JP2015517823A - 組換え微生物およびその使用 - Google Patents
組換え微生物およびその使用 Download PDFInfo
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- JP2015517823A JP2015517823A JP2015514947A JP2015514947A JP2015517823A JP 2015517823 A JP2015517823 A JP 2015517823A JP 2015514947 A JP2015514947 A JP 2015514947A JP 2015514947 A JP2015514947 A JP 2015514947A JP 2015517823 A JP2015517823 A JP 2015517823A
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- clostridium
- microorganism
- bacterium
- nucleic acid
- reductase
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Abstract
Description
本明細書で前述したとおり、本発明は、COおよび/またはCO2含有基質の発酵により、3−HPと、必要に応じて1以上の他の生成物とを生成することができる組換え微生物を提供する。
本発明は、本発明の組換え微生物を生成する際に使用する核酸および核酸コンストラクトも提供する。
本発明は、本発明の組換え微生物を用いてCOおよび/またはCO2含有基質を発酵することを含む微生物発酵による3−HPおよび必要に応じて、1以上の他の生成物を生成する方法を提供する。好ましくは、3−HPは、主要発酵生成物である。本発明の方法を用いて、産業プロセスから全大気中の炭素排出量を削減することができる。
(a)本発明の1以上の微生物の培養物を含むバイオリアクターにCOおよび/またはCO2含有基質を提供するステップと、
(b)少なくとも3−HPを生成するためにバイオリアクターで培養物を嫌気的に発酵させるステップと、
を含む。
(a)産業プロセスの結果生成されるCOおよび/またはCO2含有ガスを、ガスが大気中に放出される前に捕捉するステップと、
(b)本発明の1以上の微生物を含む培養物によって、少なくとも3−HPを生成するためにCOおよび/またはCO2含有ガスを嫌気的に発酵させるステップと、
を含む。
アセトゲンの直線的ウッド−ユングダール経路ならびに緑色非硫黄細菌および古細菌で見つかった3−HPサイクル(Thauer,2007,Science,318:1732−33)の2つのCO2固定経路を、プラットフォーム化学の3−ヒドロキシプロピオネート(3−HP)に向かう持続可能なルートを提供するために組み合わせた。このルートは、1分子の3−HPに3分子のCOまたはCO2の固定を可能にする。カルボキシドトロフ酢酸生成生物であるClostridium autoethanogenumを選択し、非硫黄光合成細菌のChloroflexus auranticusに由来する、最初のCO2固定ステップを実施する遺伝子を用いて代謝的に操作した(図1)。
微生物および増殖条件
C.autoethanogenum DSM23693は、DSMZ(ドイツ微生物および細胞培養コレクション(German Collection of Microorganisms and Cell Cultures、Inhoffenstraβe 7 B、38124 ブラウンシュヴァイク、ドイツ))から得られるC.autoethanogenum DSM10061に由来する。
標準的な組換えDNAおよび分子クローニング技術を使用した(Sambrook,J.およびRussell,D.、分子クローニング:実験室マニュアル第3版、コールド・スプリング・ハーバー研究所プレス、コールド・スプリング・ハーバー、ニューヨーク州、2001)。C.aurantiacusのマロニル補酵素A還元酵素のDNA配列を、NCBI GenBank(GI:163848165,Caur_2614;YP_001636209.1)から入手した。
プラスミドpMTL 83245−SDRを含有する組換え株を、好気的条件下で、適切な抗生物質を含むLB培地で一晩増殖させた。これらの細胞を新鮮なLB培地に接種し、初期OD600は0.1であった。細胞を対数増殖期(OD600 約0.6)に回収し、13,000×g、10分間4℃で遠心分離した。細胞ペレットを、100mMのTrisHCl(pH7.8)で2回洗浄し、プロテアーゼ阻害剤を含む同じ洗浄緩衝液に再懸濁し、100μmのガラスビーズ1.44gと混合した。チューブを氷上で5分間冷却し、その後、ミニビーズビーター(Biospec Products社)中で5000rpmで1分間叩打し、氷上に1分間置くサイクルを5サイクル行って細胞を破壊した。溶解後、試料を遠心分離(13000×g、10分間、4℃)し、上清を等分し、分析まで−80℃で保存した。抽出物のタンパク質含有量を、市販のキット(Pierce(登録商標)マイクロプレートBCAタンパク質アッセイキット−還元剤相溶性、Thermo Scientific社)を用いて決定した。
C.autoethanogenum、C.ragsdaleiおよびC.ljungdahlii由来のメチルトランスフェラーゼ遺伝子から設計した合成ハイブリッドタイプIIメチルトランスフェラーゼ遺伝子(配列番号6)を用いて、3−HP発現プラスミドpMTL83245−SDRのメチル化を大腸菌内でインビボで行った。メチルトランスフェラーゼ(配列番号6)を合成し、ベクターpGS20(ATG:biosynthetics GmbH、Merzhausen、ドイツ)中の誘導lacプロモーター(配列番号7)と融合した。
C.autoethanogenum DSM23693のコンピテントセルを作製するために、50mlの培養物(炭素源として製鋼所のガスおよび果糖を含むPETC培地(表1);37℃)を3日間連続して新鮮な培地で継代培養した。これらの細胞を用いて、40mMのDL−トレオニンを含む50mlのPETC培地に0.05のOD600nmで接種した。培養物のOD600nmが0.45に達した時に、これらの細胞を嫌気性チャンバーに移し、4℃、4700×gで回収した。この培養物を、氷冷エレクトロポレーション緩衝液(270mMのスクロース、1mMのMgCl2、7mMのリン酸ナトリウム、pH7.4)で2回洗浄し、最終的に600μlの容量の新鮮なエレクトロポレーション緩衝液に懸濁した。この混合物を、メチル化プラスミドミックスを約10μg含む0.4cmの電極ギャップを備える予め冷却したエレクトロポレーションキュベットに移した。C.autoethanogenumと比較したC.ljungdahliiのゲノム中に追加のタイプI型制限系を特定したので、1μlのタイプI制限阻害剤(EPICENTRE Biotechnologies社、Madison,WI 53713、米国)をこのプラスミド混合物に添加した。これらの細胞をプラスミドおよび制限阻害剤と混合し、以下の設定:2.5kV、600オーム、および25μFで、ジーンパルサーX細胞エレクトロポレーションシステム(Bio−Rad Labratories社、Hercules、CA 94547、米国)を用いてすぐにパルスした。時定数は3.7〜5.1msであった。再生のために、この培養物を、細胞の回収を増大させる5mlのPETC−MES培地(表1)に移した。この培養物を、チューブホルダーを備えるスペクトロニックヘリオスイプシロン分光光度計(Thermo Fisher Scientific Inc.,Waltham MA 02454,、米国)を用いて、波長600nmで監視した。成長(1回の倍増)が観察された後、この培養物を、それぞれ5μg/mlのクラリスロマイシンおよび唯一の炭素源としてヘッドスペース内に30psiの製鋼所ガスを含むPETC−MES培地で10mlまで、その後、50mlまでスケールアップした。
3−ヒドロキシプロピオネート(3−HP)および他の代謝物のHPLC分析を、35℃で動作させたRID(屈折率検出器)を備えるAgilent 1100シリーズHPLCシステムおよび35℃に保ったアミネックスHPX−87Hカラム(300×7.8mm、粒径5μm)を用いて実施した。弱酸性水(0.005MのH2SO4)を0.6ml/分の流速で移動相として用いた。タンパク質および他の細胞残渣を除去するために、400μlの試料を、1M硫酸中の1%(w/v)5−スルホサリチル酸100μlと混合し、沈殿した残渣を分離するために3分間、14,000rpmで遠心分離した。その後、上清10μlを分析のためにHPLCに注入した。
形質転換細胞における3−HPの生成
C.autoethanogenumにおけるC.aurantiacus経路遺伝子を用いたCOおよびCO2/H2からの3−HP生成:
成長実験を、ゴム栓を有する血清ボトルおよびヘッドスペース内に唯一のエネルギーおよび炭素源として30psiの製鋼所ガス(ニュージーランドのグレンブルックにあるニュージーランドスチールサイトから収集される;組成:44%CO、32%N2、22%CO2、2%H2)中50mlのPETC−MES培地(表1;果糖を含まず)中で、プラスミドpMTL83245−SDRを保有する形質転換したC.autoethanogenum DSM23693を用いて行った。HPLC分析を用いて3−HP生成を確認した。
増強したビオチン生合成を介した3−HPの生成の向上
アセチル−CoAカルボキシラーゼ(ACC)によるCO2のアセチル−CoAへの固定化は、ビオチン(ビタミンB7、ビタミンH)によって媒介される。ビオチンは、このカルボキシル化反応のための補因子として必要とされる。
律速脂肪酸生合成を介した3−HP生成の向上
マロニル−CoAは、脂肪酸生合成の前駆体でもある。3−HPの生成を増加させるために、脂肪酸生合成の速度を、3−HP生成に有利に制限することができる。マロニル−CoA:アシルキャリアタンパク質トランスアシラーゼ(FabD)[EC:2.3.1.39]は、脂肪酸鎖の伸長を開始する。この酵素をコードする遺伝子を、マロニル−CoAが天然の脂肪酸生合成のために使用されるのを防ぐためにダウンレギュレートするか、またはこの酵素の活性を低下させることができる。
Claims (16)
- COおよび/またはCO2を3−ヒドロキシプロピオネート(3−HP)に変換するプロセスであって、
培地中のカルボキシドトロフ、酢酸生成菌の培養物を含有するバイオリアクターに、ガス状のCO含有および/またはCO2含有基質を通過させ、その結果、前記細菌が前記COおよび/またはCO2を3−HPに変換する工程と、
前記バイオリアクターから前記3−HPを回収する工程と、
を含み、
前記カルボキシドトロフ酢酸生成菌が、マロニル補酵素A還元酵素を発現するように遺伝子操作されているプロセス。 - マロニル補酵素A還元酵素をコードする核酸を含む、単離された遺伝子操作カルボキシドトロフ酢酸生成菌であって、前記マロニル補酵素A還元酵素を発現し、3分子のCOまたはCO2を1分子の3−ヒドロキシプロピオネート(3−HP)に固定することができるカルボキシドトロフ酢酸生成菌。
- さらに、非硫黄光合成細菌由来のアセチル補酵素Aカルボキシラーゼをコードする核酸を含み、前記核酸がプロモーターに作動可能に連結されている、請求項2に記載の単離された遺伝子操作カルボキシドトロフ酢酸生成菌。
- Clostridium autoethanogenum、Clostridium ljungdahlii、Clostridium ragsdalei、Clostridium carboxidivorans、Clostridium drakei、Clostridium scatologenes、Clostridium aceticum、Clostridium formicoaceticum、Clostridium magnum、Butyribacterium methylotrophicum、Acetobacterium woodii、Alkalibaculum bacchii、Blautia producta、Eubacterium limosum、Moorella thermoacetica、Moorella thermautotrophica、Sporomusa ovata、Sporomusa silvacetica、Sporomusa sphaeroides、Oxobacter pfennigii、およびThermoanaerobacter kiuviからなる群から選択される、請求項2に記載の単離された遺伝子操作カルボキシドトロフ酢酸生成菌。
- C.ljundahlii、およびC.autoethanogenumからなる群から選択されるクロストリジウム種である、請求項4に記載の単離された遺伝子操作カルボキシドトロフ酢酸生成菌。
- 前記非硫黄光合成細菌が、Clostridium ljungdahlii、MetallosphaeraおよびSulfolobus種からなる群から選択される、請求項3に記載の単離された遺伝子操作カルボキシドトロフ酢酸生成菌。
- 前記非硫黄光合成細菌が、Chloroflexus auranticusである、請求項3に記載の単離された遺伝子操作カルボキシドトロフ酢酸生成菌。
- マロニル補酵素A還元酵素をコードする核酸がコドン最適化されている、請求項2に記載の単離された遺伝子操作カルボキシドトロフ酢酸生成菌。
- 請求項2に記載の単離された遺伝子操作カルボキシドトロフ酢酸生成菌を培養する方法であって、COおよび/またはCO2を含むガス状の炭素源を含む培地中で、前記細菌を培養することを含む、方法。
- 請求項2に記載の単離された遺伝子操作カルボキシドトロフ酢酸生成菌を培養する方法であって、COおよび/またはCO2を含むエネルギー源を含む培地中で、前記細菌を培養することを含む、方法。
- 前記培養が絶対嫌気性である、請求項9または10に記載の方法。
- 前記炭素源が、産業廃棄物またはオフガスを含む、請求項9または10に記載の方法。
- 前記細菌が、さらに、非硫黄光合成細菌由来のアセチル補酵素Aカルボキシラーゼをコードする外因性核酸を含み、前記核酸がプロモーターに作動可能に連結されている、請求項9または10に記載の方法。
- 前記マロニル補酵素A還元酵素が、非硫黄光合成細菌由来である、請求項1に記載のプロセス。
- 前記マロニル補酵素A還元酵素が、Chloroflexus auranticus由来のものである、請求項14に記載のプロセス。
- 前記マロニル補酵素A還元酵素が、配列番号1のヌクレオチド配列によりコードされるアミノ酸配列と少なくとも85%同一である、請求項2に記載の単離された遺伝子操作カルボキシドトロフ酢酸生成菌。
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WO2013180581A1 (en) | 2013-12-05 |
US20130323806A1 (en) | 2013-12-05 |
KR20150021081A (ko) | 2015-02-27 |
KR102098849B1 (ko) | 2020-04-09 |
CN113832199A (zh) | 2021-12-24 |
EP2855690B1 (en) | 2018-07-11 |
IN2014DN10168A (ja) | 2015-08-21 |
CN104508136A (zh) | 2015-04-08 |
JP6445970B2 (ja) | 2018-12-26 |
EP2855690A4 (en) | 2016-02-17 |
CA2874832C (en) | 2016-02-02 |
US9994878B2 (en) | 2018-06-12 |
EP2855690A1 (en) | 2015-04-08 |
CA2874832A1 (en) | 2013-12-05 |
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