JP2020506722A - 目的分子を産生するための遺伝的に最適化された微生物 - Google Patents
目的分子を産生するための遺伝的に最適化された微生物 Download PDFInfo
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Abstract
Description
定義
「組換え微生物」、「改変された微生物」および「組換え宿主細胞」という用語は、本明細書で互換的に使用され、内因性ヌクレオチド配列を発現もしくは過剰発現し、異種ヌクレオチド配列を発現するように遺伝的に改変された微生物、または内因性遺伝子の変化した発現を有する微生物を指す。「変化」とは、遺伝子の発現、または1つ以上のポリペプチドもしくはポリペプチドサブユニットをコードするRNA分子もしくは同等のRNA分子のレベル、または1つ以上のポリペプチドもしくはポリペプチドサブユニットの活性が調節されることを意味することから、発現、レベルまたは活性は、改変がない場合に観察されるものよりも高くなるかまたは低くなる。
本発明は、内因性または外因性の目的分子の産生のための遺伝的に改変された微生物を提案する。
− 機能性RuBisCO(EC4.1.1.39)の発現;
− 機能性PRK(EC2.7.1.19)の発現;
− ペントースリン酸経路の非酸化分岐の少なくとも部分的な阻害;および
− 目的分子の合成および/もしくは生物変換に関与する少なくとも1つの遺伝子の発現、ならびに/または目的分子の合成および/もしくは生物変換と競合する活性をコードする少なくとも1つの遺伝子の阻害。
本発明によれば、微生物は、機能性RuBisCOおよび機能性PRKを自然に発現することができる。これは、例えば、微細藻類および藍藻類などの光合成微生物の場合に当てはまる。
本発明によれば、ペントースリン酸経路の非酸化分岐は少なくとも部分的に阻害されていることから、微生物は、ペントースリン酸経路を介して解糖経路にもはや入ることができなくなる。
特定の一つの実施形態において、本発明による遺伝的に改変された微生物は、Entner−Doudoroff経路を有し、これは少なくとも部分的に阻害されている。主に細菌(特にグラム陰性菌)に見られるこの経路は、グルコースからピルビン酸を産生するための解糖およびペントース経路に代わるものである。より正確には、この経路は、P−グルコン酸でペントースリン酸経路に接続し、特にピルビン酸で解糖を供給する。
本発明によれば、遺伝的に改変された微生物は、目的の外因性分子の産生および/または目的の内因性分子の過剰産生を行うように形質転換されている。
a)ペントースリン酸経路および解糖を使用する野生型株と本発明による改変株との間のグルコースからの炭素固定収率の比較
(1)3グルコース+5ATP+6NADP++3H2O→5G3−P+5ADP+6NADPH+11H++3CO2
G3Pからピルビン酸形成に進むと、次の平衡に到達する:
(2)3グルコース+5ADP+6NADP++5NAD++5Pi→5ピルビン酸+5ATP+6NADPH+5NADH+11H++3CO2+2H2O
1モルのグルコースの平衡を正規化すると、次の収率が得られる:
(3)グルコース+1.67ADP+2NADP++1.67NAD++1.67Pi→1.67ピルビン酸+1.67ATP+2NADPH+1.67NADH+3.67H++CO2+0.67H2O
(5)グルコース+2ATP+2NADP++2H2O→23PG+2ADP+2NADPH+6H+
3PGからピルビン酸形成に進むと、次の平衡に到達する:
(6)グルコース+2NADP+→2ピルビン酸+2NADPH+4H+
バイオインフォマティクスによるアプローチでは、フラックス平衡解析(FBA)も実施して、本発明に従って記載される改変が異なる生合成経路の収率に与える影響をシミュレートした。
FBAシミュレーションは、OptFluxソフトウェア(Rocha et al.,BMC Syst Biol.2010 Apr 19;4:45.doi:10.1186/1752−0509−4−45)と、サッカロミセス・セレビシエ代謝モデルiMM904(Mo et al.,BMC Syst Biol.2009 Mar 25;3:37.doi:10.1186/1752−0509−37)を用いて実施した。このモデルは、(i)PRK型反応の追加、(ii)RuBisCO型反応の追加を伴う異種CO2固定経路を含む、本発明に従って記載される改善を含むように改変した。
得られた理論収率および本発明に従って提供される改善の割合を以下の表7に記載する。
CMolX/CMolGLUC:消費されたグルコースの炭素のモル数に対する、産生された分子Xの炭素のモル数
gX/gGLUC:消費されたグルコースのg数に対する、産生された分子Xのg数
サッカロミセス・セレビシエの酵母株である、市販の株CEN.PK 113−7D(GenBank:JRIV00000000)に由来するCEN.PK 1605(Mat a HIS3 leu2−3.112 trp1−289 ura3−52 MAL.28c)を、CO2損失なしでNADPHを産生し、ひいてはグルコースからのα−ファルネセン産生を改善するようにエンジニアリングする。
ペントースリン酸経路の非酸化分岐は、TAL1遺伝子とそのパラログNQM1の欠失によって不活性化した。
そのために、プラスミドpUG6(P30114)−Euroscarfに含まれるKanMXカセットに由来するG418耐性遺伝子のコーディングフェーズ(coding phase)を、オリゴヌクレオチドSdtal1−Rdtal1(表8)で増幅した。
hphMXカセット(loxP−pAgTEF1−hphMX−tAgTEF1−loxP)に由来し、プラスミドpUG75(P30671)−Euroscarfに含まれるハイグロマイシンB耐性遺伝子のコーディングフェーズを、オリゴヌクレオチドSdnqm1およびRdnqm1(表8)で増幅する。これにより、トランスアルドラーゼNQM1遺伝子座の相同組換え配列をその両端に含むΔnqm1 PCRアンプリコンを生成することが可能になる。
解糖の代替経路を作成し、株EQ−0521(CEN.PK 1605 Δtal1::kan Δnqm1::hph)がCO2を固定することによって特定の代謝産物の収率を増加させるために、株は以下を発現するように改変する:
・ リブロース−5Pを消費してリブロース−1.5bisPを与えることによってペントースリン酸経路に移植するホスホリブロキナーゼPRKをコードする遺伝子ならびに
・ I型RuBisCO(構造遺伝子RbcLおよびRbcSおよびシャペロンRbcX、GroESおよびGroELを有する)。RuBisCOは、リブロース−1.5bisPと1モルのCO2を消費して3−ホスホグリセリン酸を形成する。
EQ−0523(CEN.PK1605 Δtal1::kan Δnqm1::hph) (pFPP45+pFPP56+pFPP20)
EQ−0524(CEN.PK1605 Δtal1::kan Δnqm1::hph) (pL4+pFPP56+pFPP20)
EQ−0525(CEN.PK1605)(pL5+pFL36+pCM185)
ペントースリン酸経路の非酸化分岐がTAL1遺伝子およびNQM1遺伝子の阻害によって阻害されているサッカロミセス・セレビシエ株EQ−0524を、外因性PRKとRuBisCOを使用してCO2損失なしでNADPHを過剰産生することによりファルネセンを産生するために増殖させる。この目的株を、TAL1およびNQM1の欠失または外因性PRKとRuBisCOの添加なしで、異種ファルネセンシンターゼの添加後にファルネセンを産生する参照株EQ−0525と比較する。エルレンマイヤーフラスコでのバッチ式培養は、上記の条件下で行う。
α−ケトグルタル酸デヒドロゲナーゼ遺伝子の欠失が、グルタミン酸産生を増加させる(Usuda et al.J Biotechnol.2010 May 3;147(1):17−30.doi:10.1016/j.jbiotec.2010.02.018)ことは既に記載した。したがって、以下に記載される実験は、sucA遺伝子が欠失された大腸菌K12株MG1655で行った。この株は、大腸菌の遺伝子欠失バンク(Baba et al.Mol Syst Biol.2006;2:2006.0008)に由来し、Coli Genetic Stock CenterからJW0715−2という名称で、参照番号8786(JW0715−2:MG1655 ΔsucA::Kan)で提供される。
上記の株JW0715−2の構築に使用したものと同じ欠失ストラテジーを再利用できるようにするため、リコンビナーゼを使用して選択カセットを除去する必要があった。
edd−edaオペロンの欠失は、相同組換えと、サプライヤーのプロトコルに従ってQuick & Easy E.coli Gene Deletion Red(登録商標)/ET(登録商標)Recombination Kit(Gene Bridges)を使用することによって実施する。
1.FRT−PKG−gb2−neo−FRT耐性遺伝子発現カセットを増幅するように設計され、染色体上の欠失遺伝子座の隣接領域(位置1932065−1932115および1934604−1934654)に50ヌクレオチド以上で相同な5′配列を有し、それによりオペロン全体の両側の細菌ゲノム上にカセットの組換えアームを生成するオリゴヌクレオチド。
2.大腸菌K−12株EQ.EC002は、キットプロトコルに従ってプラスミドpRedETを用いたエレクトロポレーションにより形質転換する。得られたコロニーは、0.2%グルコース、0.0003%テトラサイクリンを含むリッチ複合培地LB寒天上で選択する。
3.液体LB中0.3%アラビノースによって1時間誘発されたRedETリコンビナーゼの存在下に第1の工程で得られたアンプリコンの形質転換。そのために、欠失カセットによるRedETを発現する細胞の2回目のエレクトロポレーションを実施し、0.2%グルコース、0.0003%テトラサイクリンを補充し、0.3%L−アラビノースおよび0.0015%カナマイシンを添加したLB寒天上でコロニーを選択する。
4.プラスミドp707−Flpe(Gene BridgesのQuick & Easy E.coli Gene Deletion Red(登録商標)/ET(登録商標)Recombination Kitに付属)を、キットプロトコルに従ってエレクトロポレーションにより形質転換する。0.2%グルコース、0.0003%テトラサイクリンを補充し、0.3%L−アラビノースを添加したLB寒天上で細胞を選択する。得られたクローンの対抗選択を、それらが0.0015%カナマイシンを補充した同じ培地でもはや増殖することができないことを検証することによって行う。
5.得られた株はEQ.EC003:MG1655 ΔsucA Δedd−edaと呼ぶ。
talA遺伝子の欠失は、相同組換えと、サプライヤーのプロトコルに従ってQuick & Easy E.coli Gene Deletion Red(登録商標)/ET(登録商標)Recombination Kit(Gene Bridges)を使用することによって実施する。
1.FRT−PKG−gb2−neo−FRT耐性遺伝子発現カセットを増幅するように設計され、欠失遺伝子座の隣接領域、すなわち遺伝子(talA)(Gene ID:947006)のコーティングフェーズに50超ヌクレオチドの相同な5′配列を有し、それにより細菌ゲノム上にカセットの組換えアームを生成するオリゴヌクレオチド。
2.大腸菌K−12株EQ.EC003は、キットプロトコルに従ってプラスミドpRedETを用いたエレクトロポレーションにより形質転換する。得られたコロニーは、0.2%グルコース、0.0003%テトラサイクリンを含むリッチ複合培地LB寒天上で選択する。
3.液体LB中0.3%アラビノースによって1時間誘発されたRedETリコンビナーゼの存在下に第1の工程で得られたアンプリコンの形質転換。そのために、欠失カセットによるRedETを発現する細胞の2回目のエレクトロポレーションを実施し、0.2%グリセロールおよび0.3%ピルビン酸、0.0003%テトラサイクリンを補充し、0.3%L−アラビノースおよび0.0015%カナマイシンを添加したLB寒天上でコロニーを選択する。
・ EQ.EC002:MG1655 ΔsucA
・ EQ.EC003:MG1655 ΔsucA Δedd−eda
・ EQ.EC020:MG1655 ΔsucA Δedd−edda ΔtalA::kan
である。
大腸菌でのI型RuBisCOの異なる成分の組換え発現のために、以下の表に記載される遺伝子を、以下の表に記載される遺伝子を含む合成オペロンとしてクローニングする。
EQ.EC020→(EQ.EC003+pZA11):MG1655 ΔsucA Δedd−eda
EQ.EC021→(EQ.EC004+pEQEC005):MG1655 ΔsucA Δedd−eda−talA::kan(RuBisCO)
EQ.EC022→(EQ.EC004+pEQEC006):MG1655 ΔsucA Δedd−edatalA::kan(RuBisCO+PRK)
EQ.EC024→(EQ.EC003+pEQEC008):MG1655 ΔsucA Δedd−eda ΔtalA::kan(PRK)
グルタミン酸産生のために、500mLのLB培養からの細胞を、20mLのMS培地(40g/Lのグルコース、1g/LのMgSO4・7H2O、20g/Lの(NH4)2SO4、1g/LのKH2PO4、10mg/LのFeSO4・7H2O、10mg/LのMnSO4・7H2O、2g/Lの酵母エキス、30g/LのCaCO3、100mg/Lのアンピシリン)にCO2雰囲気下0.1の圧力で接種する。
a)ペントースリン酸経路の非酸化分岐の阻害
還元力の増加はまた、既存の代謝経路を大幅に改善する。これは、ポリヒドロキシ酪酸(PHB)を自然に産生する細菌株ラルストニア・ユートロファ(Ralstonia eutropha)ATCC 17699(カプリアビダス・ネカトール)の場合に当てはまる。この細菌は、独立栄養条件と従属栄養条件の両方の条件下で成長することができる。
eddおよびeda遺伝子の隣接領域(eddの上流およびedaの下流)に対応する2つのPCRアンプリコンを、Srinivasan et al.(Appl Environ Microbiol.2002 Dec;68(12):5925−32)に記載されている手順に従った制限により、プラスミドpJQ200mp18Cmでクローニングする。
凍結ストックからの接種物を、グルコースの存在下に30℃で48〜96時間インキュベートしたクライオチューブから50〜100μLの割合で固体培地に広げる。RuBisCOおよびPRKをコードする遺伝子の発現は、従属栄養好気性条件下にカプリアビダス・ネカトールで維持される(Rie Shimizu et al.,Sci Rep.2015;5: 11617.Published online 2015 Jul 1.)。エルレンマイヤーフラスコでのバッチ式培養(50mLで10mL、次いで250mLで50mL)を、20g/Lグルコースの適切な培地と10%外因性CO2供給により、振盪インキュベーター(100〜200rpm、30℃)で、0.01OD620nmの最小接種量により行う。
Claims (13)
- 機能性RuBisCO酵素および機能性ホスホリブロキナーゼ(PRK)を発現し、ペントースリン酸経路の非酸化分岐が少なくとも部分的に阻害されている遺伝的に改変された微生物であって、当該微生物が、RuBisCO酵素および/またはホスホリブロキナーゼ(PRK)以外の、目的の外因性分子を産生および/または目的の内因性分子を過剰産生するように遺伝的に改変されている、微生物。
- 前記微生物が、組換えRuBisCO酵素および/またはPRKを発現するように遺伝的に改変されている、請求項1に記載の遺伝的に改変された微生物。
- 前記微生物が、リブロース−5−リン酸産生の下流の前記ペントースリン酸経路の非酸化分岐を阻害するように遺伝的に改変されている、請求項1または2に記載の遺伝的に改変された微生物。
- トランスアルドラーゼ(E.C.2.2.1.2)および/またはトランスケトラーゼ(E.C.2.2.1.1)をコードする遺伝子の発現が、少なくとも部分的に阻害されている、請求項1〜3のいずれか一項に記載の遺伝的に改変された微生物。
- 前記目的の外因性分子および/または前記目的の内因性分子が、アミノ酸、ペプチド、タンパク質、ビタミン、ステロール、フラボノイド、テルペン、テルペノイド、脂肪酸、ポリオールおよび有機酸から選択される、請求項1〜4のいずれか一項に記載の遺伝的に改変された微生物。
- 前記微生物が真核細胞であり、酵母、真菌、微細藻類または原核細胞から選択的に選らばれ、優先的には細菌である、請求項1〜5のいずれか一項に記載の遺伝的に改変された微生物。
- 前記微生物が、機能性I型またはII型RuBisCOと機能性ホスホリブロキナーゼ(PRK)を発現するように遺伝的に改変されたサッカロミセス・セレビシエ属の酵母であって、TAL1遺伝子および/またはNQM1遺伝子の発現が少なくとも部分的に阻害されている、請求項1〜6のいずれか一項に記載の遺伝的に改変された微生物。
- RuBisCO酵素および/またはホスホリブロキナーゼ(PRK)以外のもので、アミノ酸、ペプチド、タンパク質、ビタミン、ステロール、フラボノイド、テルペン、テルペノイド、脂肪酸、ポリオールおよび有機酸から選択的に選らばれる目的分子の産生または過剰産生のための、請求項1〜7のいずれか一項に記載の遺伝的に改変された微生物の使用。
- RuBisCO酵素および/またはホスホリブロキナーゼ(PRK)以外の、少なくとも1つの目的分子を産生するための生物工学的方法であって、請求項1〜8のいずれか一項に記載の遺伝的に改変された微生物を、前記微生物によって前記目的分子の合成または生物変換を可能にする条件下で培養する工程と、任意に前記目的分子を回収および/または精製する工程とを含むことを特徴とする、方法。
- 前記微生物が、前記目的分子の生物変換または合成に関与する少なくとも1つの酵素を発現するように遺伝的に改変されている、請求項9に記載の生物工学的方法。
- 前記微生物が、前記目的分子の分解に関与する酵素を少なくとも部分的に阻害するように遺伝的に改変されている、請求項9または10に記載の生物工学的方法。
- RuBisCO酵素および/またはホスホリブロキナーゼ(PRK)以外の目的分子を産生する方法であって、(i)請求項1〜8のいずれか一項に記載の組換え微生物への前記目的分子の合成または生物変換に関与する酵素をコードする少なくとも1つの配列を挿入することと、(ii)前記酵素の発現を可能にする条件下で前記微生物を培養することと、任意に(iii)前記目的分子を回収および/または精製することとを含む、方法。
- RuBisCO酵素および/またはホスホリブロキナーゼ(PRK)以外の目的分子を産生する方法であって、(i)請求項1〜8のいずれか一項に記載の組換え微生物への前記目的分子の分解に関与する酵素をコードする少なくとも1つの遺伝子の発現を阻害することと、(ii)前記酵素の発現を可能にする条件下で前記微生物を培養することと、任意に(iii)前記目的分子を回収および/または精製することとを含む、方法。
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MARY A. TICHI & F. ROBERT TABITA: "Metabolic Signals That Lead to Control of cbb Gene Expression in Rhodobacter capsulatus", JOURNAL OF BACTERIOLOGY, vol. 184, no. 7, JPN6021044782, 2002, pages 1905 - 1915, ISSN: 0004798489 * |
PENG-FEI XIA ET AL.: "Recycling Carbon Dioxide during Xylose Fermentation by Engineered Saccharomyces cerevisiae", ACS SYNTHETIC BIOLOGY, vol. 6, no. 2, JPN6021044783, 17 October 2016 (2016-10-17), pages 276 - 283, XP055405751, ISSN: 0004798490, DOI: 10.1021/acssynbio.6b00167 * |
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EP3574082A1 (fr) | 2019-12-04 |
CN110475851A (zh) | 2019-11-19 |
FR3062395A1 (fr) | 2018-08-03 |
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BR112019015411A2 (pt) | 2020-05-26 |
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