遺伝子の機能が破壊された変異株を作出した。
Example 1
Production of an amyR gene-deficient strain in Aspergillus oryzae using carbon ion beam irradiation As shown in FIG. 1, an amyR gene is selected as a target gene for which the function of Neisseria gonorrhoeae is to be disrupted, and an sC gene is selected as a marker gene Using the gene replacement disruption method, the sC gene is inserted into the amyR gene, carbon ion beam irradiation is performed, and a mutant strain in which the entire sC gene is completely deleted and the function of the amyR gene is disrupted is generated. did.
（１）マーカー遺伝子の導入
ｓＣ遺伝子はメチオニンの生合成に関わる遺伝子である一方、セレン酸が体内に取り込まれた際には、これを用いて毒性物質を生合成する経路の一部を担う。このため、ｓＣ遺伝子が正常に働く菌株はセレン酸存在下で毒性物質を生じ致死となるが、同遺伝子の機能が失われている株はセレン酸耐性を示す。
実験には、麹菌アスペルギルス・オリゼＮＳ４株を用いた。ＮＳ４株は、野生型のアスペルギルス・オリゼに紫外線照射を行うことで得られたｓＣ遺伝子の機能欠損変異株である（O.Yamada et. al. Biosci.Biotech.Biochem.61(8),1367-1369,1997）。
(1) Introduction of marker gene The sC gene is a gene involved in the biosynthesis of methionine. On the other hand, when selenate is taken into the body, it takes part of the pathway for biosynthesis of toxic substances. For this reason, a strain in which the sC gene works normally produces a toxic substance and is lethal in the presence of selenate, but a strain in which the function of the gene is lost exhibits selenate resistance.
In the experiment, Aspergillus oryzae NS4 strain was used. NS4 strain is a sC gene function-deficient mutant obtained by irradiating wild-type Aspergillus oryzae with ultraviolet light (O. Yamada et. Al. Biosci. Biotech. Biochem. 61 (8), 1367- 1369, 1997).
図２に示すように、遺伝子置換破壊法により、ＮＳ４株のａｍｙＲ遺伝子内にｓＣ遺伝子を挿入させるために用いる形質転換用のベクターを構築した。すなわち、アスペルギルス・オリゼ由来のａｍｙＲ遺伝子を含むｐＧＬ２０ベクター（K.Gomi et.al. Biosci.Biotechnol.Biochem.64(4),816-827,2000）を制限酵素ＫｐｎＩおよびＨｉｎｄＩＩＩで切断し、切断後の断片をｐＵＣ１１８ベクター（タカラバイオ）に組み込んでクローニングを行った。
ａｍｙＲ遺伝子の断片を含む上記ベクターと、アスペルギルス・ニドゥランス由来のｓＣ遺伝子の断片を含むｐＵＳＡベクター（O.Yamada et.al. J.Biosci.Bioeng.95(1),82-88,2003）のそれぞれを、制限酵素ＸｂａＩおよびＰｓｔＩで切断し、両者をライゲーションすることによって、形質転換に用いる目的とするｐＡＭＤＲベクターを作製した（図２）。
このｐＡＭＤＲベクターをプロトプラスト−ＰＥＧ法によってアスペルギルス・オリゼＮＳ４株に形質転換し、相同組換を行わせることによって、ａｍｙＲ遺伝子領域中にアスペルギルス・ニドゥランス由来のｓＣ遺伝子が挿入されている変異株（以下、ΔａｍｙＲ株という）を得た。
As shown in FIG. 2, a transformation vector used for inserting the sC gene into the amyR gene of the NS4 strain was constructed by the gene replacement disruption method. That is, the pGL20 vector (K. Gomi et.al. Biosci. Biotechnol. Biochem. 64 (4), 816-827, 2000) containing the amyR gene derived from Aspergillus oryzae was digested with restriction enzymes KpnI and HindIII. Was cloned into the pUC118 vector (Takara Bio).
Each of the above vector containing the fragment of the amyR gene and the pUSA vector (O. Yamada et.al. J. Biosci. Bioeng. 95 (1), 82-88, 2003) containing the sC gene fragment derived from Aspergillus nidulans Was cleaved with restriction enzymes XbaI and PstI, and both were ligated to produce a target pAMDR vector used for transformation (FIG. 2).
By transforming this pAMDR vector into Aspergillus oryzae NS4 strain by protoplast-PEG method and performing homologous recombination, a mutant strain in which the sC gene derived from Aspergillus nidulans is inserted into the amyR gene region (hereinafter, (Referred to as ΔamyR strain).
（２）イオンビーム照射
麹汁培地でΔａｍｙＲ株を３０℃、１週間培養し、当該菌体へ０．０５％ツィーン溶液を加え、分生子懸濁液を得た。分生子懸濁液を遠心後、得られた分生子を凍結乾燥用懸濁液（バクトペプトン１％（ｗ／ｖ、以下同様）、グリセロール１％）に懸濁した。分生子５×１０７個程度を０．２μｍのメンブレンフィルターに吸着させ、凍結乾燥を行った。凍結乾燥は所定の方法に従って行った。
イオンビーム照射は（独）日本原子力研究開発機構において行った。照射はＡＶＦサイクロトロンを用いて１２Ｃ５＋を加速し、２２０ＭｅＶのエネルギーで３００〜７００Ｇｒａｙ照射した。イオンビームの照射量については、非特許文献３に記載の範囲を参考にした。
(2) Ion beam irradiation The ΔamyR strain was cultured in a broth medium at 30 ° C. for 1 week, and 0.05% Tween solution was added to the cells to obtain a conidial suspension. After the conidia suspension was centrifuged, the obtained conidia was suspended in a suspension for lyophilization (bactopeptone 1% (w / v, the same applies hereinafter), glycerol 1%). About 5 × 10 7 conidia were adsorbed on a 0.2 μm membrane filter and lyophilized. Lyophilization was performed according to a predetermined method.
Ion beam irradiation was performed at the Japan Atomic Energy Agency. Irradiation was accelerated at 12 C 5+ using an AVF cyclotron and irradiated at 300 to 700 Gray with an energy of 220 MeV. Regarding the irradiation amount of the ion beam, the range described in Non-Patent Document 3 was referred to.
（３）変異株のスクリーニング
イオンビーム照射により生存率が１〜１０％となった麹菌の分生子約３０００００株をセレン酸培地に播種した。生育してきたコロニーをセレン酸培地（スクロース２％、硝酸ナトリウム０．３％、リン酸水素二カリウム０．１％、酢酸マグネシウム０．０５％、塩化カリウム０．０５％、メチオニン３０ｐｐｍ、トレースエレメント０．１％、セレン酸ナトリウム０．１ｍＭ；ｐＨ６．０）に２〜３回植え継ぎ変異固定を行ったところ、約３０００００株の中から４７株がセレン酸耐性株として取得された。
なお、本明細書中において「トレースエレメント」とは以下に示した組成の水溶液のことを指す。
トレースエレメント組成：硫酸鉄七水和物０．１％、硫酸亜鉛七水和物０．８８％、硫酸銅五水和物０．０４％、硫酸マンガン四水和物０．０１５％、四ホウ酸ナトリウム十水和物０．０１％、七モリブデン酸六アンモニウム四水和物０．００５％。
(3) Screening of mutant strains About 300,000 conidia of Aspergillus oryzae having a survival rate of 1 to 10% by ion beam irradiation were seeded in a selenate medium. The grown colonies were treated with selenate medium (sucrose 2%, sodium nitrate 0.3%, dipotassium hydrogen phosphate 0.1%, magnesium acetate 0.05%, potassium chloride 0.05%, methionine 30 ppm, trace element 0 .1%, sodium selenate 0.1 mM; pH 6.0) After 2 to 3 transplantation mutations were fixed, 47 strains out of about 300,000 strains were obtained as selenate resistant strains.
In this specification, “trace element” refers to an aqueous solution having the composition shown below.
Trace element composition: iron sulfate heptahydrate 0.1%, zinc sulfate heptahydrate 0.88%, copper sulfate pentahydrate 0.04%, manganese sulfate tetrahydrate 0.015%, tetraborate Sodium acetate decahydrate 0.01%, hexamolybdate hexammonium tetrahydrate 0.005%.
さらに、セレン酸からの毒性物質の生合成経路には、ｓＣ遺伝子以外にも他の遺伝子が関わっていることが知られており、他方、ｓＣ遺伝子はセレン酸以外にもクロム酸から毒性物質の生合成経路にも関与していることから、当該工程で得たセレン酸耐性株について、ｓＣ遺伝子が機能を失っていることを更に確認するために、クロム酸耐性についても更に試験した。すなわち、クロム酸培地（スクロース２％、硝酸ナトリウム０．３％、リン酸水素二カリウム０．１％、酢酸マグネシウム０．０５％、塩化カリウム０．０５％、メチオニン３０ｐｐｍ、トレースエレメント０．１％、クロム酸０．５ｍＭ；ｐＨ６．０）に植え継ぎ、感受性を示した株をｓＣ変異株とした。クロム酸に対して感受性を示した株を選抜することにより、ｓＣ遺伝子以外の遺伝子の変異によってセレン酸耐性が付与された株を除去され、ｓＣ遺伝子が機能を失っている可能性がより高い変異株を得ることができた。その結果、表１に示したように、選抜によって、ｓＣ変異株が２８株得られた。 Furthermore, it is known that other genes besides sC gene are involved in the biosynthetic pathway of toxic substances from selenate. On the other hand, sC gene is not only selenate but also chromic acid. Since it is also involved in the biosynthetic pathway, the selenate-resistant strain obtained in this step was further tested for chromic acid resistance in order to further confirm that the sC gene has lost its function. Chromic acid medium (sucrose 2%, sodium nitrate 0.3%, dipotassium hydrogen phosphate 0.1%, magnesium acetate 0.05%, potassium chloride 0.05%, methionine 30 ppm, trace element 0.1% , Chromic acid 0.5 mM; pH 6.0), and the strain showing sensitivity was designated as sC mutant. By selecting a strain showing sensitivity to chromic acid, a strain to which selenate resistance is imparted by mutation of a gene other than the sC gene is removed, and the sC gene is more likely to lose its function. I was able to get a stock. As a result, as shown in Table 1, 28 sC mutants were obtained by selection.
（４）スクリーニングによって得た菌株におけるマーカー欠損の確認
次に、得られたｓＣ変異株２８株よりゲノムを抽出し、ＰＣＲ解析を行った。
図４および５に示したように、マーカー遺伝子として挿入したアスペルギルス・ニドゥランスのｓＣ遺伝子と、宿主であるアスペルギルス・オリゼのマーカー挿入部位であるａｍｙＲ遺伝子の各々の配列を用い、プライマー１とプライマー２の２通りのプライマーセットを作成した。各プライマーの塩基配列は、表２に示したとおりである。ＰＣＲ解析にはＥｘ Ｔａｑ（タカラバイオ）を用い、反応条件は、表３の通りとした。
(4) Confirmation of marker deficiency in strains obtained by screening Next, genomes were extracted from the obtained 28 sC mutants and PCR analysis was performed.
As shown in FIGS. 4 and 5, using the sequences of Aspergillus nidulans sC gene inserted as a marker gene and amyR gene as a marker insertion site of host Aspergillus oryzae, primers 1 and 2 Two kinds of primer sets were prepared. The base sequence of each primer is as shown in Table 2. Ex Taq (Takara Bio) was used for PCR analysis, and the reaction conditions were as shown in Table 3.
プライマー１を用いたＰＣＲでは２８株中７株について、ａｍｙＲ遺伝子の５’末端配列とｓＣ遺伝子の５’末端配列が結合した１．８ｋｂＤＮＡ断片の増幅が認められず、挿入されたマーカー領域について遺伝子欠損が起きていることが考えられた（図４）。増幅が確認されなかった７株について、さらにプライマー２を用いてＰＣＲを行ったところ、７株中１株について、ａｍｙＲ遺伝子の３’末端配列とｓＣ遺伝子の３’末端配列が結合した１．１ｋｂＤＮＡ断片の増幅が認められなかった（Ｎｏ．９株）。このことから、当該株は挿入されたｓＣ遺伝子の全領域が欠損していることが考えられた（図５）。
この株についてゲノミックサザン解析を行った。プローブの標識にはＤｉｇ−Ｈｉｇｈ Ｐｒｉｍｅ（ロシュ・ダイアグノスティックス）を用い、方法は所定のプロトコルに従った。プローブには、遺伝子組換えに用いたマーカー遺伝子の全領域をカバーした、表２に示した配列を有するプライマー３で増幅されるＤＮＡ断片を用いた。ハイブリダイゼーションにはＤｉｇ Ｅａｓｙ Ｈｙｂ（ロシュ・ダイアグノスティックス）を用い、方法は所定のプロトコルに従った。その結果、ＮＳ４株およびΔａｍｙＲ株については、遺伝子組換えにより挿入されたマーカー遺伝子由来のバンドが、予想される位置に検出された。しかし、ｓＣ変異株においてはバンドの検出が認められない株があった（Ｎｏ．９株）。また、いずれの株においても、アスペルギルス・オリゼ由来の内在性ｓＣ遺伝子が検出された（図６）。
以上より、ハイブリダイゼーションは正常に行われているものの、マーカー遺伝子由来のバンドは検出されなかったことから、イオンビーム照射により、マーカー遺伝子全体がゲノム内から完全に除去されたことが確認された（Ｎｏ．９株）。
In PCR using primer 1, amplification of a 1.8 kb DNA fragment in which the 5 ′ end sequence of the amyR gene and the 5 ′ end sequence of the sC gene were combined was not observed in 7 out of 28 strains. It was considered that a defect occurred (FIG. 4). PCR was performed on 7 strains in which amplification was not confirmed, and primer 2 was used. As a result, 1.1 kb DNA in which the 3 ′ end sequence of the amyR gene and the 3 ′ end sequence of the sC gene were combined in 1 strain out of 7 strains. No fragment amplification was observed (No. 9 strain). From this, it was considered that this strain is deficient in the entire region of the inserted sC gene (FIG. 5).
Genomic Southern analysis was performed on this strain. Dig-High Prime (Roche Diagnostics) was used for the labeling of the probe, and the method followed a predetermined protocol. As the probe, a DNA fragment amplified with the primer 3 having the sequence shown in Table 2 and covering the entire region of the marker gene used for gene recombination was used. For hybridization, Dig Easy Hyb (Roche Diagnostics) was used, and the method followed a predetermined protocol. As a result, for NS4 strain and ΔamyR strain, a band derived from a marker gene inserted by gene recombination was detected at the expected position. However, there was a strain in which no band was detected in the sC mutant strain (No. 9 strain). In any strain, an endogenous sC gene derived from Aspergillus oryzae was detected (FIG. 6).
From the above, although hybridization was carried out normally, no band derived from the marker gene was detected, so it was confirmed that the entire marker gene was completely removed from the genome by ion beam irradiation ( No. 9 strain).
さらにｓＣ変異株においてａｍｙＲ遺伝子の機能が破壊されているかを調べるため、デンプン分解能の解析を行った。
ＮＳ４株、ΔａｍｙＲ株、ｓＣ変異株（Ｎｏ．９株）をそれぞれ麹汁培地に植菌し、ジャイアントコロニーを形成させた。
このジャイアントコロニーから分生子懸濁液を作成した。分生子の濃度が１０７個／ｍｌとなるように分生子懸濁液を希釈後、当該懸濁液をデンプン培地（デンプン１．０％、亜硝酸ナトリウム０．３％、リン酸水素二カリウム０．１％、硫酸マグネシウム０．０５％、塩化カリウム０．０５％、トレースエレメント０．１％、メチオニン０．１５％）に植菌し、３０℃で４日間培養した。なお、デンプン培地については培養後、ヨウ素を加え、ヨウ素デンプン反応によりハローの形成差を明確にした。
その結果、ＮＳ４株は良好な生育を示し、大きなハローを形成した一方で、ΔａｍｙＲ株、ｓＣ変異株は生育が悪く、ハローもほとんど形成されなかった（図７）。すなわち、ΔａｍｙＲ株、ｓＣ変異株（Ｎｏ．９株）はデンプン分解能を失っており、ａｍｙＲ遺伝子の機能が破壊されていることが明らかとなった。
以上のように、外来遺伝子であるアスペルギルス・ニドゥランス由来のｓＣ遺伝子全体を完全に含まず、かつ、ａｍｙＲ遺伝子の機能が破壊されたアスペルギルス・オリゼのａｍｙＲ変異株を得ることができた。
Furthermore, in order to investigate whether or not the function of the amyR gene was disrupted in the sC mutant, starch resolution was analyzed.
NS4 strain, ΔamyR strain, and sC mutant strain (No. 9 strain) were each inoculated into a broth medium to form giant colonies.
A conidial suspension was prepared from this giant colony. After diluting the conidia suspension so that the concentration of conidia is 10 7 pieces / ml, the suspension is diluted with starch medium (1.0% starch, 0.3% sodium nitrite, dipotassium hydrogen phosphate). 0.1%, magnesium sulfate 0.05%, potassium chloride 0.05%, trace element 0.1%, methionine 0.15%), and cultured at 30 ° C. for 4 days. In addition, about the starch culture medium, iodine was added after culture | cultivation and the halo formation difference was clarified by the iodine starch reaction.
As a result, NS4 strain showed good growth and formed a large halo, whereas ΔamyR strain and sC mutant strain did not grow well and almost no halo was formed (FIG. 7). That is, it was clarified that the ΔamyR strain and the sC mutant strain (No. 9 strain) have lost starch degradability and the function of the amyR gene is disrupted.
As described above, it was possible to obtain an amyR mutant of Aspergillus oryzae that does not completely contain the entire sC gene derived from the foreign gene Aspergillus nidulans and in which the function of the amyR gene was disrupted.
本発明により、糸状菌ゲノム中の目的遺伝子の機能が破壊され、かつ、目的遺伝子の機能を破壊するために用いたマーカー遺伝子全体が欠損した、食品産業上利用する上で不都合な形質を示さない有用な変異株の作出方法が提供される。 According to the present invention, the function of the target gene in the filamentous fungus genome is disrupted, and the entire marker gene used for disrupting the function of the target gene is deleted, and the trait is not unfavorable for use in the food industry. Methods for producing useful mutant strains are provided.
Claims (6)
（ａ）糸状菌ゲノム中の機能を破壊しようとする目的遺伝子内にマーカー遺伝子を挿入させる工程、
（ｂ）マーカー遺伝子を挿入した当該糸状菌にイオンビームを照射し、変異を誘発する工程、
（ｃ）イオンビーム照射後の糸状菌の中から、変異によってマーカー遺伝子の機能が欠損した株をスクリーニングする工程、
（ｄ）スクリーニングされた株において、マーカー遺伝子を含む外来遺伝子全体がイオンビーム照射によって欠損し、かつ目的遺伝子の機能が破壊されていることを確認する工程、
を含む、糸状菌ゲノム中の目的遺伝子の機能を破壊することで有用な変異株を作出する方法。 In a method of creating a mutant strain of a filamentous fungus,
(A) a step of inserting a marker gene into a target gene whose function in the genome of the filamentous fungus is to be disrupted;
(B) irradiating the filamentous fungus into which the marker gene has been inserted with an ion beam to induce mutation;
(C) screening a strain deficient in the function of the marker gene due to mutation from among filamentous fungi after ion beam irradiation;
(D) in the screened strain, confirming that the entire foreign gene including the marker gene is deleted by ion beam irradiation and that the function of the target gene is destroyed;
A method for producing a useful mutant strain by disrupting the function of a target gene in a filamentous fungus genome.
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