JP4312832B2 - In vivo recombination - Google Patents

Description

製造者により推奨されたようにBio-Rad Gene Pulser^TMを使用して調製され、そして形質転換されたエレクトロコンピテント細胞。

コンピテント細胞を、Yasbin, R.E., Wilson, G.A. and Young, F.E.（1975）Trans formation and transfection in lysogenic strains of Bacillus subtilis : evidence for selective induction of prophage in competent cells. J. Bacteriol, 121 : 296-304により記載されたように調製し、そして形質転換させた。
B. subtilis PL 1801。この株は、apr及びnpr遺伝子が破壊されているB. subtilis DN 1885である

プラスミド：
pSX120 :（EP 405901及びWO 91/00345中に記載されている）B. subtilis発現ベクター。このベクターは、野生型サビナーゼ遺伝子を含む。
pE194 :（Horinouchi, S and Weisblum, B., 1982, J. Bacteriol. 150 : 804-814）。
（WO 96/34946及びWO 92/19729中に記載されている）B. subtilis発現ベクター。このベクターは、本明細書中サビシン（Savisyn）といわれる合成サビナーゼ（Savinase）遺伝子を含む。サビシン遺伝子中には、pSX120中に含まれる野生型サビナーゼ遺伝子に比較して、多数の制限部位が導入されている。
pUC19 : Yanisch-Perron, C., Vieria, J. and Messing, J.（1985）Gene 33, 103-119。
pF64L-S65 T-GFP ;は、WO 97/11094中に記載されているF64L-S65T-GFPをコードするDNA断片をコードするE.coliプラスミドである。
pMUTIN−４−MCS :このプラスミドは、Laboratoire de Genetique Microbienne, Institut National de la Recherche Agronomique, 78352 Jouy en Josas-CEDEX, Franceから得られることができる。
溶液／培地
（Ausubel, F.M. et al.（eds.）“Current protocols in Molecular Biology”。John Wiley and Sons, 1995中に記載されるような）TY及びLB寒天。
LBPGは、0.5％グルコース及び0.05Ｍリン酸カリウム、pH7.0を補ったLB寒天である。
LBPGSKプレートは、寒天と１％のスキム・ミルクを含むLBPG培地である。
抗生物質は、６μｇ／mlのクロラムフェニコール、10μｇ／mlのカナマイシン、及び５μｇ／mlのエリスロマイシンの濃度において、異なる培地中で使用される。
（Ausubel, F.M. et al.（eds.）“Current protocols in Molecular Biology”。John Wiley and Sons, 1995中に記載されているような）TE−バッファー。
一般的分子生物学的方法
DNA操作及び形質転換を、分子生物学の標準的な方法（Sambrook et al.（1989）Molecular Cloning : A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY ; Ausubel, F.M. et al.（eds.）“Current protocols in Molecular Biology”。John Wiley and Sons, 1995 : Harwood, C.R., and Cutting, S.M.（eds.）“Molecular Biological Methods for Bacillus”。John Wiley and Sons, 1990）を使用して行った。
DNA操作のための酵素を、供給者の指示書に従って使用した。
DNA操作のための酵素
特にことわらない限り、DNA操作のための全ての酵素、例えば、制限エンドヌクレアーゼ、リガーゼ、ポリメラーゼ、PCRポリメラーゼ等は、New England Biolabs, Inc.から得られる。
湿度感受性シャッフリング・プラスミドの構築
pMB430を、以下に示すような５工程のクローニング方法において構築した。このDNA配列全体が配列番号：１内に見られる。
１）温度感受性プラスミドpE194を、pE194プラスミドのユニークClaＩ部位付近に多フローニング部位を導入し、そしてこのようにしてそのClaＩ部位を破壊することにより修飾した。この導入を、そのプラスミド全体を増幅し、そしていくつかのユニーク・クローニング部位を導入するために、プライマー＃22899と24039を使用したPCRにより行った。
このPCRを以下のように行った：
１μｌのpE194 Qiaquickプラスミド・プレップを、200μＭの各dNTP, 2.5ユニットのAmpliTaqポリメラーゼ（Perkin-Elmer, Cetus, USA）、及び100pmolの各プライマー：

を含むPCRバッファー（10mM Tris-HCl, pH8.3, 50mM KCl, 1.5mM MgCl₂, 0.Ol％（ｗ／ｖ）ゼラチン）中での50μｌのPCR増幅のために使用した。
上記PCR反応を、DNAサーマル・サイクラー（Landgraf, Germany）を使用して行った。１分間94℃での１のインキュベーション、その後の40サイクルのPCRが、１分間94℃での変性、１分間55℃でのアニーリング、及び２分間72℃での伸長を使用して行われた。増幅生成物の５μｌアリコートを、0.7％アガロース・ゲル（NuSieve, FMC）中の電気泳動により分析した。
PCR断片のサブクローニング
上記のように生成されたPCR生成物の45μｌアリコートを、製造者の指示に従ってQIAquick PCR精製キット（Qiagen, USA）を使用して精製した。精製されたDNAを、50μｌの10mM Tris-HCl, pH8.5中で溶出させた。50μｌの精製されたPCR断片を、BamHＩで消化し、0.8％低ゲル化温度のアガロース（Sea Plaque GTG, FMC）ゲル中で電気泳動にかけ、関係のある断片をそのゲルから切除し、そして製造者の指示に従ってQIAquick Gel抽出Kit（Qiagen, USA）を使用して精製した。次に単離されたDNA断片を、BamHＩ消化されたpUC191にライゲートし、そしてそのライゲーション混合物を、E. coli SJ2を形質転換させるために使用した。
細胞を、Ｘ−gal（SIGMA, USA）（５−ブロモ−４−クロロ−３−インドールイル・アルファーＤ−ガラクトピラノシド、50μｇ／ml）を補った、エリスロマイシン（200μｇ／ml）を含有するLB寒天プレート上にプレーティングした。
陽性クローンの同定及び特徴付け
形質転換された細胞を、Ｘ−gal（50μｇ／ml）を補ったエリスロマイシン（200μｇ／ml）を含むLB寒天プレート上にプレーティングし、そして一夜３７℃でインキュベートした。翌日、白色コロニーを、これらを新鮮なLB−エリスロマイシン寒天プレート上に再画線塗布することにより、レスキューし、そして一夜37℃でインキュベートした。翌日、各クローンの単一コロニーを、エリスロマイシン（200μｇ／ml）を含む液体LB培地に移し、そして250rpmで振とうしながら37℃で一夜インキュベートした。
プラスミドを、製造者の指示に従ってQIAgenプラスミド精製ミニ・キット（Qiagen, USA）を使用して上記液体培養物から抽出した。上記プラスミドの５μｌのサンプルを、BamHＩで消化した。この消化を、0.7％アガロース・ゲル（NuSieve, FMC）上の電気泳動によりチェックした。約2.7kbと3.7の２つのDNA断片の外観は、陽性クローンを示した。このクローンを、MB293と命名し、そしてこのプラスミドを、pMB293と命名した。
２）次に、pMB293をEcoRＩ消化し、そしてその3.8kb断片を0.7％アガロース・ゲル上で、製造者に従ってQiagenからのQiaQuickゲル精製キットを使用してゲル精製した。（上記PCRプライマーに、そしてpUC19のMCSに起因する）挿入された多クローニング部位をもつpE194を構成するこの断片を、ライゲーションにより円形にし、そしてB. subtilis DN1885を形質転換させるために使用し、形質転換された細胞を、５μｇ／mlのエリスロマイシンを含むLBPGプレート上で選択し、そしてインキュベーションを一夜33℃で行った。この形質転換からのクローンを再画線塗布し、そして５μｇ／mlのエリスロマイシンを含むTY中で一夜培養した。一夜の培養ブロスから細胞を単離し、そしてプラスミドをQiaquickプラスミド・キットを使用して精製した。これらのプラスミドの制限酵素消化は、上記クローンの正しさを証明した。１のこのようなクローンとプラスミドを、MB333及びpMB333と命名した。
３）次に、pMB333を、いずれのプロモーターをももたないサビジン遺伝子を含むプラスミドの構築のために温度感受性プラスミドとして使用した。この構造物を以下のように作った。
プライマー#21547と#21548及び鋳型としてpSX222を使用したPCRを、上記の条件に従って行った：
１μｌのpSX222 Qiaquickプラスミド・プレップを、200μＭの各dNTP, 2.5ユニットのAmpliTaqポリメラーゼ（Perkin-Elmer, Cetus, USA）、及び100pmolの各プライマー：

を含むPCRバッファー（10mM Tris-HCl, pH8.3, 50mM KCl, 1.5mM MgCl₂, 0.01％（ｗ／ｖ）ゼラチン）中での50μｌのPCR増幅のために使用した。
上記PCR反応を、DNAサーマル・サイクラー（Landgraf, Germany）を使用して行った。１分間94℃での１のインキュベーション、その後の40サイクルのPCRが、１分間94℃での変性、１分間55℃でのアニーリング、及び２分間72℃での伸長を使用して行われた。増幅生成物の５μｌアリコートを、0.7％アガロース・ゲル（NuSieve, FMC）中の電気泳動により分析した。
PCR断片のサブクローニング
上記のように生成されたPCR生成物の45μｌアリコートを、製造者の指示に従ってQIAquick PCR精製キット（Qiagen, USA）を使用して精製した。精製されたDNAを、50μｌの10mM Tris-HCl, pH8.5中で溶出させた。50μｌの精製されたPCR断片を、BamHＩとApaＩで消化し、0.8％低ゲル化温度のアガロース（Sea Plaque GTG, FMC）ゲル中で電気泳動にかけ、関係のある断片をそのゲルから切除し、そして製造者の指示に従ってQIAquick Gel抽出Kit（Qiagen, USA）を使用して精製した。精製された断片を、ゲル精製され、BamHＩ及びApaＩ消化されたpMB333にライゲートした。そしてそのライゲーションを、B. subtilis DN1885を形質転換させるために使用した。形質転換された細胞を、５μｇ／mlのエリスロマイシンを含むLBPG寒天プレート上にプレーティングし、そして33℃で一夜インキュベートした。クローンを同様のプレート上で再画線塗布し、そしてプラスミドを、５μｇ／mlエリスロマイシンを含むTY中で33℃でインキュベートされたクローンの一夜培養物から、製造者により記載されたようにQiaquickプラスミド・キットを使用して単離した。プラスミドを、ApaＩとBamHＩを使用した酵素消化によりチェックした。１の正しいクローンをMB339と、そしてそのプラスミドをpMB339と命名した。
４）我々は、ハイブリッド・プラスミドが形成されていることをモニタリングする簡単な方法をテストしようと欲した。従って、我々は、２つの別個のプラスミド上の２つの相同遺伝子の間の強制組換えをモニターすることを欲した。これは、GFPをコードするORFに転写的に融合されたプロモーターを欠くサビジンをもつpE194誘導体を構築することにより行われた。この方法においては、２つの相同遺伝子が組換えられず、そしてそれ故、pSX120上にその他の方法で存在するプロモーターの制御下にGFPを置かない場合には、（サビジンそしてそれ故GFP遺伝子の上流にプロモーターを欠くことにより）GFPの発現が存在しないであろう。このプラスミドを、GFP遺伝子をPCR増幅し、そしてpMB339上のサビジン遺伝子の直下流にそれを導入することにより、構築した。
本明細書中に使用するGFPコーディングDNAは元来の野生型遺伝子の誘導体であり、GFPの誘導体は、国際特許出願WO 97/11094中に記載されたように構築された突然変異体F64L-S65T-GFPのDNA構築物からクローン化した。
上記鋳型プラスミドの構築はWO 97/11094中に記載された。F64L-S65T-GFPをコードするDNA断片を、以下のように上記遺伝子をコードするE.coliプラスミドからPCR増幅した：
１μｌのpF64L-S65T-GFP Qiaquickプラスミド・プレップを、200μＭの各dNTP, 2.5ユニットのAmpliTaqポリメラーゼ（Perkin-Elmer, Cetus, USA）、及び100pmolの各プライマー：

を含むPCRバッファー（10mM Tris-HCl, pH8.3, 50mM KCl, 1.5mM MgCl₂, 0.01％（ｗ／ｖ）ゼラチン）中での50μｌのPCR増幅のために使用した。
上記PCR反応を、DNAサーマル・サイクラー（Landgraf, Germany）を使用して行った。１分間94℃での１のインキュベーション、その後の40サイクルのPCRが、１分間94℃での変性、１分間55℃でのアニーリング、及び２分間72℃での伸長を使用して行われた。増幅生成物の５μｌアリコートを、0.7％アガロース・ゲル（NuSieve, FMC）中の電気泳動により分析した。
PCR断片のサブクローニング
上記のように生成されたPCR生成物の45μｌアリコートを、製造者の指示に従ってQIAquick PCR精製キット（Qiagen, USA）を使用して精製した。精製されたDNAを、50μｌの10mM Tris-HCl, pH8.5中で溶出させた。このPCR断片とpMB339をClaＩとNsiＩで消化し、上記のようにゲル精製し、そして一緒にライゲートした。このライゲーション混合物を、DN1885を形質転換させるために使用し、形質転換された細胞を、LBPG ５μｇ／mlエリスロマイシン上にプレーティングした。18時間のインキュベーションの後、緑色の蛍光細胞が単一コロニーとして見えた。これらの中の１を分析し、そしてMB406として保存し、そして対応のプラスミドをpMB406として保存した。GFPコーディングORFはその転写を駆動する上流のプロモーターをもっていなかったので、GFP発現のための可能な説明は、サビジン−GFP転写融合物のさらに上流のプロモーター、すなわち、repF遺伝子のプロモーターからの読み進まれたということであった。
５）GFPのORF内へのいずれの読み進みをも廃止するために、転写ターミネーターを、repF遺伝子とサビジンDNAの間に挿入した。このターミネーターを、以下のプライマーと条件を使用してpMUTIN−４−MCSプラスミドから増幅した：
１μｌのpMUTIN−４−MCS Qiaquickプラスミド・プレップを、200μＭの各dNTP, 2.5ユニットのAmpliTaqポリメラーゼ（Perkin-Elmer, Cetus, USA）、及び100pmolの各プライマー：

を含むPCRバッファー（10mM Tris-HCl, pH8.3, 50mM KCl, 1.5mM MgCl₂, 0.01％（ｗ／ｖ）ゼラチン）中での50μｌのPCR増幅のために使用した。
上記PCR反応を、DNAサーマル・サイクラー（Landgraf, Germany）を使用して行った。60秒間94℃での１のインキュベーション、その後の40サイクルのPCRが、30秒間94℃での変性、30秒間55℃でのアニーリング、及び１分間72℃での伸長のサイクル・プロフィールを使用して行われた。増幅生成物の５μｌアリコートを、0.7％アガロース・ゲル（NuSieve, FMC）中の電気泳動により分析した。
増幅されたDNAを上記のように精製し、そして同じくBglII及びKpnＩ消化されたpMB406内にKpnＩ−BglII断片としてクローン化した（上記のものと同一の、ゲル精製等のための手順）。このライゲーション混合物をDN1885を形質転換させるために使用し、形質転換された細胞をLBPG ５μｇ／mlエリスロマイシン上にプレーティングした。18時間のインキュベーションの後、細胞が単一のコロニーとして見られた。これらの中の１を分析し、そしてMB430として保存し、そして同様にそのプラスミドをpMB430として保存した。このクローンは、GFPを全く発現していなかった。このことは、そのクローン化されたターミネーターが実際に予想されたように働いたことを示している。得られたプラスミドpMB430全体配列を、配列番号：１として表す。
MB432株の樹立
PL1801バチルス・サブチリスを、100μｌのコンピテントPL1801当り約１μｇの各プラスミドを使用して２つのプラスミドpMB430とpSX120で形質転換させた。この形質転換された細胞を、LBPG−５μｇ／mlエリスロマイシン上にプレーティングし、33℃で一夜インキュベートした。翌日、コロニーを、LBPG−５μｇ／mlエリスロマイシン＋６μｇ／mlクロラムフェニコール上に再画線塗布し、そして33℃で一夜インキュベートした。コロニーを、液体培養物TY−５μｇ／mlエリスロマイシン＋６μｇ／mlクロラムフェニコール中で一夜33℃でも培養し、プラスミドを培養ブロスから精製し、そしてPL1801株内の両pMB430とpSX120の存在を証明した。このクローンを保存し、そしてMB432と命名した。
実施例１
サビナーゼのズブチリシン遺伝子をコードする２つの相同配列の配列シャッフリング
ズブチリシン（subtilisin）タイプのプロテアーゼをコードする（pSX120内に含まれる）サビナーゼ野生型遺伝子を、高い相同性をもつ遺伝子のシャッフリングのための標的として選んだ。新たな制限部位を与える多数のサイレント突然変異が導入されているところの、pSX222内に含まれるサビナーゼ遺伝子（以下、サビジンという）の部位指定変異体を、サビナーゼに対するシャッフリング・パートナーとして使用した。上記２つの遺伝子を、本発明に従って２つの別個の適合性プラスミド上でクローン化した。転写活性なサビナーゼ遺伝子を、クロラムフェニコールに対する耐性をコードするpUB110起源に基づくプラスミド（pSX120、図４）上にクローン化した。サビナーゼ遺伝子を欠いたプロモーターを、同じくエリスロマイシンに対する耐性をコードする温度感受性プラスミドpE194の誘導体（pMB430）上にクローン化した。２つの転写ターミネーターを、いずれの読みをも防ぐためにサビジン遺伝子の上流にクローン化した。
pMB430中、緑色蛍光タンパク質（GFP）は、上記２つのプラスミド間のイン−クロス（in-cross）及びアウト・クロス（out-cross）に視覚的に従うことができるように、サビジン遺伝子に転写的に融合された。
２つのプラスミドpSX120とpMB430で形質転換されたB. subtilis株MB432, pL1801は、上記相同遺伝子間の２つの連続的な２重のクロス・オーバー事件を誘導するために２サイクルの温度シフトを通して採取された。相同的組換え及びプラスミド・ハイブリッド形成は、エリスロマイシンの存在下、制限的温度（42〜50℃）においてLBPGプレート上で選択された。これらの制限的条件下、温度感受性プラスミドpMB430は、サビナーゼ／サビジンの領域内での相同組換えにより安定性pSX120プラスミド内に強制される。組換えが生じている上記コロニーも、上記２つのプラスミド間の組換え及びサビジン−GFPオペロンの同時発生活性化の結果として蛍光になる（図２）。アウト・クロス事件は、２日間30℃の許容的温度でLB−ブロス中、上記クローンを培養し、そしてクロラムフェニコールとエリスロマイシンを含むLBPGプレート上で上記細胞をプレーティングすることにより、スクリーニングされた。
非蛍光性コロニーをプレート上で再画線塗布し、そして上記のような第２ラウンドの温度サイクルを通して採取した。
第２ラウンドのシャッフリングの後、プラスミド調製を20の異なるクローンから行った。コンピテントPL1801は、20のプラスミド・プレップの各々で形質転換し、そしてエリスロマイシンを補ったLBPG上でプレートした。これらのプレートを、pSX120とpMB430の両方が形質転換されていた不所望のクローンを同定するために、クロラムフェニコールとエリスロマイシンを含むLBPG上で複製した。pMB430プラスミドだけが形質転換されているクローンを、シャッフリングが生じているかどうかを同定するために、サビジン領域の配列決定について選択した。図６から、２つの遺伝子、サビナーゼとサビジンの間の組換えが生じていたことは明らかである。12の配列決定されたクローンの全てが、ある程度の組換えを示し、そしてこれらの中の４つが、２つの連続的な２重組換え事件の結果として、陽性に同定されることができる。サビナーゼとサビジン配列の交換が、シャッフルされた断片が、サビジンにとってユニークな制限部位に重複している場合にのみ観察されることができるということに注意することが重要である。２つの２重組換えのための陽性の証拠をもつ４つのクローンは、それ故、最小の数である。なぜなら、この組換えは、２つの隣接する制限部位の間で生じることができたであろうからである。
配列表
（２）配列番号：１についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：5313塩基対
（Ｂ）タイプ：核酸
（Ｃ）鎖の数：１本鎖
（Ｄ）トポロジー：直鎖状
（ii）分子タイプ： DNA
（vi）起源：
（Ａ）生物：プラスミドpMB430
（xi）配列番号：１

Field of the invention
The present invention relates to a method for in vivo recombination of homologous DNA sequences. This method is a forced artificial evolution that results in a DNA sequence encoding a polypeptide with advantageous properties.
Background of the invention
Homologous recombination between DNA sequences placed on the same plasmid is well known to those skilled in the art (Weber and Weissmann, Nucl. Acid. Res. (1983) 11, 5661-5669). EP 252666 B1 (Novo Nordisk A / S), WO 95/22625 A1 (Affymax Technologies N.V.), and WO 9101087 disclose in vivo recombination of genes placed on the same plasmid.
EP 449923 B1 (Setratech) discloses a method of recombination between genes in vivo of partially homologous DNA sequences. This recombination occurs in cells where the enzyme mismatch repair system is defective.
J. Biotechnol. (1991) 19, 221-240 discloses a method for inactivating genes in the genome of B. Amylolique facdens. This method involves integration and excision of the pE194 mutant.
In Chemical Abstracts 118: 161925 (1993), the minimum sequence length of homology required for recombination is measured. This measurement method involves the incorporation of a pE194 derivative carrying a glucose gene.
J. Bacteriol. (1982) 152,524-526 discloses plasmid pBD9, which contains two plasmids from Staphylococcus aureus, pE194 and pUB110. Other plasmids based on pE194 or pUB110 are also available in Molecular and General Genetics (1988), 213, 465-70, Molecular and General Genetics (1984), 195, 374-7, Plasmid (1981), 6, 67-77, and Genetika, (1986) 22, 2750-7.
In Yichuan Xuebao (1993), 20, 272-8 (see Chemical Abstracts 120: 1670 (1994)), Chen et. Al described a thermostable α-amylase gene from Bacillus licheniformis. The plasmids pNW102 and pE194 derivatives inserted in are disclosed. pNW102 is transformed into Bacillus subtilis strain BF 7658 and then incubated at a non-permissive temperature and subjected to homologous recombination between the α-amylase genes of pNW102 and BF 7658. The Α-Amylase made from the above recombinant exhibits the same properties as α-Amylase from Bacillus licheniformis.
However, in the related field, DNA sequences are integrated and deleted (cross-linked) to obtain genes encoding polypeptides with improved properties, such as increased stability, improved specificity or higher activity. There is no indication that it can be recombined in vivo by repeating -out).
Therefore, the problem of the present invention is to provide an improved in vivo method for new DNA sequences.
Brief Description of the Invention
The present invention is directed to an in vivo method for generating a novel DNA sequence. Novel DNA sequences with improved properties are obtained in a rapid and efficient manner from at least two homologous DNA sequences by a method for in vivo exchange of DNA between at least two vectors, each containing a homologous DNA sequence and an origin of replication. Can be made.
The method of the present invention is characterized in that the exchange of the homologous sequence is performed by repeating the integration and excision from one vector to another vector at least once in vivo.
Thus, in a first aspect, the present invention is a method for in vivo recombination of homologous DNA sequences comprising the following steps:
(A) incubating cells containing at least two DNA constructs, each containing a DNA sequence and origin of replication, under conditions that favor incorporation from one of the DNA constructs into one of the other DNA constructs; Thereby creating a hybrid DNA structure;
(B) incubating the cells under conditions that favor deletion from the DNA construct, thereby creating a new DNA construct comprising a recombinant DNA sequence and an origin of replication, respectively;
(C) repeating steps a) to b) at least once,
The method.
One of the advantages of the in vivo recombination method of the present invention is that a number of different recombination patterns can be obtained between the DNA sequences by repeating the in vivo exchange between the DNA sequences.
This is shown in FIG. 1 herein, which is an in vivo assembly between two DNA sequences, Savinase and Savisyn, performed as described in Example 1. The result of replacement is shown.
In FIG. 1, sabinase / savicin recombinant clones, such as clone numbers 3, 8, and 12, are obtained from two or more in vivo recombination events between the two sequences.
[Brief description of the drawings]
FIG. 1 shows that plasmid pTR, which is thermostable because its origin of replication works at 50 ° C., and placed on plasmid pTS, which is temperature sensitive because its origin of replication does not work at temperatures above 45 ° C. 2 Shows recombination of two DNA sequences. Under non-permissive conditions (ie at 50 ° C.), pTS was incorporated into pTR and a hybrid, plasmid was created.
FIG. 2 shows the cross out from the hybrid plasmid, forming two new DNA sequences each placed on one plasmid. Repeated crossing in and out can be achieved, for example, by temperature cycling or simply by maintaining temperature pressure and resistance to pTS (ie, Erm^R) Can be made by selecting.
FIG. 3 shows the plasmid obtained after repeated integration and crossing out.
In addition, FIGS. 1 and 2 show a DNA structure system that is suitable for in vivo measurement of hybrid structure formation and is suitable for performing forced deletion of the DNA structure.
Figure 1: DNA structure pTS:
Suitable active promoters that direct the expression of active proteins
Erm^R: Antibiotic resistance gene
DNA structure pTS:
It does not contain any active promoter to direct GFP transcription.
GFP: Green fluorescent protein
Erm in pTS^RAntibiotic resistance genes are active due to the active promoter.
The GFP gene in pTR is inactive (ie the gene is not transcribed). This is because there is no active promoter for driving the expression of the gene.
Figure 2: After forming the hybrid structure according to the present invention, GFP becomes active (driven by the promoter). This allows the formation of the hybrid structure to be measured in vivo (see below for further details).
Conversely, Erm^RThe antibiotic resistance gene becomes inactive (ie, the gene is not transcribed). This is because there is no active promoter that drives the expression of the gene. Erm for deletion of DNA hybrid structure^RThis is done by incubating the cells under antibiotic pressure, thereby forcing the DNA hybrid structure to cross out. This is because the above cells only have an Erm after crossing out of their DNA structure has occurred.^RThis is because an antibiotic resistance gene can be expressed.
Figures 4 and 5: show two vectors pSX120 (Figure 4) and pMB430 (Figure 5) used as described in Example 1 to recombine two DNA sequences sabinase and sabisin in vivo.
pSX120 consists of i) an open reading frame encoding sabinase, ii) an active promoter directing the transcription of sabinase, and iii) a gene conferring resistance to chloramphenicol (Cam^R)including.
pMB430 is i) a savidin DNA fragment, ii) a terminator (which does not result in transcription through subsequent regions), iii) a gene conferring resistance to erythromycin (Erm^RAnd iv) an open reading frame encoding green fluorescent protein (GFP).
FIG. 6: shows the results of in vivo recombination of sabinase / savidin performed as described in Example 1. This schematic shows 12 clones that have been shuffled / recombined.
Gives Sabidine a unique restriction site position horizontally.
The letter “S” indicates that the sequence at that position is identical to that of the savinase, and the letter “Z” indicates that the sequence is of savidin origin. All of the sequenced clones differ in their recombination pattern.
Definition
As used herein, the following terms have the following meanings:
The term “DNA sequence” refers to any DNA sequence that is desired to be modified according to the present invention, eg, a polypeptide, eg, an enzyme, a pharmaceutically active polypeptide, eg, insulin growth hormone / human hormone or a growth regulator. And DNA sequences encoding sequences such as promoters, transcriptional or translational regulators and other sequences such as intracellular polynucleotides and polypeptides.
The term “homologous” means that the DNA sequence has several stretches of identical nucleotides that allow recombination in vivo. The stretch within the DNA sequence is at least 15 base pairs long, more preferably at least 25 base pairs long, even more preferably at least 50 base pairs long, and most preferably at least 150 base pairs long .
The term “homologous” includes stretches of DNA sequences that are 60% or more identical to DNA sequences that differ in only one nucleotide. The stretch of DNA sequence is preferably 70% or more identical, more preferably at least 80%, especially 90% or more, and the DNA sequence stretch is 95% identical, most preferably at least 97% identical. Is more preferable.
Within the stretch of the DNA sequence of interest, the homology of the DNA sequence referred to above is measured as the degree of identity between the two sequences indicating the derivation of the first sequence from the second sequence. This homology is preferably a computer program known in the art, such as the GAP (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science, provided in the GCG program package. Drive, Madison, Wisconsin, USA 53711) (Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Bioligy, 48, 443-453). Using the GAP with the following settings for DNA sequence comparison: GAP creation penalty of 5.0, and GAP extension penalty of 0.3, the homologous DNA sequence of interest is preferably of identity as described above Indicates a degree of identity.
The term “recombined DNA sequence” means a DNA sequence that is the result of a recombination event between at least two DNA sequences. This recombination event can be a single recombination event, or a first recombination event between at least two DNA sequences, followed by a cross-over at a different site than the first recombination event. Can be out.
The term “DNA structure” means a DNA molecule, such as a vector, a plasmid, a chromosome (eg, a Bacillus subtilis chromosome), a virus, a phage, a transposon, or a genome.
The term “vector” means any linear or circular DNA molecule that can contain the DNA sequence to be recombined and an origin of replication. The term includes any vector known to those of skill in the art, such as a plasmid or phage. This DNA sequence is, although not necessarily, optionally under the control of a promoter.
The term “cell”, as used herein with respect to the in vivo recombination methods of the present invention, is intended to encompass both “cells” and “cell populations”.
The term “transferring” means that the DNA structure is introduced into the cell. DNA constructs can be obtained using techniques known in the art for direct introduction of DNA, eg, electroporation, transformation of competent cells, protoplast transformation, transfection, transduction, conjugation or ballistics. (Ballistic) can be introduced by transformation.
The term “incubating” (or culturing, proliferating growth) means that the cells are treated in such a way that their normal cell functions, in particular the functions necessary for recombination, are active. The cells can be incubated on a solid support (agar petri dishes) or the cells can be incubated under soaking conditions. The cells are preferably incubated in a test tube, such as an Eppendorf test tube. In the case of a temperature sensitive replication origin, the test tube is conveniently placed in an automatic thermocycler. In addition, the cells can be incubated in standard shake flasks.
The term “origin of replication” (ori) refers to the region where replication of a DNA structure is initiated. With respect to the origin of replication, the term “functional” means that replication can be initiated from the origin. The term “non-functional” means that replication does not begin under certain conditions, or that the initiation of replication is, for example, a DNA construct with a marker gene and ori under appropriate selection pressure. It is meant to be reduced to a level that is not sufficient for the survival of the containing cells.
The term “temperature sensitive” refers to an origin of replication or a vector that cannot replicate in a non-permissive condition, eg, at an elevated temperature, ie, still allowing the growth of its parent cell. It means that.
The term “temperature tolerance” means that with respect to an origin of replication or vector, the vector can replicate at an elevated temperature.
The term “DNA library” is a library containing a number of different DNA sequences. DNA libraries can be prepared according to standard procedures known in the art, for example by in vitro DNA shuffling / DNA recombination (WO 95/17413, WO 95/22625) and / or error-prone PCR and cassette mutagenesis. Can be made.
As used herein, a DNA library can include as little as 2-20 different sequences, for example, a library in which only one amino acid has been altered.
In addition, a DNA library can contain a number of different DNA sequences, such as 10^TenUp to different sequences can be included.
Detailed Description of the Invention
The present invention is a method for in vivo recombination of homologous DNA sequences comprising the following steps:
(A) i) at least one origin of replication is non-functional, and ii) the cell under selective pressure that allows the cell to grow only if it contains a DNA construct with said non-functional origin. Under certain conditions, incubate cells containing at least two DNA structures, each containing a DNA sequence and an origin of replication (ie, from one in the DNA structure into one in the other DNA structure). Supporting the integration of DNA, thereby creating a hybrid DNA structure);
(B) changing from the above conditions to a condition where fewer origins of replication are non-functional than in step a) (ie, supporting crossing out from the hybrid DNA construct);
(C) repeating steps a) to b) at least once,
Including the method.
The recombined DNA sequence will encode a novel polypeptide with new properties. A selection or screening system can then be incorporated into the methods of the present invention to select and / or screen for the desired property encoded by one or more of the recombinant DNA sequences.
The number of repeats in step (c) can be one repeat, or it can be more, for example, 5-10 repeats, or 50-100 repeats or even more.
By incubating the cells under conditions such that the origin of replication of the DNA structure is non-functional, integration is forced (or preferred for DNA structure survival), and again the DNA structure It is preferred to force cross-out by incubating the cells under conditions such that the origin of replication is functional.
In another preferred embodiment of the routine shown above in steps (a) to (c), under steady conditions (eg, constant temperature and selective pressure) where the natural cross-in and cross-out occur, It is preferred to maintain the cells. That is, the shuffling procedure in the cells is repeated without changing the above conditions repeatedly.
In many DNA constructs, such as many Bacillus DNA constructs (vectors) (Bacillus subtitis and other Gram-positive bacteria, Sonensheim et al., 1993, American Society for Microbiology, Washington, DC) The origin of replication includes ori (+) and ori (−). ori (+) controls where replication of the first DNA strand begins, and ori (-) controls the starting point for replication on the second strand (Bacillus subtilis and other Gram positives) Bacteria, Sonensheim et al., 1993, American Society for Microbiology, Washington DC).
In one embodiment of the method of the invention, at least one DNA construct does not contain ori (−) (ie only contains ori (+)). Preferably, an ori (-) free DNA construct is a DNA construct whose origin (i.e., ori (+) here) is functional during the formation of a hybrid construct performed according to the present invention. It is.
One advantage of the above aspect of the invention is that when only ori (+) is functional in the DNA structure, the DNA structure comprises both ori (+) and ori (−). This means that it is in a single-stranded state for a relatively long period of time compared to the object. This extended single-stranded state facilitates the formation of a hybrid DNA structure according to the method of the present invention.
Preferably, the ori (-) free DNA construct as described above is a Bacillus construct, more preferably a Bacillus vector.
To illustrate the above concept, there is an example of a modified pTR (TR: thermostable) Bacillus vector that does not contain ori (−) (see FIG. 1). When the hybridization is performed at 50 ° C., only the origin of pTR is functional (the origin of pTS (TS: temperature sensitivity) is not functional) (see FIGS. 1 and 2). The DNA construct pTR contains only ori (+) and is therefore in a single-stranded state for a relatively long period of time, which facilitates recombination events and subsequent hybridization with the pTS DNA construct (See FIGS. 1 and 2).
More preferably, one DNA construct is a vector that can replicate under certain (permissive) conditions and cannot replicate under other (non-permissive) conditions. The vector can be, for example, one that is temperature sensitive with respect to replication. Thus, the vector can be one that cannot replicate at elevated temperatures, further allowing its parental cell to grow. The cells are first incubated at a temperature that allows replication of the vector, and then incubated after the integration into other DNA constructs can occur, and the vector is Incubate at a temperature that does not allow replication of the vector so that it is lost from the cells.
The vector can further include a selectable marker. In this case, incubation at non-permissive temperatures should be performed under selective conditions to ensure that only cells containing the integrated vector (including its DNA sequence and selectable marker) will survive. Can do.
Furthermore, it is preferable to construct a system that can measure the formation of the hybrid structure in vivo.
Preferably, this is done by constructing a system in which proteins that can be screened or selected in vivo are expressed only after the hybrid structure is formed.
Preferably, the screenable protein is a protein that is fluorescent under suitable exposure, and in particular, the fluorescent protein is green fluorescent protein (GFP) or a variant thereof.
Green fluorescent protein (GFP) or variants thereof, and how GFP is activated to fluoresce has been described in the art and for further details (Crameri et al. Nature Biotechnology 14: 315-319 (1996); and Cormack et al. GENE 173: 33-38 (1996)).
One of the advantages of using a screenable protein as described above that is fluorescent under suitable exposure, and in particular GFP or a variant thereof, is for selectively picking up cells containing the hybrid construct. This is possible, for example, by fluorescence-activated cell sorting (FACS). This is in accordance with the first aspect of the present invention and can remove the background of cells that do not contain the hybrid structure after step a).
FACS is a well-known screening technique and see (Cormack et al. “FACS-optimized mutants of the green fluorescent protein (GFP)” GENE 173: 33-38 (1996)) for further details. be able to.
To illustrate a suitable system that allows in vivo measurement of the formation of the hybrid structure, an example of a suitable system is shown in FIGS.
Instead of using screenable proteins as discussed above, it is advantageous to use a selectable marker such that the selectable marker is active in transcription only when the hybrid structure is formed. be able to.
Furthermore, it is preferable that the crossing out from the hybrid DNA structure in step b) of the first aspect of the present invention is a forced crossing out case.
Forced crossing out from the above hybrid structure:
i) constructing a system in which the DNA hybrid construct does not express a selectable marker and at least one of the at least two DNA constructs in step a) of the first aspect can express the selectable marker And
ii) Since the cell can express the marker gene only after the crossing out of the DNA hybrid structure has occurred, the cell is incubated under selectable conditions, thereby Force a cross-out,
Can be done.
Preferably, the selectable marker gene described above is on a DNA structure whose origin of replication is non-functional during forced integration of the DNA structure into the DNA hybrid structure performed as described above. Expressed and preferably the selectable marker gene is an antibiotic resistance gene. See below for further description of suitable selectable marker genes.
To illustrate the concept of forced crossing out from the hybrid DNA structure, an example of a suitable system is shown in FIGS.
A selectable marker is any marker known in the art, for example, a gene that encodes a product that confers antibiotic resistance to a cell, prototrophy to an auxotrophic strain, or complements a host defect (Eg, the dal gene introduced into the dal-strain; B. Diderichsen (1986), Bacillus: Molecular Genetics and Biotechnology Applications, AT Gene san and JA Hoch, Eds., Academic Press, pp. 35 See -46). A selectable marker can be, for example, a plasmid excised from a known source or present on a vector and used, for example, for the construction of a DNA construct to be used in the methods of the invention.
This selection can be accomplished by culturing the cells under selective pressure for the selectable marker encoded by the marker gene.
The DNA construct (where the vector is incorporated) can be any of the types described above, eg, with respect to the marker optionally carried by the vector to be used in the methods of the invention. A gene encoding a selectable marker. Thus, the marker gene encodes resistance to antibiotics such as kanamycin, tetracycline, ampicillin, erythromycin, chloramphenicol, or resistance to various heavy metals such as selenate, antimony or arsenate. Can do.
In preferred embodiments, the cells used in the method are defective or temporarily inactivated in the enzyme mismatch repair system or have a reduced level of mismatch repair. In the synthesis of DNA, errors can occur and the resulting non-complementary base pairs are referred to as mismatches. There is a process for calibration of errors (mismatches) in DNA. In E. coli, these errors are due to the fact that the third enzyme (MutU) unwinds two DNA strands and the fourth enzyme (MutH) is subsequently sequenced by a methylated DNA ( GATC) is detected very fast and accurately by two enzymes (MutS and MutL) that make it possible to cleave the newly synthesized strand on itself. For a more thorough explanation see EP 449923 (Setratech), the contents of which are incorporated herein by reference.
In other preferred embodiments, the cells used in the process are damaged or blocked by a major recombinant enzyme such as AddAB. Strains damaged by the AddAB gene show a higher frequency of homologous recombination. Both legitimate and illegitimate recombination frequencies are increased. However, in particular, recombination frequencies for illegitimate recombination are supported when working with strains that have been damaged or blocked in AddAB ATP-dependent nucleases (Meima, R; Haijema, BJ; Dijkstra, H; Venema, G; Bron, S (1997) Role of enzymes of homologous recombination in illegitimate plasmid recombination in Bacillus-subtilis. JOURNAL OF BACTERIOLOGY Vol. 179, No. 4 pp. 1219-1229).
In another preferred embodiment, the cells used in the above method overexpress one of the key recombinant enzymes, recA. This recA enzyme is involved in homologous recombination by initiating the regeneration of ssDNA to form the heteroduplex molecules necessary for the recombination event, and initiates DNA strand exchange. (PROGRESS IN NUCLEIC ACID RESEARCH AND MOLECULAR BIOLOGY Vol. 56, pp. 129-223 (1997)). Overexpression of recA can be achieved either by classical mutagenesis of the host strain or by cloning of the highly expressed recA gene.
In another preferred embodiment, the cells used in the above method are subjected to UV, X-ray or DNA damaging agents to increase the frequency of recombination between two homologous genes to be shuffled. . Stresses such as UV, X-ray or DNA damaging agents are known to induce recombinant enzymes through the SOS response and to activate enzymes involved in the recombination process such as recA. Yes. (BACILLUS SUBTILIS AND OTHER GRAM-POSITIVE BACTERIA, pp. 529-537 (1993)).
In another preferred embodiment, the pTR-based plasmid used as one carrier in the gene to be shuffled is a low-copy version of pTR or pWV01, (MOLECULAR AND GENERAL GENETICS Vol. 249, No. 1 pp. 43-50 (1995)), pTA1060, (JOURNAL OF BACTERIOLOGY Vol. 142, No. 1 pp. 315-8 (1980)) or pAMb1, (JOURNAL OF BACTERIOLOGY Vol. 157, No. 2 pp. 445- 53 (1984)) and is replaced by any of the other B. subtilis compatible low-copy plasmids. In a further preferred embodiment, one of the DNA constructs containing one of the genes to be shuffled is placed on the cell stain and is another DNA construct of the subject of interest to be recombined. Those containing other genes are plasmids. When carrying out the in vivo recombination method of the invention, the plasmid will be recombined with a DNA construct on the chromosome.
The plasmid can be, for example, a temperature sensitive plasmid.
The advantage of using a low-copy pTR, or system in which one of the DNA constructs is located on the chromosome as described above, is that the hybrid DNA construct after recombination of two homologous genes is It will be the dominant form of the gene in the cell. The high-copy background of the non-recombinant pTR plasmid inevitably reverts the gene hybrid formed in the first round to the wild type after the second round of recombination, and hence shuffling of the two genes Will be blocked. When the chromosome is used as a carrier in one of the above genes, the wild type background cannot coexist when pTS is integrated via a homologous gene.
The cells used in the above method are preferably cells suitable for large-scale production of recombinant DNA sequence products. The cell is preferably a bacterial cell, more preferably a Bacillus cell, and most preferably a Bacillus subtilis cell.
The DNA sequence to be recombined can be isolated / cloned from the organism containing the sequence in a manner known per se. For example, a suitable oligonucleotide probe can be prepared based on a first DNA sequence of interest to obtain a second sequence that is homologous to the first sequence. Alternatively, the DNA sequence can be amplified by PCR based on primers designed according to the knowledge of the conserved region.
In a different embodiment of the present content, the term “a set of homologous DNA sequences (“ set A ”,“ set B ”,“ set C ”, etc.)”, for example, of two or more different sets of homologous DNA sequences A set of homologous DNA sequences (“Set A”, “Set B”, “Set C”, etc.) and of the above homologous sequences (“Set A”, “Set B”, “Set C”, etc.) The present invention relates to a method according to the present invention such that each is inserted into a different DNA structure (“DNA structure A”, “DNA structure B”, “DNA structure C”, etc.) according to the present invention. Used with respect to one aspect of
This term is used to explain that the in vivo recombination methods of the invention can be used to recombine many “sets” of homologous DNA sequences. Each individual “set” of homologous DNA sequences can individually contain from one to many individual homologous DNA sequences (eg, a “set” comprising a DNA library).
Next, each of the above sets of homologous DNA sequences (“Set A”, “Set B”, “Set C”, etc.) is different from the DNA structure (“DNA structure A”, “DNA structure B” according to the present invention). "," DNA structure C ", etc.). Each of the different DNA constructs described above preferably differ according to the present invention in important parameters such as different origins of replication, different selectable markers, etc.
Next, all of the DNA structures (“DNA structure A”, “DNA structure B”, “DNA structure C”, etc.) can be introduced into cells according to the present invention, and When performing the method for in vivo recombination of the present invention, each individual DNA construct (“DNA construct A”, “DNA construct B”, “DNA construct C”, etc.) According to the present invention, it can be recombined randomly. After repeating the method of the present invention 2 to 3 times, this is due to the different recombined DNA sequences recombined randomly between all “sets” of the homologous DNA sequences used in the method. The DNA population will be given within a cell population, including many different combinations.
Further, the term “a set of homologous DNA sequences (“ set A ”,“ set B ”,“ set C ”, etc.) is used herein to refer to 1, 2 to 3, and / or many (eg, Large DNA library) To illustrate one of the advantages of the in vivo recombination method of the present invention, which is the flexibility of the system for recombining both sets of homologous DNA sequences, including individual homologous DNA sequences Used for.
Accordingly, one aspect of the present invention relates to a method according to the present invention wherein each of the above sets of homologous DNA sequences comprises only one DNA sequence (ie recombination of two or more homologous sequences).
Further, in embodiments of the invention, the homologous DNA sequence to be recombined is obtained from a pre-made DNA library and / or from an individual different pre-made library.
Homologous DNA obtained from the above DNA library is inserted into a vector according to the present invention (that is, DNA representing the DNA library is ligated (inserted) into the vector).
As described herein, the methods of the invention comprise at least two DNA constructs (eg, vectors) that differ in methods that permit forced in vivo recombination of the vectors.
Homologous DNA representing a DNA library can be inserted into only one and / or both of the different vectors.
That is, for example, a homologous DNA representing one DNA library is inserted (ligated) into one of the different vectors, and a homologous DNA representing another DNA library is inserted into another vector. In some cases, this will allow rapid and efficient recombination between two different libraries by the in vivo recombination method according to the invention. To illustrate this concept, DNA representing one library can be inserted into the pTR vector (see Figure 1), and DNA representing the other library is inserted into the pTS vector. (See FIG. 1). Subsequently, repeating the in vivo recombination event performed by the method according to the present invention 2-3 times will give a cell population representative of a novel library that is a combination of the two libraries described above.
Thus, at least one of the set of homologous DNA sequences is obtained from a pre-made DNA library, and at least one of the set of homologous DNA sequences is a DNA sequence that is homologous to the DNA library. A method according to the invention (ie resulting in a method for in vivo recombination of said DNA library into a DNA sequence homologous to said DNA library); or
In a method according to the present invention wherein the set of homologous DNA sequences is obtained from the same pre-made DNA library (i.e. resulting in a method for in vivo recombination of the DNA library); or
The method according to the invention, wherein the set of homologous DNA sequences is obtained from at least two different pre-made DNA libraries (ie resulting in a method for in vivo recombination of at least two different DNA libraries) Related to.
In order to obtain further recombination between the DNA sequences, it is preferred to isolate the DNA construct after the first round of in vivo recombination of the DNA sequences performed according to the present invention. The isolated construct is then retransformed into the cell and a further process round of in vivo recombination is then performed.
The advantages of the above process are illustrated by the following examples:
Set A is a vector A in which DNA sequences a and b are individually introduced into the vector A (ie, the population of vectors A includes a vector having the DNA sequence a and a vector having the sequence b. ); And
Set B is a vector B in which the DNA sequences c and d are individually introduced into the vector B (that is, the population of vectors B includes a vector having the DNA sequence c and a vector having the sequence d. ).
Vectors A and B are then transformed into the cell population and recombined in vivo according to the present invention. This will give recombination between the DNA sequences ac, ad, bc, bd. Vectors A and B containing the recombined sequence are isolated and retransformed into the cell population. In vivo recombination according to the invention will then give all possible recombination of the four (a, b, c, d) DNA sequences, eg abc, abdc, etc.
In a further aspect, the present invention is a method according to the present invention, wherein two sets of homologous DNA sequences (“Set A” and “Set B”) are recombined in vivo, comprising the following steps:
a) inserting the sequence from “Set A” into one vector containing the thermostable origin of replication and the gene for the first marker;
b) inserting the sequence from “Set B” into one vector containing the temperature sensitive origin of replication and the gene for the second marker;
c) introducing the two vectors into the cell;
d) optionally incubating said cells at a temperature at which both origins of replication are functional under selective pressure that favors cells carrying both marker genes;
e) shifting the temperature to a temperature where only the heat-resistant replication origin is functional;
f) Repeat steps d) to e) at least once;
In said method comprising or
A method according to the present invention wherein two sets of homologous DNA sequences ("Set A" and Set B ") are recombined in vivo, comprising the following steps:
a) inserting the sequence from “Set A” into a vector containing the thermostable origin of replication and the gene for the first marker;
b) inserting the sequence from “Set B” into one vector containing the temperature sensitive origin of replication and the gene for the second marker;
c) introducing the two vectors into the cell;
d) incubating the cells at a temperature at which only the thermostable origin of replication is functional, optionally under selective pressure that favors cells with both marker genes;
e) optionally shifting the temperature to a temperature at which both origins of replication are functional;
f) Repeat d) to e) at least once,
The method.
In a further aspect, the present invention is a method for producing one or more recombinant proteins having a desired biological activity:
a) performing a method for in vivo recombination of homologous DNA sequences, wherein at least one of the DNA constructs is an expression vector (this method is performed according to the present invention as described herein) );
b) expressing a number of different recombinant polypeptides encoded by a number of different recombinant DNA sequences from step a); and
c) Screen or select many different recombinant proteins from step b) in a suitable screening or selection system for one or more recombinant polypeptides having the desired activity.
The method.
Preferably, the expression vector in step a) above is during the forced hybrid DNA formation performed according to the method for in vivo recombination of homologous DNA sequences according to the invention (ie the incorporation of the structures into each other). A DNA construct that cannot replicate (eg, has a non-functional origin of replication). This will improve the probability that the expression vector will contain the recombined homologous DNA after the crossing out event of the hybrid structure according to the present invention. Other DNA constructs that can replicate under the conditions used to force the formation of the hybrid structure only when the expression vector can survive This is because it is actually recombined.
After the method for in vivo recombination has been carried out (above step a)) and before the expression and screening steps b) and c), it is advantageous to mainly create a cell population containing only the expression vector. (Ie, other DNA constructs are partially or nearly completely removed from the cell population). This is particularly advantageous when the expression vector is unable to replicate during the formation of the forced hybrid DNA construct as described above. This is because such cell populations will advantageously have a very limited background of the non-recombinant DNA sequence of interest, which is in the subsequent screening step (step c above)).
Mainly creating a cell population that contains only the expression vector is in accordance with standard procedures known in the art, for example, under conditions that selectively favor replication of the expression vector relative to other DNA constructs. This can be done by culturing the cells; or by selectively purifying the expression vector from the cells and then introducing it into a new cell population.
Expression of the recombinant polypeptide encoded by the recombination sequence in step a) above can be performed by use of a standard expression vector and / or a standard expression vector according to the invention It can be modified to include elements that are particularly suitable for in vivo recombination methods.
A suitable screening or selection system for screening or selecting many different recombinant proteins from step b) above will depend on its desired biological activity.
A number of screening or selection systems suitable for screening or selecting for a desired biological activity have been described in the art. These examples are
Describes a screening system for screening for subtilisin variants with calcium-independent stability, Strauberg et al. (Biotechnology 13: 669-673 (1995));
Describes a screening assay to screen for proteases with enhanced thermostability, Bryan et al. (Proteins 1: 326-334 (1986)); and
PCT-DK 96/00322, which describes a screening assay to screen for lipases with improved cleaning performance in laundry detergents
It is.
Preferred embodiments of the invention include screening or selection of recombinant proteins where the desired biological activity is improved performance in dishwashing or laundry detergents. Examples of suitable dishwashing or laundry detergents are disclosed in PCT / DK96 / 00322 and WO 95/30011.
The present invention also comprises a set (at least two) vectors comprising at least two different origins of replication capable of functioning in the same cell under permissive conditions, and at least two homologous DNA sequences encoding the polypeptide. Including. In a preferred embodiment, at least one origin of replication is temperature sensitive. The vector can preferably be a plasmid.
The present invention further comprises at least:
a) a vector A comprising a DNA sequence encoding a polypeptide and an origin of replication; and
b) a vector B comprising a DNA sequence homologous to the DNA sequence in vector A, and a replication origin different from the replication origin in vector A;
A vector system comprising
Polypeptides wherein the DNA sequence has biological activity, in particular enzymes, and in particular enzymes such as amylases, lipases, cutinases, cellulases, oxidases, phytases, and proteases,
Is preferably encoded.
The present invention is a novel, recombinant DNA sequence which is produced by the method according to the present invention or which encodes a recombinant protein having the desired biological activity, wherein the desired organism A recombinant protein having a biological activity comprises said DNA sequence, characterized in that it is made by a method for one or more recombinant proteins having a desired biological activity according to the present invention.
The invention further relates to a process for the production of a polypeptide, in particular an enzyme, i) culturing cells expressing the polypeptide encoded by the recombinant DNA sequence according to claim 38, and ii) said expression A process comprising purifying the produced polypeptide.
The invention further encompasses novel polypeptides, in particular enzymes, characterized in that they are produced by the method according to the invention.
The invention further encompasses a polypeptide, in particular an enzyme, characterized in that it is encoded by a recombinant DNA sequence according to the invention.
The polypeptides of the present invention include peptides and proteins, and optionally their glycosylated forms. Examples of these are enzymes, pharmaceutically active polypeptides, such as analogs of human or mammalian polypeptides, such as insulin, growth hormone, human hormones or growth regulators.
The invention is explained in more detail in the following examples. The examples are not intended to limit the scope of the invention in any way.
Example
Materials and methods
stock

Bio-Rad Gene Pulser as recommended by the manufacturer^TMElectrocompetent cells prepared and transformed using

According to Yasbin, RE, Wilson, GA and Young, FE (1975) Trans formation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells. J. Bacteriol, 121: 296-304 Prepared and transformed as described.
B. subtilis PL 1801. This strain is B. subtilis DN 1885 in which the apr and npr genes are disrupted

Plasmid:
pSX120: B. subtilis expression vector (described in EP 405901 and WO 91/00345). This vector contains the wild type sabinase gene.
pE194: (Horinouchi, S and Weisblum, B., 1982, J. Bacteriol.150 : 804-814).
B. subtilis expression vector (described in WO 96/34946 and WO 92/19729). This vector contains a synthetic Savinase gene referred to herein as Savisyn. A number of restriction sites have been introduced into the savicin gene as compared to the wild-type sabinase gene contained in pSX120.
pUC19: Yanisch-Perron, C., Vieria, J. and Messing, J. (1985) Gene 33, 103-119.
pF64L-S65 T-GFP; is an E. coli plasmid encoding a DNA fragment encoding F64L-S65T-GFP described in WO 97/11094.
pMUTIN-4-MCS: This plasmid can be obtained from Laboratoire de Genetique Microbienne, Institut National de la Recherche Agronomique, 78352 Jouy en Josas-CEDEX, France.
Solution / medium
TY and LB agar (Ausubel, F.M. et al. (Eds.) “Current protocols in Molecular Biology” as described in John Wiley and Sons, 1995).
LBPG is LB agar supplemented with 0.5% glucose and 0.05M potassium phosphate, pH 7.0.
The LBPGSK plate is an LBPG medium containing agar and 1% skim milk.
Antibiotics are used in different media at concentrations of 6 μg / ml chloramphenicol, 10 μg / ml kanamycin, and 5 μg / ml erythromycin.
TE-buffer (as described in Ausubel, F.M. et al. (Eds.) “Current protocols in Molecular Biology”. John Wiley and Sons, 1995).
General molecular biology method
DNA manipulation and transformation are performed using standard methods of molecular biology (Sambrook et al. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, FM et al. (Eds .) “Current protocols in Molecular Biology”, John Wiley and Sons, 1995: Harwood, CR, and Cutting, SM (eds.) “Molecular Biological Methods for Bacillus”, John Wiley and Sons, 1990) .
Enzymes for DNA manipulation were used according to the supplier's instructions.
Enzymes for DNA manipulation
Unless otherwise stated, all enzymes for DNA manipulation, such as restriction endonucleases, ligases, polymerases, PCR polymerases, etc. are obtained from New England Biolabs, Inc.
Construction of humidity-sensitive shuffling plasmid
pMB430 was constructed in a 5-step cloning method as shown below. The entire DNA sequence is found within SEQ ID NO: 1.
1) The temperature sensitive plasmid pE194 was modified by introducing a multi-flowing site near the unique ClaI site of the pE194 plasmid and thus destroying the ClaI site. This introduction was performed by PCR using primers # 22899 and 24039 to amplify the entire plasmid and introduce several unique cloning sites.
This PCR was performed as follows:
1 μl of pE194 Qiaquick plasmid prep, 200 μM of each dNTP, 2.5 units of AmpliTaq polymerase (Perkin-Elmer, Cetus, USA), and 100 pmol of each primer:

Containing PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.01% (w / v) gelatin) was used for 50 μl PCR amplification.
The PCR reaction was performed using a DNA thermal cycler (Landgraf, Germany). One incubation at 94 ° C for 1 minute followed by 40 cycles of PCR was performed using denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute, and extension at 72 ° C for 2 minutes. A 5 μl aliquot of the amplified product was analyzed by electrophoresis in a 0.7% agarose gel (NuSieve, FMC).
Subcloning of PCR fragments
A 45 μl aliquot of the PCR product generated as described above was purified using a QIAquick PCR purification kit (Qiagen, USA) according to the manufacturer's instructions. The purified DNA was eluted in 50 μl of 10 mM Tris-HCl, pH 8.5. 50 μl of the purified PCR fragment was digested with BamHI, electrophoresed in a 0.8% low gelling temperature agarose (Sea Plaque GTG, FMC) gel, the relevant fragments were excised from the gel, and the manufacturer Purified using QIAquick Gel Extraction Kit (Qiagen, USA) according to instructions. The isolated DNA fragment was then ligated into BamHI digested pUC191 and the ligation mixture was used to transform E. coli SJ2.
Cells contain erythromycin (200 μg / ml) supplemented with X-gal (SIGMA, USA) (5-bromo-4-chloro-3-indoleyl alpha-D-galactopyranoside, 50 μg / ml). Plated on LB agar plates.
Identification and characterization of positive clones
Transformed cells were plated on LB agar plates containing erythromycin (200 μg / ml) supplemented with X-gal (50 μg / ml) and incubated overnight at 37 ° C. The next day, white colonies were rescued by re-streaching them onto fresh LB-erythromycin agar plates and incubated overnight at 37 ° C. The next day, a single colony of each clone was transferred to liquid LB medium containing erythromycin (200 μg / ml) and incubated overnight at 37 ° C. with shaking at 250 rpm.
Plasmid was extracted from the liquid culture using the QIAgen plasmid purification mini kit (Qiagen, USA) according to the manufacturer's instructions. A 5 μl sample of the plasmid was digested with BamHI. This digestion was checked by electrophoresis on a 0.7% agarose gel (NuSieve, FMC). Appearance of two DNA fragments of approximately 2.7 kb and 3.7 indicated positive clones. This clone was named MB293 and this plasmid was named pMB293.
2) pMB293 was then EcoRI digested and the 3.8 kb fragment was gel purified on a 0.7% agarose gel using the QiaQuick gel purification kit from Qiagen according to the manufacturer. This fragment constituting pE194 with the inserted multiple cloning site (due to the PCR primer and MCS of pUC19) was circularized by ligation and used to transform B. subtilis DN1885 The transformed cells were selected on LBPG plates containing 5 μg / ml erythromycin and incubation was performed overnight at 33 ° C. Clones from this transformation were re-streaked and cultured overnight in TY containing 5 μg / ml erythromycin. Cells were isolated from the overnight culture broth and the plasmid was purified using the Qiaquick plasmid kit. Restriction enzyme digestion of these plasmids proved the correctness of the clone. One such clone and plasmid were designated MB333 and pMB333.
3) pMB333 was then used as a temperature sensitive plasmid for the construction of a plasmid containing the savidin gene without any promoter. This structure was made as follows.
PCR using primers # 21547 and # 21548 and pSX222 as template was performed according to the above conditions:
1 μl of pSX222 Qiaquick plasmid prep, 200 μM of each dNTP, 2.5 units of AmpliTaq polymerase (Perkin-Elmer, Cetus, USA), and 100 pmol of each primer:

Containing PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.01% (w / v) gelatin) for 50 μl PCR amplification.
The PCR reaction was performed using a DNA thermal cycler (Landgraf, Germany). One incubation at 94 ° C for 1 minute followed by 40 cycles of PCR was performed using denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute, and extension at 72 ° C for 2 minutes. A 5 μl aliquot of the amplified product was analyzed by electrophoresis in a 0.7% agarose gel (NuSieve, FMC).
Subcloning of PCR fragments
A 45 μl aliquot of the PCR product generated as described above was purified using a QIAquick PCR purification kit (Qiagen, USA) according to the manufacturer's instructions. The purified DNA was eluted in 50 μl of 10 mM Tris-HCl, pH 8.5. 50 μl of the purified PCR fragment was digested with BamHI and ApaI, electrophoresed in a 0.8% low gelling temperature agarose (Sea Plaque GTG, FMC) gel, the relevant fragments were excised from the gel, and Purified using QIAquick Gel Extraction Kit (Qiagen, USA) according to manufacturer's instructions. The purified fragment was ligated to gel purified and BamHI and ApaI digested pMB333. The ligation was then used to transform B. subtilis DN1885. Transformed cells were plated on LBPG agar plates containing 5 μg / ml erythromycin and incubated overnight at 33 ° C. Clones were re-streaked on the same plate and plasmids were obtained from overnight cultures of clones incubated at 33 ° C. in TY containing 5 μg / ml erythromycin from Qiaquick plasmid plasmids as described by the manufacturer. Isolated using kit. The plasmid was checked by enzymatic digestion using ApaI and BamHI. One correct clone was named MB339 and the plasmid was named pMB339.
4) We wanted to test a simple way to monitor the formation of hybrid plasmids. We therefore wanted to monitor forced recombination between two homologous genes on two separate plasmids. This was done by constructing a pE194 derivative with savidin lacking a promoter that was transcriptionally fused to the ORF encoding GFP. In this method, two homologous genes are not recombined and, therefore, if GFP is not placed under the control of a promoter that is otherwise present on pSX120 (upstream of the savidin and hence the GFP gene. GFP expression will be absent (by lacking a promoter). This plasmid was constructed by PCR amplification of the GFP gene and introducing it directly downstream of the Savidin gene on pMB339.
As used herein, the GFP coding DNA is a derivative of the original wild type gene, and the derivative of GFP is a mutant F64L-S65T constructed as described in International Patent Application WO 97/11094. -Cloned from a DNA construct of GFP.
The construction of the template plasmid was described in WO 97/11094. A DNA fragment encoding F64L-S65T-GFP was PCR amplified from the E. coli plasmid encoding the gene as follows:
1 μl of pF64L-S65T-GFP Qiaquick plasmid prep, 200 μM of each dNTP, 2.5 units of AmpliTaq polymerase (Perkin-Elmer, Cetus, USA), and 100 pmol of each primer:

Containing PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.01% (w / v) gelatin) for 50 μl PCR amplification.
The PCR reaction was performed using a DNA thermal cycler (Landgraf, Germany). One incubation at 94 ° C for 1 minute followed by 40 cycles of PCR was performed using denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute, and extension at 72 ° C for 2 minutes. A 5 μl aliquot of the amplified product was analyzed by electrophoresis in a 0.7% agarose gel (NuSieve, FMC).
Subcloning of PCR fragments
A 45 μl aliquot of the PCR product generated as described above was purified using a QIAquick PCR purification kit (Qiagen, USA) according to the manufacturer's instructions. The purified DNA was eluted in 50 μl of 10 mM Tris-HCl, pH 8.5. This PCR fragment and pMB339 were digested with ClaI and NsiI, gel purified as above, and ligated together. This ligation mixture was used to transform DN1885 and the transformed cells were plated on LBPG 5 μg / ml erythromycin. After 18 hours of incubation, green fluorescent cells appeared as a single colony. One of these was analyzed and stored as MB406 and the corresponding plasmid was stored as pMB406. Since the GFP-encoding ORF did not have an upstream promoter driving its transcription, a possible explanation for GFP expression is reading from the promoter further upstream of the savidin-GFP transcription fusion, ie, the promoter of the repF gene. It was that it was rare.
5) To abolish any reading of GFP into the ORF, a transcription terminator was inserted between the repF gene and the savidin DNA. This terminator was amplified from the pMUTIN-4-MCS plasmid using the following primers and conditions:
1 μl of pMUTIN-4-MCS Qiaquick plasmid prep, 200 μM of each dNTP, 2.5 units of AmpliTaq polymerase (Perkin-Elmer, Cetus, USA), and 100 pmol of each primer:

Containing PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.01% (w / v) gelatin) for 50 μl PCR amplification.
The PCR reaction was performed using a DNA thermal cycler (Landgraf, Germany). One incubation at 94 ° C for 60 seconds, followed by 40 cycles of PCR using a cycle profile of denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and extension at 72 ° C for 1 minute It was conducted. A 5 μl aliquot of the amplified product was analyzed by electrophoresis in a 0.7% agarose gel (NuSieve, FMC).
The amplified DNA was purified as above and cloned as a KpnI-BglII fragment into pMB406, also digested with BglII and KpnI (same procedure for gel purification etc. as above). This ligation mixture was used to transform DN1885 and the transformed cells were plated on LBPG 5 μg / ml erythromycin. After 18 hours of incubation, the cells were seen as a single colony. One of these was analyzed and stored as MB430, and the plasmid was similarly stored as pMB430. This clone did not express any GFP. This indicates that the cloned terminator worked as expected in practice. The entire sequence of the obtained plasmid pMB430 is represented as SEQ ID NO: 1.
Establishment of MB432 strain
PL1801 Bacillus subtilis was transformed with two plasmids pMB430 and pSX120 using approximately 1 μg of each plasmid per 100 μl of competent PL1801. The transformed cells were plated on LBPG-5 μg / ml erythromycin and incubated overnight at 33 ° C. The next day, colonies were re-streaked on LBPG-5 μg / ml erythromycin + 6 μg / ml chloramphenicol and incubated overnight at 33 ° C. Colonies were grown overnight in liquid culture TY-5 μg / ml erythromycin + 6 μg / ml chloramphenicol at 33 ° C., the plasmid was purified from the culture broth, and the presence of both pMB430 and pSX120 in the PL1801 strain was demonstrated. . This clone was saved and named MB432.
Example 1
Sequence shuffling of two homologous sequences encoding the subtilisin gene of sabinase
A sabinase wild type gene (contained within pSX120) encoding a subtilisin type protease was chosen as a target for shuffling of highly homologous genes. A site-directed variant of the sabinase gene (hereinafter referred to as savidin) contained in pSX222, where a number of silent mutations giving new restriction sites were introduced, was used as a shuffling partner for sabinase. The two genes were cloned on two separate compatible plasmids according to the present invention. A transcriptionally active sabinase gene was cloned on a plasmid based on pUB110 origin (pSX120, FIG. 4) encoding resistance to chloramphenicol. A promoter lacking the sabinase gene was cloned on a derivative of temperature sensitive plasmid pE194 (pMB430) which also encodes resistance to erythromycin. Two transcription terminators were cloned upstream of the savidin gene to prevent any reading.
In pMB430, green fluorescent protein (GFP) is transcriptionally transferred to the savidin gene so that it can visually follow the in-cross and out-cross between the two plasmids. It was fused.
B. subtilis strains MB432, pL1801 transformed with two plasmids pSX120 and pMB430 were taken through a two-cycle temperature shift to induce two consecutive double crossover events between the homologous genes. It was. Homologous recombination and plasmid hybridization were selected on LBPG plates at the limiting temperature (42-50 ° C.) in the presence of erythromycin. Under these restrictive conditions, the temperature sensitive plasmid pMB430 is forced into the stable pSX120 plasmid by homologous recombination within the sabinase / savidin region. The colonies that have undergone recombination also become fluorescent as a result of recombination between the two plasmids and the simultaneous activation of the savidin-GFP operon (FIG. 2). Out cross events were screened by culturing the clones in LB-broth at a permissive temperature of 30 ° C. for 2 days and plating the cells on LBPG plates containing chloramphenicol and erythromycin. It was.
Non-fluorescent colonies were re-streaked on the plates and picked through a second round temperature cycle as described above.
After the second round of shuffling, plasmid preparations were made from 20 different clones. Competent PL1801 was transformed with each of the 20 plasmid preps and plated on LBPG supplemented with erythromycin. These plates were replicated on LBPG containing chloramphenicol and erythromycin to identify unwanted clones in which both pSX120 and pMB430 were transformed. Clones in which only the pMB430 plasmid was transformed were selected for sequencing of the savidin region to identify whether shuffling occurred. From FIG. 6, it is clear that recombination between the two genes, sabinase and savidin occurred. All twelve sequenced clones show some degree of recombination, and four of these can be positively identified as a result of two consecutive double recombination events. It is important to note that the exchange of sabinase and savidin sequences can only be observed if the shuffled fragment overlaps with a unique restriction site for savidin. The four clones with positive evidence for two double recombination are therefore the smallest number. This is because this recombination could have occurred between two adjacent restriction sites.
Sequence listing
(2) Information about SEQ ID NO: 1:
(I) Sequence features:
(A) Length: 5313 base pairs
(B) Type: Nucleic acid
(C) Number of chains: 1 single chain
(D) Topology: Linear
(Ii) Molecular type: DNA
(Vi) Origin:
(A) Organism: Plasmid pMB430
(Xi) SEQ ID NO: 1

Claims (28)

A method for in vivo recombination of homologous DNA sequences comprising the following steps:
(A) a cell containing at least two DNA constructs, each containing a DNA sequence and an origin of replication ; i) at least one origin of replication is non-functional; and ii) the cell has the non-functional origin Incubating under the condition that it is under a selective pressure that only allows growth of the cell when it contains a DNA structure, thereby allowing one of the DNA structures to be within one of the other DNA structures. Support integration into and thereby form a hybrid DNA construct;
(B) changing from the above conditions to a condition where fewer origins of replication are non-functional than in step a), thereby crossing out from the hybrid DNA construct and replicating with the recombinant DNA sequence Support the formation of DNA structures including the origin;
(C) repeating steps a) to b) at least once,
Including said method. 2. Method according to claim 1, characterized in that at least one DNA structure is a chromosome, preferably a bacterial chromosome, more preferably a Bacillus chromosome, in particular a Bacillus subtilis chromosome. 3. The at least one DNA construct is a vector, preferably a bacterial vector, more preferably a vector capable of replicating in a Bacillus cell, in particular a Bacillus subtilis cell. the method of. 4. The method of claim 3, wherein at least two of the DNA constructs are two different vectors. A method for in vivo recombination of homologous DNA sequences comprising the following steps:
a) inserting the homologous DNA sequence into at least two different vectors containing different origins of replication;
b) introducing the vector into the cell;
The c) the cell, i) at least one origin of replication is non-functional, and ii) selective pressure for the cells to only permit the growth of the cell if it contains DNA structure having the non-functional origin under the conditions, incubated as in, thereby supporting the integration into the one of the 1 from the other DNA structures of the DNA structure, thereby forming a hybrid DNA structure;
d) changing from the above conditions to a condition where fewer origins of replication are non-functional than in step c), thereby crossing out from the hybrid DNA construct, and the recombinant DNA sequence and origin of replication. Supporting the formation of DNA structures comprising:
e) Repeat multiple times as appropriate, but at least once, steps c) to d),
Including said method. 6. At least one origin of replication is selected in such a way that the replication rate of the vector can be chemically or physically regulated. The method described in 1. 7. A method according to claim 6, characterized in that at least one origin of replication is temperature sensitive. The method according to claim 7, characterized in that the replication rate of at least one vector can be regulated by a temperature shift. 9. A method according to any one of claims 1 to 8, characterized in that the vector comprises a gene for a marker, preferably a selectable marker, in particular an antibiotic marker. 10. The method according to claim 9, characterized in that the vector contains genes for different markers. The method according to any one of claims 1 to 10, wherein the at least one DNA structure does not contain ori (-) and contains only ori (+). 12. The DNA structure which does not contain ori (−) is functional at its origin during the formation of a hybrid DNA structure carried out according to the method of any one of claims 1-11. The method according to claim 11, which is a DNA construct of The formation of the hybrid structure is measured in vivo, preferably by having a system of DNA structures, where a selectable protein in vivo is expressed only after the hybrid structure is formed. A method according to any one of claims 1-12. 14. The method of claim 13, wherein the selectable protein is a protein that is fluorescent under suitable exposure, and in particular the fluorescent protein is green fluorescent protein (GFP) or a variant thereof. 15. A method according to any one of the preceding claims, wherein the crossing out from the hybrid structure is a forced crossing out. Forced crossing out from the hybrid structure:
i) the DNA hybrid construct does not express a selectable marker and at least one of the at least two DNA constructs of any one of claims 1-15 expresses the selectable marker; Build a system that can
ii) Since the cells can express the marker gene only after the crossing out of the DNA hybrid structure has occurred, the cells are incubated under selectable conditions, thereby Force a cross-out,
16. The method of claim 15, wherein The selectable marker in step i) is on the DNA construct where its origin of replication is non-functional during forced integration of the DNA construct into the DNA hybrid construct performed as described above. 17. The method of claim 16, wherein the method is expressed in and preferably the selectable marker gene is an antibiotic resistance gene. 18. A method according to any one of claims 1 to 17, wherein the homologous DNA sequence comprises a DNA sequence from at least one pre- made DNA library. 19. The homologous DNA sequence is made from two or more different sets of homologous DNA sequences, and each of the sets of homologous sequences is inserted into a different DNA structure according to any one of claims 1-18. 19. The method according to any one of claims 1 to 18, wherein: At least one of the set of homologous DNA sequences is obtained from a pre-made DNA library, and at least one of the set of homologous DNA sequences is a DNA sequence that is homologous to the DNA library 20. The method of claim 19. 20. The method of claim 19, wherein the set of homologous DNA sequences is obtained from the same pre-made DNA library. 20. The method of claim 19, wherein the set of homologous DNA sequences is obtained from at least two different pre- made DNA libraries. 20. The method of claim 19, wherein each set of homologous DNA sequences comprises only one DNA sequence. The cells containing the DNA construct, characterized in that with that is defective in the enzymatic mismatch repair system or temporarily being deactivated or reduced level of mismatch modification of claims 1 to 23 The method according to any one of the above. 1. The cell according to claim 1, characterized in that it is a Gram-positive cell, for example a Staphylococcus cell, a Streptococcus cell, a Bacillus cell, or in particular a Bacillus subtilis cell. the method according to any one of 1-24. 25. A method according to any one of claims 1 to 24 , characterized in that the cell is a Gram negative cell, preferably an Escherichia cell, in particular an E. coli cell. Said homologous DNA sequence, a polypeptide, especially an enzyme, and in particular enzymes, for example, amylase, lipase, cutinase, cellulase, oxidase, wherein the encoding phytase, and a protease, any claim 1 to 26 The method according to claim 1. The following steps :
a) performing the method for in vivo recombination of homologous DNA sequences according to any one of claims 1 to 27 , wherein at least one of said DNA constructs is an expression vector;
b) expressing many different recombinant polypeptides encoded by many different recombinant DNA sequences from step a); and c) preferred for one or more recombinant polypeptides having the desired activity Screen or select many different recombinant proteins from step b) in a simple screening or selection system,
A process for producing one or more recombinant proteins having a desired biological activity .

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