JP6806297B2 - 半連続培養法 - Google Patents
半連続培養法 Download PDFInfo
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- JP6806297B2 JP6806297B2 JP2017513131A JP2017513131A JP6806297B2 JP 6806297 B2 JP6806297 B2 JP 6806297B2 JP 2017513131 A JP2017513131 A JP 2017513131A JP 2017513131 A JP2017513131 A JP 2017513131A JP 6806297 B2 JP6806297 B2 JP 6806297B2
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N1/12—Unicellular algae; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12P7/6409—Fatty acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Description
本出願は、その全体が参照によって本明細書に組み入れられる2014年10月16日に出願された米国仮特許出願番号62/064,668に対する優先権を主張する。
本明細書に記載されている方法には微生物の集団から脂質を抽出することが含まれる。本明細書に記載されている微生物の集団は、藻類(たとえば、微細藻類)、真菌類(酵母を含む)、細菌または原生生物であることができる。任意で、微生物には、Thraustochytriales目のThraustochytrids、さらに具体的には、Thraustochytrium属のThraustochytrialesが挙げられる。任意で、微生物の集団には、その全体が参照によって本明細書に組み入れられる米国特許第5,340,594号及び同第5,340,742号にて記載されたようなThraustochytrialesが挙げられる。微生物は、その全体が参照によって本明細書に組み入れられる米国特許第8,163,515号にて記載されたようなATCC受入番号PTA−6245(すなわち、ONC−T18)として寄託されているThraustochytrium種のようなThraustochytrium種であることができる。従って、微生物は、配列番号1に対して少なくとも95%、96%、97%、98%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%以上(たとえば、100%を含む)同一である18srRNAを有することができる。
本明細書で提供されるのは微生物を培養する方法である。該方法には、第1の炭素対窒素の比を有する培地における1以上の微生物の容器を提供することと;培養物が閾値指標に達するまで微生物を培養することと;容器にて培養物の大半を維持しながら、培養物の一部を回収することと;微生物を伴った培養物の大半を含有する容器に第2の炭素対窒素の比を有する新鮮な培地を加えることとが含まれる。
提供される方法は、当該技術で既知の方法に従って微生物を培養する追加の工程を含む、またはそれと併せて使用することができる。たとえば、Thraustochytrid、たとえば、Thraustochytrium sp.は、方法の各工程またはその中で使用される組成物についてその全体が参照によって本明細書に組み入れられる米国特許公開2009/0117194または2012/0244584にて記載された方法に従って培養することができる。微生物は増殖培地(「培養培地」としても知られる)で増殖させる。本明細書に記載されている微生物を培養することにおける使用に種々の培地のいずれもが好適である。任意で、培地は微生物のために炭素源及び窒素源を含む種々の栄養成分を供給する。
任意で、得られたバイオマスを殺菌してバイオマスに存在する望ましくない物質を不活化する。たとえば、バイオマスを殺菌して化合物を分解する物質を不活化することができる。バイオマスは、発酵培地に存在することができ、または殺菌工程のために発酵培地から単離することができる。殺菌工程は、バイオマス及び/または発酵培地を高温に加熱することによって行うことができる。たとえば、バイオマス及び/または発酵培地を約50℃〜約95℃(たとえば、約55℃〜約90℃または約65℃〜約80℃)の温度に加熱することができる。任意で、バイオマス及び/または発酵培地を約30分間〜約120分間(たとえば、約45分間〜約90分間または約55分間〜約75分間)加熱することができる。殺菌は、当業者に既知のような好適な加熱手段を用いて、たとえば、直接蒸気圧入法によって行うことができる。
任意で、当業者に現在知られるものを含む種々の方法に従ってバイオマスを回収することができる。たとえば、任意で細胞バイオマスの回収を加速するための沈降剤(たとえば、リン酸ナトリウムまたは塩化カルシウム)と共に、遠心分離(たとえば、固形物−排出遠心分離)または濾過(たとえば、クロスフロー濾過)を用いて発酵培地からバイオマスを回収することができる。
提供される方法には任意で、当業者に現在知られるものを含む種々の方法を用いてバイオマスまたは微生物に由来するポリ不飽和脂肪酸を単離することが含まれる。たとえば、ポリ不飽和脂肪酸を単離する方法は、その全体が参照によって本明細書に組み入れられる米国特許第8,163,515号に記載されている。任意で、ポリ不飽和脂肪酸の単離に先立って培地は滅菌されない。任意で滅菌は温度の上昇を含む。任意で、微生物によって産生され、提供される方法から単離されるポリ不飽和脂肪酸は中鎖脂肪酸である。任意で、1以上のポリ不飽和脂肪酸は、α−リノレン酸、アラキドン酸、ドコサヘキサエン酸、ドコサペンタエン酸、エイコサペンタエン酸、γ−リノレン酸、リノール酸、リノレン酸、及びそれらの組み合わせから成る群から選択される。
本明細書に記載されている方法に従って産生されるポリ不飽和脂肪酸(PUFA)及び他の脂質を含む油はその生物学的、栄養学的または化学的特性を活用する種々の応用のいずれかで利用することができる。従って、提供される方法には任意で、回収された培養物から油を単離することが含まれる。任意で、油を用いて、燃料、たとえば、バイオ燃料を製造する。任意で、油は、医薬品、食品、サプリメント、動物飼料添加剤、化粧品等で使用することができる。本明細書に記載されている方法に従って産生される脂質は他の化合物の製造における中間体としても使用することができる。
微生物による油の産生の分野では、従属栄養の暗所発酵は、工程の効率性と生成物の収率という点で光合成独立栄養の微生物培養よりも一般に優れていると見なされている。しかしながら、それは高い固定資本費(容器に基づく発酵プラントを建設するコストは開放池と管路の型の培養系の資本コストよりも一般にはるかに高い)によって妨げられることが多い。本明細書に記載されているような半連続の生産工程を用いて、低い操作コスト(生産発酵槽容器方向転換手順と容器滅菌手順を最小化する)によって高い全体的な生産性を達成することができる。このことは、固定資本投資(発酵槽及び関連する資本設備)のさらに良好な利用及びさらに高い年間生産能を意味する。
Claims (33)
- Thraustochytrid微生物の培養方法であって、
(a)30:1〜60:1の第1の炭素対窒素のモル比を含む培地にて1以上のThraustochytrid微生物を含む容器を提供する工程と、
(b)培養物が閾値指標に達するまで前記Thraustochytrid微生物を培養する工程と、
(c)前記容器における前記培養物の大半を維持しながら、前記培養物の一部を回収する工程であって、回収された前記一部が前記培養物の5%〜20%を含む工程と、
(d)前記微生物を含む前記培養物の大半を含む前記容器に、前記第1の炭素対窒素のモル比よりも高い、60:1〜95:1である第2の炭素対窒素のモル比を含む新鮮な培地を、前記培養物の前記回収された一部と同一容量で加える工程とを含み、
前記閾値指標が、光学密度(OD)、細胞濃度、培養培地における栄養素の濃度、バイオマス生産性、油生産性、またはそれらの組み合わせから成る群から選択される、前記培養方法。 - 前記第1の炭素対窒素のモル比が40:1〜50:1である請求項1に記載の方法。
- 前記第2の炭素対窒素のモル比が70:1〜95:1である請求項1又は2に記載の方法。
- 前記第2の炭素対窒素のモル比が80:1〜90:1である請求項1又は2に記載の方法。
- 回収された前記一部が前記培養物容量の5〜20%を含む請求項1〜3のいずれか1項に記載の方法。
- 回収された前記一部が前記培養物容量の10%を含む請求項5に記載の方法。
- 前記栄養素が炭素または窒素である請求項1〜6のいずれか一項に記載の方法。
- (i)前記培養物が閾値指標に達するまで前記微生物を培養する工程と、(ii)前記容器にて前記培養物の大半を維持しながら、前記培養物の一部を回収する工程と、(iii)前記微生物を含む前記培養物の大半を含む前記容器に前記第2の炭素対窒素の比を含む新鮮な培地を加える工程とを繰り返すことを含む、請求項1〜7のいずれか1項に記載の方法。
- 前記炭素が、脂肪酸、脂質、グリセロール、トリグリセロール、炭水化物、ポリオール及びアミノ糖から成る群から選択される炭素源である請求項1〜8のいずれか1項に記載の方法。
- 前記炭素源がグルコース、フルクトースまたはグリセロールである請求項9に記載の方法。
- 前記窒素が、アンモニウム溶液、アンモニウム塩またはアミン塩、ペプトン、トリプトン、酵母抽出物、麦芽抽出物、魚粉、グルタミン酸ナトリウム、ダイズ抽出物、カザミノ酸、及び醸造カスから成る群から選択される窒素源である請求項1〜10のいずれか1項に記載の方法。
- 前記窒素源が硫酸アンモニウムである請求項11に記載の方法。
- 前記回収された培養物から油を単離することをさらに含む請求項1〜12のいずれか1項に記載の方法。
- 前記油が、α−リノレン酸、アラキドン酸、ドコサヘキサエン酸、ドコサペンタエン酸、エイコサペンタエン酸、γ−リノレン酸、リノール酸、リノレン酸、及びそれらの組み合わせから成る群から選択される脂肪酸を含む請求項13に記載の方法。
- 前記油がトリグリセリドを含む請求項13に記載の方法。
- 前記油が、パルミチン酸(C16:0)、ミリスチン酸(C14:0)、パルミトレイン酸(C16:1(n−7))、シス−バクセン酸(C18:1(n−7))、ドコサペンタエン酸(C22:5(n−6))、ドコサヘキサエン酸(C22:6(n−3))、及びそれらの組み合わせから成る群から選択される脂肪酸を含む請求項13に記載の方法。
- 前記単離された油におけるドコサヘキサエン酸の濃度が、前記単離された油中の脂肪酸総濃度の20%以下である請求項13に記載の方法。
- 前記培養物が高レベルのバイオマス生産性で維持される請求項1〜17のいずれか1項に記載の方法。
- 前記バイオマス生産性が60g/L−d〜180g/L−dを含む請求項18に記載の方法。
- 前記バイオマス生産性が80g/L−d〜130g/L−dを含む請求項18に記載の方法。
- 前記バイオマス生産性が90g/L−d〜100g/L−dを含む請求項18に記載の方法。
- 油生産性が35g/L−d〜130g/L−dを含む請求項1〜21のいずれか一項に記載の方法。
- 油生産性が60g/L−d〜90g/L−dを含む請求項1〜21のいずれか一項に記載の方法。
- 油生産性が70g/L−d〜80g/L−dを含む請求項1〜21のいずれか一項に記載の方法。
- 微生物の前記培養物が、60%〜85%の油を含むバイオマスを含む請求項1〜24のいずれか1項に記載の方法。
- 微生物の前記培養物が、65%〜75%の油を含むバイオマスを含む請求項1〜24のいずれか1項に記載の方法。
- 前記培養物が長時間にわたって維持される請求項1〜26のいずれか1項に記載の方法。
- 前記培養物が、数時間、数日、数週間または数カ月の期間維持される請求項27に記載の方法。
- 前記培養物が少なくとも150〜500時間維持される請求項27に記載の方法。
- 前記培養物が少なくとも250時間維持される請求項27に記載の方法。
- 前記培養物が、1、2、3、4、または5週間維持される請求項27に記載の方法。
- 前記Thraustochytrid微生物がThraustochytrium属のものである請求項1〜31のいずれか1項に記載の方法。
- 前記Thraustochytrid微生物が、ATCC受入番号PTA−6245として寄託されたThraustochytrium種である請求項1〜31のいずれか1項に記載の方法。
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