JP2014522635A - シゾキトリウム属(Schizochytriumsp.)の突然変異誘導の方法及びそれから産生された変異体 - Google Patents
シゾキトリウム属(Schizochytriumsp.)の突然変異誘導の方法及びそれから産生された変異体 Download PDFInfo
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- JP2014522635A JP2014522635A JP2014516178A JP2014516178A JP2014522635A JP 2014522635 A JP2014522635 A JP 2014522635A JP 2014516178 A JP2014516178 A JP 2014516178A JP 2014516178 A JP2014516178 A JP 2014516178A JP 2014522635 A JP2014522635 A JP 2014522635A
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- schizochytrium
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Abstract
【選択図】なし
Description
シゾキトリウム・リマシナム(Schizochytrium limacinum)SR21(これは以降の実施例において対照株又は未処置株としても使用される)を皿の上で平板培養し、紫外線照射にそれぞれ、0秒(対照)、30秒、40秒、50秒、60秒、70秒、80秒、90秒、及び100秒(実験群)間暴露した。照射後、皿を暗所に24時間保持し、各皿上のコロニーの数を数えた。対照群におけるコロニーの数を100%と規定し、かつそれに基づいて各実験群における致死率を計算した(図1)。図1に見られるように、照射期間が延びるにつれてシゾキトリウム属(Schizochytrium sp.)においてより高い致死率が観測され、容量依存性の効果が示された。具体的には、70〜90秒の照射継続は(結果的にシゾキトリウム属(Schizochytrium sp.)における致死率は60%〜80%であり)、それはシゾキトリウム属(Schizochytrium sp.)における突然変異誘導を誘発するために使用された。
A.培養媒体であって、
グルコース 55g/L
酵母抽出物 10g/L
グルタミン酸ナトリウム 5g/L
塩化ナトリウム 2.4g/L
にが塩 4.0g/L
硫酸アンモニウム 4.0g/L
水 残部
からなる培養媒体、
B.培養温度:22〜27℃、及び
C.初期pH:5.0〜7.0
である。
キザロホップエチルをシゾキトリウム属(Schizochytrium sp.)を培養する媒体の中に濃度0μモル/L、10μモル/L、30μモル/L、50μモル/L、70μモル/L、80μモル/L、及び90μモル/Lで添加した。対照群(0μモル/L)におけるコロニーの数を100%として規定した。各群におけるコロニーを数え、かつ対照群におけるコロニーの数に基いて致死率を計算した(図2)。図2に見られるように、実験の範囲内でシゾキトリウム属(Schizochytrium sp.)致死率とキザロホップの濃度との間に正の相関関係が観察された。耐性株を選択するために使用したキザロホップの濃度は、50μモル/L〜80μモル/Lに設定した。
A.培養媒体であって、
グルコース 60g/L
酵母抽出物 15g/L
グルタミン酸ナトリウム 4g/L
塩化ナトリウム 3g/L
にが塩 5.0g/L
硫酸アンモニウム 5.0g/L
水 残部
からなる培養媒体、
B.培養温度:22〜27℃、及び
C.初期pH:5.0〜7.0
である。
実施例2における選定及び実証の後に、200種を超えるキザロホップ耐性のシゾキトリウム属(Schizochytrium sp.)株を得た。次にこれらのキザロホップ耐性のシゾキトリウム属(Schizochytrium sp.)株をより高い成長速度及びDHA含量に関して2つの巡回に対して選抜した。第1の巡回後で20〜50種の株を選別し、かつ第2の巡回後では3種の株を選別した。その3種の株の各々の成長速度及びDHA含量は、対照の(未処置の)株よりも10%を超えて高かった。
A.培養媒体であって、
グルコース 60g/L
酵母抽出物 15g/L
グルタミン酸ナトリウム 4g/L
塩化ナトリウム 3g/L
にが塩 5.0g/L
硫酸アンモニウム 5.0g/L
水 残部
からなる培養媒体、
B.培養温度:22〜27℃、
C.初期pH:5.0〜7.0
である。
A.培養媒体であって、
グルコース 60g/L
酵母抽出物 15g/L
グルタミン酸ナトリウム 4g/L
塩化ナトリウム 3g/L
にが塩 5.0g/L
硫酸アンモニウム 5.0g/L
水 残部
からなる培養媒体、
B.培養温度:22〜27℃、
C.初期pH:5.0〜7.0、
D.1VVMの通気速度及び70〜100rpmの回転速度を有した50Lの発酵槽、
E.培養期間:4〜7日
である。
グルコースの濃度以外は、培養条件は上記の一般的な条件のものと同一である。
窒素供給源以外は、培養条件は上記の一般的な条件と同一である。
pH値以外は、培養条件は上記の一般的な条件のものと同一である。
50L発酵槽において培養した対照(未処置)株と株321−1、2010−0321、及び303−11との成長速度及びDHAの蓄積を評価するために実施例7を実施した(図4)。株は上記で説明したような一般的条件下で培養した。対照株と比較して、株321−1では9.5%増加したバイオマス、及び16.7%増加したDHA含量を、それぞれ示した。株2010−0321に関しては、バイオマス及びDHA含量はそれぞれ、6.7%及び48.1%増加した。株303−11については、バイオマスは対照株とほぼ同じであり、DHA含量は僅か7.9%増加した。3種の突然変異株の間で総合的に比較すると、2010−0321が最も優れ、特にDHA含量に関して最も優れていた。
表4及び表5は、タンパク質、アミノ酸及び脂肪酸の組成を含む生化学的組成における、対照(未処置)株と株2010−0321との間の相違を示した。タンパク質の含量はケルダール法によって決定した。アミノ酸の含量は、アミノ酸分析装置によって定量した。脂肪の含量は脂質分析装置によって分析した。
全RNAを溶解法によって試験株から抽出した。cDNAに逆転写した後で、18S RNA遺伝子を順方向プライマー5’−CCAACCTGGTTGATCCTGCCAGTA−3’(配列番号3)及び逆方向プライマー5’−CCTTGTTACGACTTCACCTTCCTCT−3’(配列番号4)で増幅した。単位複製配列(アンプリコン)を回収し、大腸菌(E.coli)DH5α コンピテント細胞を形質転換するために使用した。陽性のコロニーを配列決定用に選択した。対照(未処置)と株2010−0321との18SRNA遺伝子の配列をBlastソフトウエアで整列化及び比較した。結果を図5に示した。
Claims (17)
- 2010−0321として識別され、かつCCTCC M 2011024の寄託参照番号を有して中国典型培養物保蔵センターにおいて寄託されているシゾキトリウム属(Schizochytrium sp.)株の変異体。
- 前記変異体が、未処置のシゾキトリウム属(Schizochytrium sp.)株と比較して、増加したDHA含量及び/又は向上した成長速度の特性を有する、請求項1に記載の変異体。
- 前記変異体が、未処置のシゾキトリウム属(Schizochytrium sp.)株と比較して、異なったタンパク質含量、アミノ酸組成の異なるタンパク質及び/又は異なった脂肪酸組成を有する、請求項1又は2に記載の変異体。
- シゾキトリウム属(Schizochytrium sp.)株の変異体を産生する方法であって、
前記シゾキトリウム属(Schizochytrium sp.)株を紫外線照射に暴露させシゾキトリウム属(Schizochytrium sp.)株において突然変異誘導を誘発し、突然変異株を産生すること、
前記突然変異株をアセチル補酵素Aカルボキシラーゼ阻害剤と接触させること、
未処置シゾキトリウム属(Schizochytrium sp.)株と比較して、増加したDHA含量及び/又は向上した成長速度を有するシゾキトリウム属(Schizochytrium sp.)株の変異体を選択すること、を含む方法。 - 前記アセチル補酵素Aカルボキシラーゼ阻害剤がキザロホップである、請求項4に記載の方法。
- 前記変異体が増加したDHA含量及び向上した成長速度を有する、請求項4又は5に記載の方法。
- 前記変異体が異なったタンパク質含量、アミノ酸組成の異なるタンパク質及び/又は異なった脂肪酸組成を有する、請求項4〜6のいずれか一項に記載の方法。
- 前記シゾキトリウム属(Schizochytrium sp.)株が、紫外線照射に約30秒〜約100秒、又は約70秒〜90秒の間で暴露される、請求項4〜7のいずれか一項に記載の方法。
- 前記キザロホップが、約10μモル/L〜約90μモル/L、又は約50μモル/L〜約80μモル/Lの範囲内の濃度において使用される、請求項4〜8のいずれか一項に記載の方法。
- シゾキトリウム属(Schizochytrium sp.)株の前記変異体が、2010−0321として識別され、かつCCTCC M 2011024の寄託参照番号を有して中国典型培養物保蔵センターにおいて寄託されている株である、請求項4〜9のいずれか一項に記載の方法。
- DHAを産生する方法であって、
請求項1〜3のいずれか一項に記載の、又は請求項4〜10のいずれか一項に記載の方法によって産生された、シゾキトリウム属(Schizochytrium sp.)株の変異体を、DHAを産生するためのシゾキトリウム属(Schizochytrium sp.)株を培養するのに好適な条件下で培養すること、及び任意選択で
シゾキトリウム属(Schizochytrium sp.)株の前記培養された変異体のバイオマス、又はシゾキトリウム属(Schizochytrium sp.)株の培養媒体からDHAを収集すること、を含む、方法。 - 前記条件が、
約50〜70g/Lの炭素供給源、及び10〜20g/Lの窒素供給源を含む培養媒体、
約20℃〜約28℃、又は約22℃〜約27℃の培養温度、
及び/又は
pH:3〜10、例えば4〜8又は5〜7など、
を含む、請求項11に記載の方法。 - 前記培養媒体が、g/Lで
グルコースを 50〜70、
酵母抽出物を 10〜30、
グルタミン酸ナトリウムを 10〜20
塩化ナトリウムを 2.4〜4.0、
にが塩を 3.0〜6.0、
硫酸アンモニウムを 3.0〜6.0、
含む、請求項11又は12に記載の方法。 - 請求項11〜13のいずれか一項に記載の方法によって産生したバイオマス。
- 請求項14に記載の前記バイオマス又は前記バイオマスから抽出されたDHAを含む食品製品であって、前記食品製品が乳児、子供、若者及び成人に食べさせることを意図した製品を含む、又は前記食品製品が乳製品である、食品製品。
- 請求項1〜3のいずれか一項に記載の変異体、又は請求項4〜13のいずれか一項に記載の方法であって、前記変異体により、3〜7日にわたる培養又は5日の培養で産生されたDHAの量が、未処置シゾキトリウム属(Schizochytrium sp.)株によって産生されたDHAの量よりも、少なくとも約10、20、25、30、35、40%、又は41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70%高い、変異体又は方法。
- 請求項1〜3及び16のいずれか一項に記載の変異体、又は請求項4〜13及び16のいずれか一項に記載の方法であって、3〜7日にわたる培養又は5日の培養で、シゾキトリウム属(Schizochytrium sp.)株の前記変異体の前記成長速度が、未処置のシゾキトリウム属(Schizochytrium sp.)株の前記成長速度よりも、少なくとも約3、4、5、6、7、8、9、10%高い、変異体又は方法。
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FR3001736B1 (fr) | 2013-02-06 | 2016-03-04 | Roquette Freres | Biomasse de la microalgue schizochytrium mangrovei et son procede de preparation |
CN104450529A (zh) * | 2013-09-25 | 2015-03-25 | 郭星 | 一种裂殖壶菌菌株及其诱变方法和应用 |
CN104312929B (zh) * | 2014-10-27 | 2017-10-17 | 山东百龙创园生物科技股份有限公司 | 一株高产dha裂殖壶菌菌株及其培养方法与应用 |
FR3031984B1 (fr) * | 2015-01-27 | 2019-05-24 | Roquette Freres | Procede d'enrichissement de la biomasse de microalgues du genre traustochytrium en dha et en acides amines arg et glu |
FR3038913B1 (fr) * | 2015-07-17 | 2020-05-01 | Fermentalg | Biomasse de thraustochytrides, procede de culture et utilisations |
FR3038914B1 (fr) * | 2015-07-17 | 2020-03-13 | Fermentalg | Biomasse de thraustochytrides, procede de culture et utilisations |
CN105558305B (zh) * | 2015-12-10 | 2019-12-03 | 新奥科技发展有限公司 | 裂殖壶菌突变株及其应用 |
CN108587916B (zh) * | 2018-05-23 | 2021-09-14 | 昆明理工大学 | 一种共培养单针藻在中性条件下快速絮凝的方法 |
CN108707630B (zh) * | 2018-06-12 | 2020-10-16 | 厦门大学 | 一种提高裂殖壶菌中epa含量的调控方法与应用 |
CN112481348A (zh) * | 2019-09-11 | 2021-03-12 | 天津大学青岛海洋技术研究院 | 一种高产dha裂殖壶菌突变菌株的筛选方法 |
CN111235035A (zh) * | 2019-12-30 | 2020-06-05 | 嘉必优生物技术(武汉)股份有限公司 | 一种裂殖壶菌突变株及其用于制备二十二碳六烯酸油脂的方法和应用 |
KR102614551B1 (ko) * | 2020-12-07 | 2023-12-15 | 씨제이제일제당 주식회사 | 단일 미세조류로부터 단백질 및 오메가-3 지방산을 포함하는 바이오매스를 제조하는 방법 및 이에 의해 제조된 바이오매스 |
CN114164122B (zh) * | 2021-12-02 | 2023-06-20 | 嘉必优生物技术(武汉)股份有限公司 | 一种高产epa的裂殖壶菌及其应用 |
CN114621983B (zh) * | 2022-05-13 | 2022-08-16 | 南京师范大学 | 提高裂殖壶菌的dha产量的方法和微生物油脂的制备方法 |
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CN103827289A (zh) | 2014-05-28 |
CN102839129A (zh) | 2012-12-26 |
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JP5894666B2 (ja) | 2016-03-30 |
CN103827289B (zh) | 2017-07-21 |
WO2012175027A1 (en) | 2012-12-27 |
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