JP5637711B2 - 組み換え微生物及びこれを用いた脂肪族ポリエステルの製造方法 - Google Patents
組み換え微生物及びこれを用いた脂肪族ポリエステルの製造方法 Download PDFInfo
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- JP5637711B2 JP5637711B2 JP2010069688A JP2010069688A JP5637711B2 JP 5637711 B2 JP5637711 B2 JP 5637711B2 JP 2010069688 A JP2010069688 A JP 2010069688A JP 2010069688 A JP2010069688 A JP 2010069688A JP 5637711 B2 JP5637711 B2 JP 5637711B2
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- lactic acid
- gene
- pha synthase
- derived
- protein
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Description
(a)配列番号6、8、10、12、14、16又は18に示すアミノ酸配列を含むタンパク質コードする遺伝子
(b)配列番号6、8、10、12、14、16又は18に示すアミノ酸配列において1又は複数個のアミノ酸が置換、欠失又は付加されたアミノ酸配列を含み、上記活性を有するタンパク質をコードする遺伝子
(c)配列番号5、7、9、11、13、15又は17に示す塩基配列に対する相補的な塩基配列を有するポリヌクレオチドに対してストリンジェントな条件でハイブリダイズし、上記活性を有するタンパク質をコードする遺伝子
(a)配列番号6、8、10、12、14、16又は18に示すアミノ酸配列を含むタンパク質コードする遺伝子
(b)配列番号6、8、10、12、14、16又は18に示すアミノ酸配列において1又は複数個のアミノ酸が置換、欠失又は付加されたアミノ酸配列を含み、上記活性を有するタンパク質をコードする遺伝子
(c)配列番号5、7、9、11、13、15又は17に示す塩基配列に対する相補的な塩基配列を有するポリヌクレオチドに対してストリンジェントな条件でハイブリダイズし、上記活性を有するタンパク質をコードする遺伝子
本発明において、プロピオニルCoAトランスフェラーゼ遺伝子(以下、pct遺伝子と称する)としては、特に限定されず、乳酸を乳酸CoAに変換する活性を有するタンパク質をコードする遺伝子であれば如何なる遺伝子を使用することができる。すなわち、pct遺伝子は、プロピオニルCoAトランスフェラーゼ活性を有するタンパク質をコードする如何なる遺伝子を使用することができる。プロピオニルCoAトランスフェラーゼ活性とは、プロピオン酸にCoAが転移される反応を触媒する活性を意味する。すなわち、適当なCoA基質からプロピオン酸にCoAが転移される反応を触媒する活性をプロピオニルCoAトランスフェラーゼ活性と称する。このプロピオニルCoAトランスフェラーゼは、プロピオン酸のみならず乳酸に対してもCoA基質からCoAを転移することができる。
本発明では、ポリヒドロキシアルカン酸シンターゼ遺伝子(PHAシンターゼ遺伝子とも称される)としては、Alcanivorax borkumensis由来の遺伝子、Hyphomonas neptunium由来の遺伝子、Rhodobacter sphaeroides由来の遺伝子、Rhizobium etli由来の遺伝子、Pseudomonas sp.由来の遺伝子及びHaloarcula marismortui由来の遺伝子から選ばれる少なくとも1以上の遺伝子を使用する。特に、PHAシンターゼ遺伝子としては、Alcanivorax borkumensis由来の遺伝子及び/又はHyphomonas neptunium由来の遺伝子を使用することが好ましい。なお、PHAシンターゼ遺伝子とは、ヒドロキシアシルCoAを基質としてポリヒドロキシアルカン酸を合成する活性を有するタンパク質をコードする遺伝子である。
本発明において、宿主微生物としては、例えば、シュードモナス・エスピー(Pseudomonas sp.)61-3株などのシュードモナス(Pseudomonas)属細菌、R.ユートロファなどのラルストニア(Ralstonia)属細菌、バチルス・ズブチリス(Bacillus subtilis)などのバチルス(Bacillus)属細菌、大腸菌(Escherichia coli)などのエシェリヒア(Escherichia)属細菌、コリネバクテリウム(Corynebacterium)属細菌、サッカロマイセス・セレビシー(Saccharomyces cerevisiae)などのサッカロマイセス(Saccharomyces)属酵母、カンジダ・マルトーサ(Candida maltosa)などのカンジダ(Candida)属酵母などを挙げることができる。宿主微生物としては、特に大腸菌を使用することが好ましい。
上述したpct遺伝子及びPHAシンターゼ遺伝子を宿主微生物に導入して得られる組み換え微生物を、炭素源を含む培地で培養し、培養物中に脂肪族ポリエステルのオリゴマーを生成蓄積させ、その後、脂肪族ポリエステルのオリゴマーを回収することで、目的とする脂肪族ポリエステルのオリゴマーを製造することができる。この組み換え微生物は、糖の代謝経路によって糖から乳酸を合成し、pct遺伝子によりコードされるプロピオニルCoAトランスフェラーゼが乳酸を乳酸CoAに変換する。そして、この組み換え微生物は、PHAシンターゼ遺伝子によりコードされるPHAシンターゼが乳酸CoAを基質として構成単位として乳酸を含む脂肪族ポリエステルのオリゴマーを合成する。当該オリゴマーとしては、構成単位が乳酸のみからなるポリ乳酸(ホモポリマー)であってもよいし、構成単位として乳酸と乳酸以外のヒドロキシアルカン酸とからなる乳酸系共重合体であってもよい。また、培地中に生産するオリゴマーとしては、主として二量体から五量体である。ここで「主として」とは、培地に含まれる脂肪族ポリエステル成分のうち50%以上、好ましくは70%以上、より好ましくは90%以上が上記オリゴマーであるとこと意味する。
本実施例では、種々のPHAシンターゼ遺伝子について、Megasphaera elsdenii由来のpct遺伝子とともに発現させた場合の乳酸オリゴマーの生産性を評価した。
本実施例では、実施例1で作製したAlcanivorax borkumensis由来のPHAシンターゼ遺伝子(No. 12)を導入した組み換え大腸菌を使用し、培地の種類による乳酸オリゴマーの生産性の相違を検討した。
本実施例では、実施例1で作製したAlcanivorax borkumensis由来のPHAシンターゼ遺伝子(No. 12)を導入した組み換え大腸菌を使用し、培地時間と乳酸オリゴマーの生産性との関係を検討した。
Claims (5)
- 宿主微生物に対して、乳酸を乳酸CoAに変換する活性を有するタンパク質をコードする遺伝子と、ヒドロキシアシルCoAを基質としてポリヒドロキシアルカン酸を合成する活性を有するタンパク質をコードする、以下の(a)又は(b)に示すAlcanivorax borkumensis由来の遺伝子とを導入してなる組み換え微生物を培養し、
4量体及び5量体を含む乳酸オリゴマーを細胞外に産生させ、培地から4量体及び5量体を含む乳酸オリゴマーを回収することを特徴とする乳酸オリゴマーの製造方法。
(a)配列番号6に示すアミノ酸配列を含むタンパク質をコードする遺伝子
(b)配列番号6に示すアミノ酸配列において1又は複数個のアミノ酸が置換、欠失又は付加されたアミノ酸配列を含み、ヒドロキシアシルCoAを基質としてポリヒドロキシアルカン酸を合成する活性を有するタンパク質をコードする遺伝子 - 上記培地は最小培地であることを特徴とする請求項1記載の乳酸オリゴマーの製造方法。
- 上記組み換え微生物を48時間以上培養した後、上記4量体及び5量体を含む乳酸オリゴマーを回収することを特徴とする請求項1記載の乳酸オリゴマーの製造方法。
- 宿主微生物に対して、乳酸を乳酸CoAに変換する活性を有するタンパク質をコードする遺伝子と、ヒドロキシアシルCoAを基質としてポリヒドロキシアルカン酸を合成する活性を有するタンパク質をコードする遺伝子であって、以下の(a)又は(b)に示すAlcanivorax borkumensis由来の遺伝子を導入してなる組み換え微生物。
(a)配列番号6に示すアミノ酸配列を含むタンパク質をコードする遺伝子
(b)配列番号6に示すアミノ酸配列において1又は複数個のアミノ酸が置換、欠失又は付加されたアミノ酸配列を含み、ヒドロキシアシルCoAを基質としてポリヒドロキシアルカン酸を合成する活性を有するタンパク質をコードする遺伝子 - 上記宿主微生物は大腸菌であることを特徴とする請求項4記載の組み換え微生物。
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EP (1) | EP2551349A4 (ja) |
JP (1) | JP5637711B2 (ja) |
KR (1) | KR101458495B1 (ja) |
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US8802402B2 (en) * | 2010-07-14 | 2014-08-12 | Toyota Jidosha Kabushiki Kaisha | Mutant polyhydroxyalkanoic acid synthase gene and method for producing aliphatic polyester using the same |
JP7149473B2 (ja) * | 2016-03-29 | 2022-10-07 | 学校法人東京農業大学 | アルコール終止ポリエステル及びポリエステルの製造方法 |
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DE60016555T2 (de) * | 1999-08-09 | 2005-12-29 | Japan Science And Technology Agency, Kawaguchi | Verfahren zur herstellung von copolymerisierten polyestern |
CA2429039A1 (en) | 2000-11-20 | 2002-05-30 | Cargill, Incorporated | 3-hydroxypropionic acid and other organic compounds |
KR100656529B1 (ko) | 2004-04-08 | 2006-12-13 | 한국원자력연구소 | 재조합 대장균 및 상기 대장균을 이용한 폴리하이드록시부티레이트의 대량 생산방법 |
KR100979694B1 (ko) | 2005-05-24 | 2010-09-02 | 한국과학기술원 | 폴리락테이트 또는 그 공중합체 생성능을 가지는 세포 또는식물 및 이를 이용한 폴리락테이트 또는 그 공중합체의제조방법 |
EP1752532A1 (en) * | 2005-08-09 | 2007-02-14 | Helmholtz-Zentrum für Infektionsforschung GmbH | Extracellular polyhydroxyalkanoates produced by genetically engineered microorganisms |
CN105754966A (zh) | 2006-11-23 | 2016-07-13 | Lg化学株式会社 | 源自假单胞菌6-19的pha合酶的突变体和使用该突变体制备乳酸酯均聚物或共聚物的方法 |
KR101045572B1 (ko) * | 2007-08-14 | 2011-07-01 | 주식회사 엘지화학 | Clostridium propionicum 유래의프로피오닐―CoA 트랜스퍼라아제 변이체 및 상기변이체를 이용한 락테이트 중합체 또는 락테이트공중합체의 제조방법 |
KR101037354B1 (ko) | 2007-09-07 | 2011-05-26 | 한국과학기술원 | 수크로스로부터 폴리락틱산 또는 폴리락틱산 공중합체를제조할 수 있는 재조합 미생물 및 이러한 미생물을이용하여 수크로스로부터 폴리락틱산 또는 락틱산공중합체를 제조하는 방법 |
WO2009031762A2 (en) * | 2007-09-07 | 2009-03-12 | Lg Chem, Ltd. | Recombinant microorganism capable of producing polylactate or polylactate copolymer from sucrose and method for producing polylactate or polylactate copolymer from sucrose using the same |
KR20090078925A (ko) * | 2008-01-16 | 2009-07-21 | 주식회사 엘지화학 | 폴리락테이트 또는 그 공중합체 생성능을 가지는 재조합미생물 및 이를 이용한 폴리락테이트 또는 그 공중합체의제조방법 |
JP2010069688A (ja) | 2008-09-17 | 2010-04-02 | Seiko Epson Corp | 液体噴射ヘッド及び液体噴射装置 |
WO2010050470A1 (ja) * | 2008-10-27 | 2010-05-06 | トヨタ自動車株式会社 | 組み換え微生物を用いたポリ乳酸の製造方法 |
KR101211767B1 (ko) | 2009-06-30 | 2012-12-12 | 한국과학기술원 | 폴리하이드록시알카노에이트 합성효소 변이체를 이용한 락테이트 중합체 및 락테이트 공중합체의 제조방법 |
JP2011024503A (ja) * | 2009-07-27 | 2011-02-10 | Toyota Motor Corp | 組み換え微生物及びこれを用いた脂肪族ポリエステルの製造方法 |
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- 2011-03-24 EP EP11759476.2A patent/EP2551349A4/en not_active Withdrawn
- 2011-03-24 KR KR1020127027752A patent/KR101458495B1/ko active IP Right Grant
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EP2551349A8 (en) | 2013-03-27 |
EP2551349A4 (en) | 2014-03-26 |
JP2011200153A (ja) | 2011-10-13 |
KR20130009817A (ko) | 2013-01-23 |
US20130045516A1 (en) | 2013-02-21 |
CN102844439B (zh) | 2014-06-04 |
EP2551349A1 (en) | 2013-01-30 |
KR101458495B1 (ko) | 2014-11-07 |
WO2011118671A1 (ja) | 2011-09-29 |
CN102844439A (zh) | 2012-12-26 |
US8980615B2 (en) | 2015-03-17 |
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