JP5134953B2 - 改良された酵素 - Google Patents
改良された酵素 Download PDFInfo
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- JP5134953B2 JP5134953B2 JP2007519719A JP2007519719A JP5134953B2 JP 5134953 B2 JP5134953 B2 JP 5134953B2 JP 2007519719 A JP2007519719 A JP 2007519719A JP 2007519719 A JP2007519719 A JP 2007519719A JP 5134953 B2 JP5134953 B2 JP 5134953B2
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- Prior art keywords
- gtp cyclohydrolase
- amino acid
- gch2
- enzyme
- modified
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Description
(i)修飾されたGTPシクロヒドロラーゼIIのアミノ酸配列は、対応する修飾されていないGTPシクロヒドロラーゼIIのアミノ酸配列と比較する場合、少なくとも1つの変異を含有し、および
(ii)少なくとも1つの変異は、配列番号2に示される枯草菌(Bacillus subtilis)GTPシクロヒドロラーゼIIのアミノ酸配列の261、270、276、279、308および347位に対応するアミノ酸位置からなる群から選択される1つもしくはそれ以上のアミノ酸位置に存在する。
(i)修飾された酵素の比活性は、対応する修飾されていない酵素と比較して増加し、および
(ii)修飾された酵素のアミノ酸配列は、配列番号2の261、270、276、279、308および/または347位に対応するアミノ酸位置において1、2、3、4、5、または6つの変異を含む1つもしくはそれ以上の変異を含んでなる。
(i)修飾された酵素の比活性は、対応する修飾されていない酵素と比較して増加し、および
(ii)修飾された酵素のアミノ酸配列は、配列番号2の261、270、276、279、308および/または347位に対応するアミノ酸位置において1、2、3、4、5、または6つの変異を含む1つもしくはそれ以上の変異を含んでなる。
(a)本発明の宿主細胞を、適切な培地において、前記宿主細胞において修飾されたGTPシクロヒドロラーゼIIの発現を可能にする条件下で培養すること;および
(b)場合により、培地から産物(リボフラビン、リボフラビン前駆体、FMN、FAD、または1つもしくはそれ以上のそれらの誘導体)を分離すること。
(a)本発明の宿主細胞を、本発明の修飾されたGTPシクロヒドロラーゼIIの発現を可能にする条件下で培養すること;および
(b)細胞または培地から修飾されたGTPシクロヒドロラーゼIIを回収すること。
(a)望ましくは、増加すべきである比活性を伴う第1のGTPシクロヒドロラーゼIIをコードするポリヌクレオチドを提供すること;
(b)第1のGTPシクロヒドロラーゼIIと比較する場合、変異型ポリヌクレオチド配列が、1つもしくはそれ以上の変異を含んでなる修飾されたGTPシクロヒドロラーゼIIをコードするように、1つもしくはそれ以上の変異をポリヌクレオチド配列に導入することであって、ここで、1つもしくはそれ以上の変異は、配列番号2の261位、270位、276位、279位、308位および/または347位に対応するアミノ酸位置において1、2、3、4、5、または6つの変異を含む;
(c)場合により、変異型ポリヌクレオチドをベクターまたはプラスミドに挿入すること;
(d)ポリヌクレオチドまたはベクターもしくはプラスミドを適切な宿主細胞に導入すること;ならびに
(e)修飾されたGTPシクロヒドロラーゼIIの発現を可能にする条件下で宿主細胞を培養すること。
(a)望ましくは、増加すべきである比活性を伴う第1のGTPシクロヒドロラーゼIIをコードするポリヌクレオチドを提供すること;
(b)比活性に対する効果を有する位置を提供すること;
(c)(b)において規定される野生型GTPシクロヒドロラーゼIIの所定のアミノ酸の置き換えのための至適アミノ酸を規定し、変異型ポリヌクレオチド配列が新規のGTPシクロヒドロラーゼIIをコードするように、1つもしくはそれ以上の変異を、(b)において規定される位置で(a)のポリヌクレオチド配列に導入すること;
(d)場合により、変異型ポリヌクレオチドをベクターまたはプラスミドに挿入すること;
(d)ポリヌクレオチドまたはベクターもしくはプラスミドを適切な宿主細胞に導入すること;ならびに
(e)修飾されたGTPシクロヒドロラーゼIIの発現を可能にする条件下で宿主細胞を培養すること。
GTPシクロヒドロラーゼII活性を測定するために使用される酵素アッセイは、リッツ(Ritz)ら(J.Biol.Chem.276,22273−22277,2001年)から適応した。最終アッセイ緩衝液は、50mM Tris/HCl、pH8.5、10mM MgCl2、7.5mMメルカプトエタノール、2.5mM GTPおよび0.1mg/mlウシ血清アルブミンを含有した。精製(実施例5を参照のこと)後、酵素を、50mM Tris/HCl、pH8.5、10mM MgCl2、7.5mMメルカプトエタノール、および10%グリセロールを含有する緩衝液中に保持した。基質を酵素に添加し、GTPが吸収を示さない310nmでの吸収を、20〜30分間、追跡した。最終反応混合物は、枯草菌(B.subtilis)または表1もしくは2に示される変異体の1つ由来の0.02〜0.04mg/mlの間のGTPシクロヒドロラーゼIIを含有した。DRAPPに対する6.28[mM−1 cm−1]の吸収係数を、活性の算出に使用した。タンパク質の決定は、Bio−Rad由来のProtein Assay(カタログ番号500−0002,Bio−Rad Laboratories AG,Nenzlingerweg2,CH−4153Reinach、瑞国)で実施した。
至適アッセイは、酵素濃度との直線性および時間との直線性のような多くの要件を満たすべきである。実施例1に記載の条件および22μgの酵素を使用して、310nmでの吸収の増加を、25分間、追跡した。どの範囲で、アッセイが、酵素濃度で直線であるかを試験するために、増加する酵素濃度(0〜40μgのHis6−タグ化RibA)に対するアッセイの依存性について試験した。アッセイは、25分間、枯草菌(B.subtilis)由来の0〜40μgのHis6−タグ化RibAで、直線であることが証明された。
枯草菌(B.subtilis)を、30℃で、Veal Infusion Broth(Becton Dickinson、Sparks、メリーランド州21152、米国)中、1晩、増殖させた。1.5ml培養物を1.5mlチューブに移し、遠心分離した。
GTPシクロヒドロラーゼIIおよびDHBPシンターゼをコードする枯草菌(B.subtilis)のribA遺伝子(配列番号1)のクローニングを、PCRによって行った。枯草菌(B.subtilis)のゲノムDNAを、実施例3に従って単離した。このDNAまたはribA遺伝子の変異型をコードするテンプレートの100ngを、プライマーRibA1S(配列番号27)およびRibA1AS(配列番号28)を使用するPCRに使用した。次のPCR条件を使用した:DNAポリメラーゼと共に供給される適切な緩衝液中の2μMの各プライマー、0.2mMの各ヌクレオチド、2.5UのプルーフリーディングDNAポリメラーゼ(Stratagene、Gebouw California、1101CB Amsterdam Zuidoost、蘭国)、および100ngのゲノムDNA。
工程1:95℃で3分間
工程2:95℃で30秒間
工程3:52℃で30秒間
工程4:72℃で60秒間
工程2〜4を30回反復した。
変異型酵素の作製を、上に記載および当業者に公知である方法を使用して、実施した。さらに調べた枯草菌(B.subtilis)由来のRibAの変異体を、表1に示す。すべての変異遺伝子を、実施例4に記載のpQE60ベクターにクローニングした。すべての最終構築物は、N末端His6−タグを含有した。
以下の実施例では、RibA Y196C、A276T、A282T(PCR III)、RibA Y196C、A276T、Q279R、A282T、K308R、M347I(構築物C)、およびRibA Y196C、A276T、Q279R、A282T、K308R、F325Y、M347I(構築物E)の変異型ribAポリヌクレオチド配列を、最初に、強力な構成性プロモーターPvegIを含有するベクターに導入し、次いで、大腸菌(E.coli)においてさらに操作した。ポリヌクレオチド配列およびフランキングベクター配列による天然のコンピテント枯草菌(B.subtilis)微生物の形質転換により、変異型ribAを過剰発現する枯草菌(B.subtilis)株を生じた。標準的な組換えDNA技術を、ポリヌクレオチド配列および枯草菌(B.subtilis)株の構築に使用した。例えば、サンブルック(Sambrook)ら、Molecular Cloning.A Laboratory Manual(第2版)、Cold Spring Harbor Laboratory Press (1989年)、ならびにハーウッド(Harwood)およびカッティング(Cutting)、Molecular Biology Methods for Bacillus,John Wiley and Sons(1990年)を参照のこと。
GTPシクロヒドロラーゼIIの比活性に影響を及ぼす変異のインビボ効果について試験するために、枯草菌(Bacillus subtilis)GTPシクロヒドロラーゼII(RibA)変異PCR III、構築物C、または構築物Eを、株RB50::(pRF69)n::(pRF93)m(パーキンス(Perkins)ら、J.Ind.Microbiol.Biotechnol.22、8−18、1999年)のようなリボフラビン過剰産生枯草菌(B.subtilis)株に、例えば、sacB遺伝子座において導入した。リボフラビンの産生を、ribA遺伝子における変異の有無のみが異なる枯草菌(B.subtilis)の2つの組換え株において直接比較した。バチルス(Bacillus)株の培養を、実施例8に記載のとおりに行った。
リボフラビン産生を、PvegIプロモーターによって駆動されるGTPシクロヒドロラーゼII変異体PCR III、構築物C、または構築物EがsacB遺伝子座に組込まれた(実施例6を参照のこと)リボフラビン過剰産生枯草菌(B.subtilis)株RB50::(pRF69)n::(pRF93)mの流加培養において試験した。株の発酵は、405370号明細書に記載のとおりに行った。
リボフラビンの決定のために、以下の分析方法を使用することができる(ブレットゼル(Bretzel)ら、J.Ind.Microbiol.Biotechnol.22、19−26、1999年)。
SWISS−PROTおよびTrEMBL(図1を参照のこと)のような標準的なデータベースを使用するプログラムBLASTNによって見出される92の異なるGTPシクロヒドロラーゼII酵素の複数のアミノ酸配列アラインメントを、次のパラメータ:ギャップサクセイペナルティ(gap creation penalty)8、ギャップ伸長ペナルティ(gap extension penalty)2、およびブロサム62.cmpマトリックス(blosum62.cmp matrix)(デフォルトパラメータ)を使用するプログラム「PILEUP」(GCG Wisconsin Package、バージョン10.2、Accelrys Inc.、9685 Scranton Road、San Diego、カリフォルニア州92121−3752、米国)により算出した。
(1)gch2_bacsu:SWISS−PROT:gch2_bacsu(枯草菌(Bacillus subtilis))
(2)gch2_cangu:geneseqp:aay69776(カンジダ・ギリエルモンディ(Candida guilliermondii))
(3)gch2_ashgo:TrEMBL:CAA02912:(アシブヤ・ゴッシピィ(Ashbya gossypii)エレモセシウム・ゴシピィ(Eremothecium gosypii))
(4)gch2_酵母:SWISS−PROT:gch2_酵母(サッカロミセス・セレビシエ(Saccharomyces cerevisiae))
(5)gch2_neucr:TrEMBL:Q871B3(アカパンカビ(Neurospora crassa))
(6)gch2_schpo:TrEMBL:Q9P7M9(シゾサッカロミセス・ポンベ(Schizosaccharomyces pombe))
(7)gch2_arcfu:SWISS−PROT:gch2_arcfu(アーケグロバス・フルギダス(Archaeoglobus fulgidus))
(8)gch2_strco:SWISS−PROT:gch2_strco(ストレプトミセス・コエリカラー(Streptomyces coelicolor))
(9)gch2_helpj:SWISS−PROT:gch2_helpj(ヘリコバクター・ピロリ(Helicobacter pylori)J99)
(10)gch2_helpy:SWISS−PROT:gch2_helpy(ヘリコバクター・ピロリ(Helicobacter pylori))
(11)gch2_pyrfu:TrEMBL:Q8U4L7(パイロコッカス・フリオサス(Pyrococcus furiosus))
(12)gch2_thema:SWISS−PROT:gch2_thema(サーモトガ・マリティマ(Thermotoga maritima))
(13)gch2_chlmu:SWISS−PROT:gch2_chlmu(クラミジア・ムリダルム(Chlamydia muridarum))
(14)gch2_chltr:SWISS−PROT:gch2_chltr(クラミジア・トラコマチス(Chlamydia trachomatis)
(15)gch2_chlca:TrEMBL:AAP05635(クラミジア・キャビエ(Chlamydia caviae)GPIC)
(16)gch2_chlpn:SWISS−PROT:gch2_chlpn(クラミジア・ニューモニエ(Chlamydia pneumoniae))
(17)gch2_arath:SWISS−PROT:gch2_arath(シロイヌナズナ(Arabidopsis thaliana))
(18)gch2_lyces:TrEMBL:CAC09119(トマト(Lycopersicum esculentum))
(19)gch2_orysa:TrEMBL:AAO72560(オリザ・サティバム(Oryza sativum))
(20)gch2_alceu:TrEMBL:Q9F184(アルカリゲネス・ユートロファス(Alcaligenes eutrophus))
(21)gch2_neima:SWISS−PROT:gch2_neima(髄膜炎菌(Neisseria meningitidis)(血清型A))
(22)gch2_neimb:SWISS−PROT:gch2_neimb(髄膜炎菌(Neisseria meningitidis)(血清型B))
(23)gch2_psepk:SWISS−PROT:gch2_psepk(シュードモナス・プチダ(Pseudomonas putida)(株KT2440))
(24)gch2_psesm:SWISS−PROT:gch2_psesm(シュードモナス・シリンゲ(Pseudomonas syringae)(pv.トマト))
(25)gch2_actac:TrEMBL:Q9JRR0(アクチノバシラス・アクチノマイセテムコミタンス(Actinobacillus actinomycetemcomitans)(ヘモフィルス・アクチノマイセテムコミタンス(Haemophilus actinomycetemcomitans)))
(26)gch2_haein:SWISS−PROT:gch2_pasmugch2_haein(インフルエンザ菌(Haemophilus influenzae))
(27)gch2_pasmu:SWISS−PROT:(パスツレラ菌(Pasteurella multocida))
(28)gch2_ecO6:TrEMBL:Q8FHU5(大腸菌(Escherichia coli)O6)
(29)gch2_ecoli:SWISS−PROT:gch2_ecoli(大腸菌(Escherichia coli))
(30)gch2_salty:TrEMBL:Q8XFY7(ネズミチフス菌(Salmonella typhimurium))
(31)gch2_yerpe:TrEMBL:Q8ZEF0(ペスト菌(Yersinia pestis))
(32)gch2_bucai:SWISS−PROT:gch2_bucai(ブフネラ・アフィディコラ(Buchnera aphidicola)(亜種エンドウヒゲナガアブラムシ(Acyrthosiphon pisum))(エンドウヒゲナガアブラムシ(Acyrthosiphon pisum)共生細菌))
(33)gch2_bucap:SWISS−PROT:gch2_bucap(ブフネラ・アフィディコラ(Buchnera aphidicola)(亜種ムギミドリアブラムシ(Schizaphis graminum)))
(34)gch2_wigbr:SWISS−PROT:gch2_wigbr(ウィグルスウォルシア・グロスインディア・ブレビパルピス(Wigglesworthia glossinidia brevipalpis))
(35)gch2_bucbp:SWISS−PROT:gch2_wigbr(ブフネラ・アフィディコラ(Buchnera aphidicola)(亜種バイゾンギア・ピスタシエ(Baizongia pistaciae)))
(36)gch2_mycle:TrEMBL:Q9CCP4(らい菌(Mycobacterium leprae))
(37)gch2_myctu:SWISS−PROT:gch2_myctu(ヒト型結核菌(Mycobacterium tuberculosis))
(38)gch2_coref:TrEMBL:Q8FT57(コリネ菌(Corynebacterium efficiens))
(39)gch2_corgl:GENESEQP:AAB79913(コリネバクテリウム・グルタミカム(Corynebacterium glutamicum))
(40)gch2_coram:SWISS−PROT:gch2_coram(コリネバクテリウム・アンモニアゲネス(Corynebacterium ammoniagenes)(ブレビバクテリウム・アンモニアゲネス(Brevibacterium ammoniagenes)))
(41)gch2_staau:TrEMBL:Q8NW14(黄色ブドウ球菌(Staphylococcus aureus)(株MW2))
(42)gch2_staep:GENESEQP:ABP40248(表皮ブドウ球菌(Staphylococcus epidermidis))
(43)gch2_actpl:SWISS−PROT:gch2_actpl(アクチノバチルス・プルロニューモニエ(Actinobacillus pleuropneumoniae))
(44)gch2_lacla:TrEMBL:Q9CGU7(ラクトコッカス・ラクチス(Lactococcus lactis)(亜種ラクティス(lactis))(ストレプトコッカス・ラクティス(Streptococcus lactis)))
(45)gch2_stcag:TrEMBL:Q8E658(ストレプトコッカス・アガラクティエ(Streptococcus agalactiae)(血清型III))
(46)gch2_stcpn:TrEMBL:Q8DRF1(肺炎球菌(Streptococcus pneumoniae)(株ATCCBAA255/R6))
(47)gch2_cloac:TrEMBL:Q97LG9(Clostridium acetobutylicum)
(48)gch2_fusnu:TrEMBL:Q8RIR1(フソバクテリウム・ナクレタム(Fusobacterium nucleatum)(亜種ナクレタム(nucleatum)))
(49)gch2_anasp:TrEMBL:Q8RIR1(アナベナ(Anabaena)sp.(株PCC7120))
(50)gch2_syny3:SWISS−PROT:gch2_syny3(シネコシスティス(Synechocystis)sp.(株PCC6803))
(51)gch2_synel:TrEMBL:Q8DI64シネココッカス・エロンガータス(Synechococcus elongatus)(サーモシネココッカス・エロンガータス(Thermosynechococcus elongatus))
(52)gch2_bacam:SWISS−PROT:gch2_bacam(バチルス・アミロリクエファシエンス(Bacillus amyloliquefaciens))
(53)gch2_bacce:TrEMBL:AAP11030(セレウス菌(Bacillus cereus)ATCC14579)
(54)gcn2_bacha:TrEMBL:Q9KCL5(バチルス・ハロデュランス(Bacillus halodurans))
(55)gch2_clope:TrEMBL:Q8XMX0(ウェルシュ菌(Clostridium perfringens))
(56)gch2_clote:TrEMBL:Q897Q8(破傷風菌(Clostridium tetani))
(57)gch2_chlte:TrEMBL:Q8KC35(クロロビウム・テピダム(Chlorobium tepidum))
(58)gch2_aquae:SWISS−PROT:gch2_aquae(アクイフェックス・エオリカス(Aquifex aeolicus))
(59)gch2_lepin:TrEMBL:Q8F701(レプトスピラ・インテロガンス(Leptospira interrogans))
(60)gch2_deira:TrEMBL:Q9RXZ9(デイノコッカス・ラディオデュランス(Deinococcus radiodurans))
(61)gch2_bacth:TrEMBL:Q8A528(バクテロイデス・テタイオタオミクロン(Bacteroides thetaiotaomicron))
(62)gch2_caucr:TrEMBL:Q9A9S5(カウロバクター・クレセンタス(Caulobacter crescentus))
(63)gch2_coxbu:TrEMBL:AAO90191(コクシエラ・バーネッティ(Coxiella burnetii)RSA493)
(64)gch2_rhiet:TrEMBL:Q8KL38(リゾビウム・エティリ(Rhizobium etli))
(65)gch2_lacpl:TrEMBL:Q88X17(ラクトバチルス・プランタラム(Lactobacillus plantarum))
(66)gch2_psegl:TrEMBL:Q8RS38(シュードモナス・グルメ(Pseudomonas glumae))
(67)gch2_strav:TrEMBL:BAC71833(ストレプトミセス・アバーミチリス(Streptomyces avermitilis))
(68)gch2_phopo:SWISS−PROT:gch2_phopo(フォトバクテリウム・ホスホレウム(Photobacterium phosphoreum))
(69)gch2_azobr:SWISS−PROT:gch2_azobr(アゾスピリラム・ブラシレンセ(Azospirillum brasilense))
(70)gch2_agrtu:TrEMBL:Q8UHC9(アグロバクテリム・ツメファシエンス(Agrobacterium tumefaciens)(株C58/ATCC33970))
(71)gch2_rhime:TrEMBL:Q92RH2(リゾビウム・メリロティ(Rhizobium meliloti)(シノリゾビウム・メリロティ(Sinorhizobium meliloti)))
(72)gch2_brume:TrEMBL:Q8YFL5(マルタ熱菌(Brucella melitensis))
(73)gch2_brusu:TrEMBL:Q8G298(ブタ流産菌(Brucella suis))
(74)gch2_rhilo:TrEMBL:Q985Z3(リゾビウム・ロッティ(Rhizobium loti)(メソルヒゾビウム・ロッティ(Mesorhizobium loti)))
(75)gch2_braja:TrEMBL:Q89RZ7(ブラジリゾビウム・ジャポニクム(Bradyrhizobium japonicum))
(76)gch2_niteu:TrEMBL:CAD86468(ニトロソモナス・エウロパエア(Nitrosomonas europaea)ATCC19718)
(77)gch2_ralso:TrEMBL:Q8Y1H7(ラルストニア・ソラナセアラム(Ralstonia solanacearum)(シュードモナス・ソラナセアラム(Pseudomonas solanacearum)))
(78)gch2_neime:TrEMBL:Q9JZ77(髄膜炎菌(Neisseria meningitidis)(血清型B、見出された第2の酵素))
(79)gch2_xanax:TrEMBL:Q8PPD7(キサントモナス・アキソノポディス(Xanthomonas axonopodis)(pv.シライ(citri)))
(80)gch2_xanca:TrEMBL:Q8PCM8(キサントモナス・キャンペストリス(Xanthomonas campestris)(pv.キャンペストリス(campestris)))
(81)gch2_vibpa:TrEMBL:Q87RU5(腸炎ビブリオ菌(Vibrio parahaemolyticus))
(82)gch2_vibvu:TrEMBL:Q8DF98(ビブリオ・バルニフィカス(Vibrio vulnificus))
(83)gch2_vibch:TrEMBL:Q9KPU3(コレラ菌(Vibrio cholerae))
(84)gch2_vibfi:TrEMBL:Q8G9G5(ビブリオ・フィシェリ(Vibrio fischeri))
(85)gch2_sheon:TrEMBL:Q8EBP2(シェワネラ・オネイデンシス(Shewanella oneidensis))
(86)gch2_phoph:TrEMBL:Q8G9H7(フォトバクテリウム・ホスホレウム(Photobacterium phosphoreum))
(87)ribb_phole:TrEMBL:Q93E93(フォトバクテリウム・レイオグナティ(Photobacterium leiognathi))
(88)gch2_psepu:TrEMBL:Q88GB1(シュードモナス・プチダ(Pseudomonas putida)(株KT2440、見出された第2の酵素))
(89)gch2_psesy:TrEMBL:Q882G0(シュードモナス・シリンゲ(Pseudomonas syringae)(pv.トマト、見出された第2の酵素))
(90)gch2_pseae:TrEMBL:Q9HWX4(シュードモナス・エアルギノサ(Pseudomonas aeruginosa))
(91)ribb_dehmu:SWISS−PROT:ribb_dehmu(デハロスピリラム・マルチボランス)
(92)gch2_xylfa:TrEMBL:Q87D69(キシレラ・ファスチディオサ(Xylella fastidiosa)(株テメキュラ1/ATCC700964))
Claims (7)
- 修飾されたGTPシクロヒドロラーゼIIであって、対応する修飾されていない酵素と比較して少なくとも10%高い比活性を示し、修飾された酵素のアミノ酸配列は、配列番号18および22からなる群から選択される、修飾されたGTPシクロヒドロラーゼII。
- 請求項1に記載の修飾されたGTPシクロヒドロラーゼIIをコードするヌクレオチド配列を含んでなるポリヌクレオチド。
- 請求項1に記載の修飾されたGTPシクロヒドロラーゼIIを含んでなる、宿主細胞。
- 枯草菌(Bacillus subtilis)である、請求項3に記載の宿主細胞。
- 適切な培地において請求項3または4に記載の宿主細胞を培養することを含んでなる、リボフラビン、FMNまたはFADを産生するための方法。
- (a)配列番号2の修飾されていないGTPシクロヒドロラーゼIIを準備すること、
および
(b)196、276、279、282、308、325および/または347位のアミノ酸位置におけるアミノ酸置換を導入して配列番号18または22の修飾されたGTPシクロヒドロラーゼIIをもたらすこと、
を含んでなる、請求項1に記載の修飾されたGTPシクロヒドロラーゼIIを産生するための方法。 - リボフラビン、FMNまたはFADの産生のための請求項1に記載の修飾されたGTPシクロヒドロラーゼIIまたは請求項3または4に記載の宿主細胞の使用。
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