JP4318638B2 - プロセス制御として特定の酸素取り込み速度を用いた発酵プロセス。 - Google Patents
プロセス制御として特定の酸素取り込み速度を用いた発酵プロセス。 Download PDFInfo
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Description
Porro et al., "Development of metabolically engineered Saccharonayces cefevisiae cells for the production of lactic acid", Biotechnol. Prog. 1995 May-Jun; 11 (3): 294-8. Porro et al.,"Replacement of a metabolic pathway for large-scale production of lactic acid from engineered yeasts", App. Environ. Microbiol. 1999 Sep: 65 (9): 4211-5. Bianchi et al. ,"Efficient homolactic fermentation by Klzyveroonyces lactis strains defective in pyruvate utilization and transformed with the heterologous LDH gene", App. Environ. Microbiol. 2001 Dec; 67 (12) 5621-5.
a) 発酵の生産期の間、前記OURを測定する工程と、
b) 発酵の前記生産期の間、飽和の1%未満のDOを維持しながら、所定の範囲内に、前記OURが維持されるような通気条件に調整する工程とを含む。
a) 発酵微生物が炭水化物を所望の発酵産物に発酵させるOUR値の至的範囲を決定する工程、
b) 細胞が生育および繁殖(reproduce)し、培地のDOを飽和の1%未満に減少させ、細胞が少なくとも10 mmol O2/g(細胞の乾燥重量)/h(mmol O2/gdw/h)である特定の酸素取り込み速度を示すように、前記培地に通気しながら、前記工学酵母細胞を、前記細胞が代謝可能である炭水化物および1以上の栄養素を含む培地で生育させる工程、
および、それから、
c)前記至的範囲内の特定の酸素取り込み速度(OUR)での培養を提供するのに十分な微好気条件を含む発酵条件下、緩衝培地で微生物を培養する工程。
a) 細胞が生育および繁殖(reproduce)し、培地の溶存酸素濃度を飽和の1%未満に減少させ、細胞が少なくとも10 mmol O2/g(細胞の乾燥重量)/h(mmol O2/gdw/h)である特定の酸素取り込み速度を示すように、前記培地に通気しながら、破壊されたPDC経路および前記細胞に所望の発酵産物を生産させることを可能にする外来遺伝子を有する工学酵母細胞を、前記細胞が代謝可能である炭水化物を含む培地で生育させる工程、および、
b) 約0.8〜約3.0mmol O2/gdw/hの特定の酸素取り込み速度(OUR)での培養を提供するのに十分な微好気条件を含む発酵条件下、緩衝培地で細胞を培養する工程。
生産期において、一般的な細胞の生命力および健康を促進するために、少ない酸素量を代謝することが必要と思われる。このような生物の実験は、一般的にPDC破壊を有し、特に、特定の発酵産物を生産することを可能とする外来遺伝子を有する、工学設計された酵母を含む。完全な嫌気条件において、これらの細胞が示す基質消費速度と発酵産物生産速度は、通常非常に低い。加えて、所望の発酵産物の収率も悪い。微好気条件下、特に特定の株に依存するあるOUR値において、細胞はより迅速に基質を代謝することができる。これは、基質消費速度および所望の発酵産物生産速度が増加する結果となる。しかしながら、酸素消費がある値を超えて増加すると、基質が二酸化炭素に変換されて所望の産物の収率が減少する。これに加えて、ある値を超えてOURが増加すると生産速度は横ばいの傾向もしくは減少の傾向となり、収率のロスが速度上昇により補償されなくなる。したがって、生産期の間ある範囲にOURを維持することは、収率と生産速度との経済的に最適なバランスを達成することとなる。
lically engineeredSaccharo7nyces cerevisiae cells for the production of lacticacid", BiotechiioL Prog. 1995 May−Jun; 11
(3): 294−8、 Porro et al. ,"Replacement o
f a metabolic pathway for large−scale production of lactic acid from engineered yeasts", App.Enviroiz. Microbiol. 1999 S
ep: 65 (9): 4211−5、およびBianchi et al. ,"E
fficient homolactic fermentation by Kluyverimyces lactis strains defective in pyruvate utilaization and transdormed with the heterologous LDH gene", App. Enviro
n. Microbiol. 2001 Dec; 67(12)5621−5、WO 00/71738、WO02/42471、PCT/US02/16223、および2002年5月20日に出願された米国仮出願No.60/384,333等に開示されている。前記細胞は、また、クラブトリー陰性表現型を示すことが好ましい。これは、高濃度グルコースの存在下や高い生育速度の通気条件において呼吸および生育できるためである。
A. 収率が、少なくとも30g(発酵産物)/g(基質)、好ましくは少なくとも40g(発酵産物)/g(基質)、より好ましくは少なくとも60g(発酵産物)/g(基質)、さらに好ましくは75g(発酵産物)/g(基質)である。理論上の望ましい収率は100%であるが、実用上の収率の限界は約98%である。
B. 特定の生産力が、少なくとも0.1g(発酵産物)/g(細胞)/時間、好ましくは少なくとも0.3 g(発酵産物)/g(細胞)/時間、より好ましくは少なくとも0.4 g(発酵産物)/g(細胞)/時間、特に約0.5 g(発酵産物)/g(細胞)/時間である。特定の生産力は、できるだけ高いことが好ましい。
C. 滴定値(発酵産物の最大濃度)が、少なくとも15g/L(発酵培地)であり、好ましくは少なくとも20g/Lであり、より好ましくは40g/Lであり、さらに好ましくは80g/Lであり、また、150g/Lまでであり、好ましくは約120g/Lまでである。乳酸の場合、高濃度乳酸溶液(例えば、約150g/L以上)は、約35℃以下の温度で、非常に粘性またはゲル化する傾向にあるため、発酵培地の温度は、すぐに達成しうる適定の高値に影響する。約35-50℃のような高い発酵温度は、ゲル化または粘度が上昇することなく、高い滴定値を可能とする。
最大乳酸滴定量 106±3.1 g/kg
グルコース消費速度 1.2±0.05 g/gdw/h
乳酸生産速度 1.1±0.04 g/gdw/h
乳酸収率(生産期) 0.92±0.03 g(乳酸)/g(グルコース)
% 回収炭素 99%±3.0
乳酸の光学純度 >99.9
最大乳酸滴定量 111 g/kg
グルコース消費速度 0.94 g/gdw/h
乳酸生産速度 0.83 g/gdw/h
乳酸収率(生産期) 0.89 g(乳酸)/g(グルコース)
% 回収炭素 95.6%
乳酸の光学純度 >99.9
特性 実施例No.
3A 3B 3C
保持時間:DO=0 1hr 0 >1.5hr
OUR:生産期(mmol/gdw/h) 2.1 1.8 1.4
最大乳酸滴定量(g/kg) 112.3 84 94
グルコース消費速度(g/gdw/h) 1.22 1.06 0.40
乳酸生産速度(g/gdw/h) 1.20 0.93 0.32
乳酸収率(生産期、g/g) 0.89 0.84 0.79
% 回収炭素(生産期) 105 104.3 98
乳酸の光学純度(%) >99.9 >99.9 >99.9
Claims (5)
- 発酵微生物、及び前記微生物により発酵可能な炭水化物を含む発酵培地において発酵プロセスを行う方法であって、
前記発酵培地が、多量の溶存酸素(DO)を有し、前記発酵が、特定の酸素取り込み速度(OUR)を示し、
a)発酵の生産期の間、前記OURを測定する工程、
b)発酵の前記生産期の間、飽和の1%未満にDOを維持しながら、前記OURが0.8〜3.0mmol O 2 /gdw/hの範囲に維持され、DOが10μmol O2/L未満に維持されるような通気条件に調整する工程を含み、
前記微生物が、破壊されたPDC経路、および、機能性外来遺伝子である乳酸デヒドロゲナーゼ遺伝子を有する酵母細胞である、発酵プロセスを行う方法。 - 前記酵母細胞が、Kluyveromyces属またはCandida属である、請求項1記載の方法。
- 以下の工程を含む発酵プロセス。
a)細胞が生育および繁殖(reproduce)しているときに、培地の溶存酸素濃度をゼロに減少させ、細胞が少なくとも10 mmol O2/g(細胞の乾燥重量)/h(mmol O2/gdw/h)である特定の酸素取り込み速度(OUR)を示すように、前記培地に通気しながら、破壊されたPDC経路、および、細胞に発酵産物を産生させることを可能にする外来性の乳酸デヒドロゲナーゼ遺伝子を有する酵母細胞を、前記細胞が代謝可能である炭水化物を含む培地で生育させる工程、および、その後、
b)0.8〜3.0mmol O 2 /gdw/hの特定の酸素取り込み速度(OUR)での培養を提供するのに十分な微好気条件を含む発酵条件下、緩衝培地で細胞を培養する工程。 - 前記酵母細胞が、Kluyveromyces属またはCandida属である、請求項3記載の発酵プロセス。
- 工程a)における前記OURが、少なくとも18mmol O2/gdw/hである、請求項3又は4に記載の発酵プロセス。
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US38433302P | 2002-05-30 | 2002-05-30 | |
PCT/US2003/016825 WO2003102200A2 (en) | 2002-05-30 | 2003-05-30 | Fermentation process using specific oxygen uptake rates as a process control |
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JP2004510437A Expired - Fee Related JP4318638B2 (ja) | 2002-05-30 | 2003-05-30 | プロセス制御として特定の酸素取り込み速度を用いた発酵プロセス。 |
JP2004510394A Pending JP2005528106A (ja) | 2002-05-30 | 2003-05-30 | 酵母における乳酸の生産方法及びその材料 |
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IT1294728B1 (it) * | 1997-09-12 | 1999-04-12 | Biopolo S C A R L | Ceppi di lievito per la riproduzione di acido lattico |
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US7141410B2 (en) * | 2000-11-22 | 2006-11-28 | Natureworks Llc | Methods and materials for the production of organic products in cells of Candida species |
US7232664B2 (en) | 2002-05-30 | 2007-06-19 | Natureworks Llc | Fermentation process using specific oxygen uptake rates as a process control |
CN1922198B (zh) | 2003-05-02 | 2012-07-11 | 卡吉尔公司 | 基因修饰酵母物种和使用基因修饰酵母的发酵方法 |
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JP4806904B2 (ja) * | 2004-07-09 | 2011-11-02 | トヨタ自動車株式会社 | 乳酸生産方法 |
JP2006296377A (ja) * | 2005-04-25 | 2006-11-02 | Toyota Central Res & Dev Lab Inc | 有機酸生産用形質転換体 |
EP1885842A4 (en) | 2005-06-02 | 2009-06-10 | Cargill Inc | GENETICALLY MODIFIED YEAST OF ISSATCHENKIAORIENTALIS SPECIES AND CLOSELY BINDED SPECIES AND PROCESSES OF FERMENTATION USING THE SAME |
DK1760156T3 (da) | 2005-08-10 | 2013-03-18 | Univ Florida | Materialer og fremgangsmåder til effektiv mælkesyrefremstilling |
JP4692173B2 (ja) * | 2005-09-13 | 2011-06-01 | 東レ株式会社 | D−乳酸デヒドロゲナーゼ活性を有するポリペプチド、これをコードする遺伝子およびd−乳酸の製造方法 |
US7718405B2 (en) * | 2005-09-19 | 2010-05-18 | American Air Liquide, Inc. | Use of pure oxygen in viscous fermentation processes |
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