JP2015519918A - 組み換え微生物およびそれにより作製されるバイオディーゼル - Google Patents
組み換え微生物およびそれにより作製されるバイオディーゼル Download PDFInfo
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- JP2015519918A JP2015519918A JP2015518359A JP2015518359A JP2015519918A JP 2015519918 A JP2015519918 A JP 2015519918A JP 2015518359 A JP2015518359 A JP 2015518359A JP 2015518359 A JP2015518359 A JP 2015518359A JP 2015519918 A JP2015519918 A JP 2015519918A
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- clostridium
- microorganism
- biodiesel
- nucleic acid
- plasmid
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Abstract
Description
a.本発明の第1の態様の1つ以上の微生物の培養物を含むバイオリアクターに、COを含む物質を提供するステップと、
b.バイオリアクター中でこの培養物を嫌気的に発酵し、バイオディーゼルを産生するステップと
を含む。
a.工業用工程の結果として生成されたCO含有ガスを捕獲するステップと、
b.本発明の第1の態様の1つ以上の微生物を含む培養物により、CO含有ガスを嫌気的に発酵してバイオディーゼルを産生するステップと
を含む。
本発明に記載されるように、「目的微生物」は、発現構築物/ベクターに含まれる遺伝子が、シャトル微生物から発現され、かつシャトル微生物と異なる微生物である。
発酵を触媒する微生物の増殖の速度、上昇した産物濃度での増殖速度および/または産物の産生速度、消費した物質容量あたり産生された所望の産物の容量、所望の産物の産生速度または産生レベル、および、他の発酵産物と比較して産生される所望の産物の相対比率のうち、1つ以上を増加させることが含まれる。
本明細書に前述されるように、本発明は、COを含む物質の発酵により、バイオディーゼルおよび任意に1つ以上の他の産物を産生できる組み換え微生物を提供する。
本発明はまた、本発明の組み換え微生物を産生する際に使用する1つ以上の核酸または核酸構築物を提供する。
1つ以上の外因性核酸は、裸の核酸として親微生物に送達されてもよく、または形質転換工程を促進するため、1つ以上の薬剤で製剤化されてもよい(たとえば、リポソーム結合核酸、この核酸が含まれる生物)。この1つ以上の核酸は、適宜、DNA、RNA、またはその組み合わせであってもよく、ある実施形態では、制限阻害剤を使用してもよい(たとえば、N.E. et al. (2000) Microbial. Molec. Biol. Rev. 64, 412.)参照)。
(i)本明細書に記載される発現構築物/ベクターおよび(ii)メチルトランスフェラーゼ遺伝子を含むメチル化構築物/ベクターのシャトル微生物への導入;
メチルトランスフェラーゼ遺伝子の発現;
シャトル微生物から1つ以上の構築物/ベクターの単離;および
目的微生物への1つ以上の構築物/ベクターの導入
を含む、方法により産生される。
本発明は、本発明の組み換え微生物を使用して、COを含む物質を発酵することを含む微生物発酵により、バイオディーゼルおよび任意の1つ以上の他の産物の産生方法を提供する。本発明の方法を使用して、工業工程からの合計大気炭素排出を低減してもよい。
a.本発明の1つ以上の培養物を含むバイオリアクターにCOを含む物質を提供するステップと、
b.バイオリアクター中の培養物を嫌気的に発酵して少なくともバイオディーゼルを産生するステップと
を含む。
a.工業工程の結果として生成されたCO含有ガスを捕獲するステップと、
b.本発明の1つ以上の微生物を含む培養物により、CO含有ガスを嫌気的に発酵してバイオディーゼルを産生するステップと
を含む。
本発明を、以下の非限定的な実施例を参照して説明する。
酢酸産生酸化炭素資化性のClostridium autoethanogenumを、バイオディーゼル脂肪酸アシルエステル、ブタン酸ブチルエステル(FABE)の産生のために、Acinetobacter baylyiの非特異的アシルトランスフェラーゼで改変した。ブタノール産生を、Clostridium autoethanogenumの遺伝子改変株を使用して以前に例証した(WO 2012/053905)。
すべてのサブクローニングステップを、以前に記載される標準的な株および増殖条件を使用してE. coli中で実施した(Sambrook et al, Molecular Cloning: A laboratory Manual, Cold Spring Harbour Labrotary Press, Cold Spring Harbour, 1989; Ausubel et al, Current protocols in molecular biology, John Wiley & Sons, Ltd., Hoboken, 1987)。
標準的な組み換えDNAおよび分子クローニング技術を本発明に使用し、これらの技術は、Sambrook et al, 1989およびAusubel et al, 1987により記載される。Acinetobacter baylyiの非特異的アシルトランスフェラーゼ(YP_045555.1; Gene ID: 2879218)は最適化コドンであり、合成されていた(配列番号1)。
FAEE発現プラスミドpMTL85245−atfのメチル化を、C.autoethanogenum, C. ragsdalei および C.ljungdahlii由来のメチルトランスフェラーゼから設計し、合成したハイブリッドタイプIIメチルトランスフェラーゼ(配列番号12)を使用してE.coliでin vivoで実施した。このメチルトランスフェラーゼを、ベクターpGS20 (配列番号:14)中の誘導型のlacプロモーター(配列番号13)に融合した。
完全な形質転換の実験の間、C.autoethanogenum DSM23693を、1g/Lの酵母抽出物および10g/lのフルクトース、ならびに炭素供給源としての30psi製鋼廃ガス(ニュージーランドのGlenbrookの工場地帯で収集、組成:44% CO、32% N2、22% CO2、2% H2)を補充したPETC培地(表1)で増殖した。
DNAの転換を確認するために、プラスミドミニプレップを、Zyppy plasmid miniprep kit(Zymo)を使用して10mlの培養物容量から実施した。単離プラスミドの質が、クロストリジウムのエキソヌクレアーゼの活性により制限溶解に対して十分ではないため[Burchhardt および Durre, 1990]、PCRを、表2に得られる単離プラスミドおよびオリゴヌクレオチドを用いて実施し、プラスミドの存在を確認した。PCRを、iNtRON Maximise Premix PCR kit(Intron Bio Technologies)を使用し、以下の、最初の変性(94℃、2分間)、35サイクルの変性(94℃、20秒間)、アニーリング(55℃、20秒間)、および伸長(72℃、60秒間)、条件で、最終伸長ステップ(72℃、5分間)の前に実施した。
FAEE産生を例証するために、PETC培地を調製し、発現プラスミドpMTL85245−atfを有するC.autoethanogenum株を用いて接種した。50mLPETC培地(表1)の入った血清ビンを、製鋼廃ガス(ニュージーランドのGlenbrookの工場地帯で収集、組成:44% CO、32% N2、22% CO2、2% H2)由来の30psiのCO含有ガス流で加圧し、5日間培養した。同一の実験を、プラスミドを有さない野生型C.autoethanogenum株を用いて実行した。
Abrini, J., Naveau, H., & Nyns, E. J. (1994). Clostridium autoethanogenum, sp. nov., an anaerobic bacterium that produces ethanol from carbon monoxide. Archives of microbiology, 161(4), 345-351.
Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez−Garayzabal, J., Garcia, P., Cai, J., et al. (1994). The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. International journal of systematic bacteriology, 44(4), 812-26.
Herbert, M., O’Keeffe, T. a., Purdy, D., Elmore, M., & Minton, N. P. (2003). Gene transfer into Clostridium difficile CD630 and characterisation of its methylase genes. FEMS Microbiology Letters, 229(1), 103-110.
Jennert, K. C., Tardif, C., Young, D. I., & Young, M. (2000). Gene transfer to Clostridium cellulolyticum ATCC 35319. Microbiology (Reading, England), 146 Pt 12, 3071-80.
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Kopke, M., Mihalcea, C., Liew, F., Tizard, J. H., Ali, M. S., Conolly, J. J., Al−Sinawi, B., et al. (2011). 2,3−Butanediol Production By Acetogenic Bacteria, an Alternative Route To Chemical Synthesis, Using Industrial Waste Gas. Applied and environmental microbiology, 77(15), 5467-75.
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Claims (16)
- 非特異的なアセチルトランスフェラーゼ(ワックスエステルシンターゼ/アシル補酵素A:ジアシルグリセロール アシルトランスフェラーゼ)をコードする外因性核酸を含む、遺伝子改変した酸化炭素資化性細菌。
- クロストリジウム(Clostidium)である、請求項1に記載の細菌。
- 前記外因性非特異的アセチルトランスフェラーゼが、Acinetobacter baylyiの非特異的アセチルトランスフェラーゼである、請求項1に記載の細菌。
- 前記核酸はプラスミドである、請求項1に記載の細菌。
- C.autoethanogenumである、請求項1に記載の細菌。
- C.ljundahliiである、請求項1に記載の細菌。
- 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1に記載の細菌。
- 前記非特異的アセチルトランスフェラーゼをコードする核酸が、C.autoethanogenumに最適化されるコドンである、請求項5に記載の細菌。
- COおよび/またはCO2をバイオディーゼルに変換する工程であって、前記工程が、
気体のCO含有物質および/またはCO2含有物質を、培養媒体中の請求項1に記載の酸化炭素資化性酢酸産生細菌の培養物を含むバイオリアクターに通し、これにより、前記細菌が前記COおよび/またはCO2を前記バイオディーゼルに変換することと、
前記バイオリアクターから前記バイオディーゼルを回収することと
を含む、工程。 - 前記物質が、工業廃ガスを含む、請求項9に記載の工程。
- 前記培養物が厳密に嫌気性である、請求項9に記載の工程。
- 前記バイオディーゼルが、脂肪酸エチルエステルを含む、請求項9に記載の工程。
- 前記バイオディーゼルが、脂肪酸ブチルエステルを含む、請求項9に記載の工程。
- 酸化炭素資化性酢酸産生細菌を複製するプラスミドであって、前記プラスミドが、非特異的アセチルトランスフェラーゼ(ワックスエステルシンターゼ/アシル補酵素A:ジアシルグリセロール アシルトランスフェラーゼ)をコードする外因性核酸を含む、プラスミド。
- 前記非特異的アセチルトランスフェラーゼをコードする核酸が、C.autoethanogenumに最適化されたコドンである、請求項14に記載のプラスミド。
- メチル化されている、請求項14に記載のプラスミド。
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MY164882A (en) | 2018-01-30 |
PL2864470T5 (pl) | 2021-05-17 |
PL2864470T3 (pl) | 2018-05-30 |
KR20150023651A (ko) | 2015-03-05 |
ES2655214T5 (es) | 2021-10-11 |
BR112014031876B1 (pt) | 2022-05-10 |
IN2014DN10507A (ja) | 2015-08-21 |
EP2864470A1 (en) | 2015-04-29 |
BR112014031876A2 (pt) | 2017-08-01 |
US20130344547A1 (en) | 2013-12-26 |
CN104640977A (zh) | 2015-05-20 |
KR102097844B1 (ko) | 2020-04-06 |
JP6466836B2 (ja) | 2019-02-06 |
EP2864470A4 (en) | 2015-12-16 |
CN104640977B (zh) | 2019-04-12 |
CA2876178A1 (en) | 2013-12-27 |
EP2864470B1 (en) | 2017-10-04 |
CA2876178C (en) | 2017-02-28 |
US9347076B2 (en) | 2016-05-24 |
EP2864470B2 (en) | 2020-12-23 |
WO2013191567A1 (en) | 2013-12-27 |
NO2953671T3 (ja) | 2018-03-17 |
ES2655214T3 (es) | 2018-02-19 |
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