JP2019519242A - 細菌ヘモグロビンライブラリーを生成するための方法およびその使用 - Google Patents
細菌ヘモグロビンライブラリーを生成するための方法およびその使用 Download PDFInfo
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Abstract
Description
本願は、2016年6月30日に出願された米国仮出願第62/356,934号の優先権の利益を主張し、その全体が、すべての目的のために参照によって本明細書に組み込まれる。
配列表に関する記述
以下の用語は、当業者であればよく理解していると考えられるが、以下の定義を、本開示の主題の説明を容易にするために示す。
概要
プロモーター
原核生物ヘモグロビン遺伝子
原核生物ヘモグロビン遺伝子の変異形態の生成
原核生物ヘモグロビン遺伝子を含むライブラリーの生成
プラスミドのアセンブリング/クローニング
宿主細胞の形質転換
選択された配列のループアウト
宿主微生物
細胞発酵および培養
産物回収および定量
選択判断基準および目標
(実施例1)
細菌ヘモグロビンライブラリーの生成のための細菌ヘモグロビンライブラリーを用いたCorynebacteriumの形質転換
E.coli中へのアセンブルされたクローンの形質転換
Corynebacterium中へのアセンブルされたクローンの形質転換
Corynebacterium中の個々の細菌ヘモグロビン構築物の評価
発酵条件下での個々の細菌ヘモグロビン構築物の評定
(実施例2)
異種細菌ヘモグロビン構築物を用いたCorynebacteriumの形質転換:第2の発酵最終産物を産生させるための発酵条件下で成長させたVhb01を発現するCorynebacteria形質転換体の評定。
参照による組込み
Claims (46)
- 第1のプロモーターポリヌクレオチドに機能的に連結した異種細菌ヘモグロビン遺伝子を含む宿主細胞であって、該第1のプロモーターポリヌクレオチドが、配列番号1、配列番号2、配列番号3、配列番号4、配列番号5、配列番号6、配列番号7、および配列番号8から選択される配列を含む、宿主細胞。
- 前記細菌ヘモグロビン遺伝子が、配列番号12、配列番号10、配列番号11、配列番号9、配列番号13、配列番号14、配列番号15、配列番号16、配列番号17、配列番号18、配列番号19、および配列番号20から選択されるヌクレオチド配列を有する遺伝子である、請求項1に記載の宿主細胞。
- 前記細菌ヘモグロビン遺伝子が、配列番号26、配列番号24、配列番号25、配列番号23、配列番号27、配列番号28、配列番号29、配列番号30、配列番号31、配列番号32、配列番号33および配列番号34から選択されるアミノ酸配列を有するポリペプチドをコードする、請求項1に記載の宿主細胞。
- 前記細菌ヘモグロビン遺伝子が、表2に列挙された微生物の株、種、または亜種に由来する、請求項1に記載の宿主細胞。
- 前記細菌ヘモグロビン遺伝子が、細菌フラボヘモグロビン遺伝子である、請求項1に記載の宿主細胞。
- 前記細菌フラボヘモグロビン遺伝子が、表2に列挙された微生物の株、種、または亜種に由来する、請求項5に記載の宿主細胞。
- Corynebacterium属に属する、請求項1から6までのいずれか一項に記載の宿主細胞。
- Corynebacterium glutamicumである、請求項1から7までのいずれか一項に記載の宿主細胞。
- 生体分子を産生させる方法であって、請求項1から8までのいずれか一項に記載の宿主細胞を該生体分子の産生に適した条件下で培養するステップを含む方法。
- 前記生体分子が、低分子、アミノ酸、ヌクレオチド、有機酸、またはアルコールである、請求項9に記載の方法。
- 前記アミノ酸が、リシン、グルタミン酸、チロシン、フェニルアラニン、トリプトファン、アスパラギン酸、アスパラギン、トレオニン、イソロイシン、またはメチオニンである、請求項10に記載の方法。
- 前記有機酸が、スクシネート、ラクテートまたはピルベートである、請求項10に記載の方法。
- 前記アルコールが、エタノールまたはイソブタノールである、請求項10に記載の方法。
- 生体分子の産生を増加させることが可能な微生物を生成するための方法であって、
a)宿主微生物を遺伝子改変するステップであって、該改変するステップは、細菌ヘモグロビン遺伝子のライブラリー由来の細菌ヘモグロビン遺伝子を、該宿主微生物のゲノムに導入することを含み、細菌ヘモグロビン遺伝子の該ライブラリー由来の細菌ヘモグロビン遺伝子のそれぞれは、配列番号1、配列番号2、配列番号3、配列番号4、配列番号5、配列番号6、配列番号7、および配列番号8から選択されるヌクレオチド配列を含むプロモーターに機能的に連結しており、該改変は、該細菌ヘモグロビン遺伝子を発現する該宿主微生物の株を生成させる、ステップと;
b)該宿主微生物の複数の株が生成するまで、ステップa)を複数ラウンド繰り返すステップであって、該宿主微生物の該複数の株の各株は、細菌ヘモグロビン遺伝子の該ライブラリー由来の別々の細菌ヘモグロビン遺伝子を発現する、ステップと;
c)該宿主微生物の該複数の株の各株を、発酵条件下で、炭素源と接触させるステップと;
d)対照微生物から産生される生体分子の量と比較して増加した量の該生体分子を産生する該宿主微生物の各株を選択するステップであって、該対照微生物は、細菌ヘモグロビン遺伝子の該ライブラリー由来の細菌ヘモグロビン遺伝子を発現しない、ステップと
を含む方法。 - 細菌ヘモグロビン遺伝子の前記ライブラリーが、配列番号12、配列番号10、配列番号11、配列番号9、配列番号13、配列番号14、配列番号15、配列番号16、配列番号17、配列番号18、配列番号19、および配列番号20から選択されるヌクレオチド配列を有する1種または複数種の細菌ヘモグロビン遺伝子を含む、請求項14に記載の方法。
- 細菌ヘモグロビン遺伝子の前記ライブラリーが、配列番号26、配列番号24、配列番号25、配列番号23、配列番号27、配列番号28、配列番号29、配列番号30、配列番号31、配列番号32、配列番号33および配列番号34から選択される1種または複数種のポリペプチド配列をコードする1種または複数種の細菌ヘモグロビン遺伝子を含む、請求項14に記載の方法。
- 細菌ヘモグロビン遺伝子の前記ライブラリーが、表2に列挙された微生物の株、種、または亜種に由来する1種または複数種の細菌ヘモグロビン遺伝子を含む、請求項14に記載の方法。
- 前記細菌ヘモグロビン遺伝子が、細菌フラボヘモグロビン遺伝子である、請求項14に記載の方法。
- 細菌フラボヘモグロビン遺伝子のライブラリーが、表2に列挙された微生物の株、種、または亜種に由来する1種または複数種の細菌フラボヘモグロビン遺伝子を含む、請求項18に記載の方法。
- ヘモグロビンの前記ライブラリー内の前記細菌ヘモグロビンの少なくとも1種が、細菌フラボヘモグロビンである、請求項14に記載の方法。
- 細菌ヘモグロビン遺伝子の前記ライブラリーが、表2に列挙された微生物の株、種、または亜種に由来する1種または複数種の細菌ヘモグロビン遺伝子および表2に列挙された微生物の株、種、または亜種に由来する1種または複数種の細菌フラボヘモグロビン遺伝子を含む、請求項20に記載の方法。
- 前記宿主微生物が、Corynebacterium属に属する、請求項14から21までのいずれか一項に記載の方法。
- 前記宿主微生物が、Corynebacterium glutamicumである、請求項14から22までのいずれか一項に記載の方法。
- 前記導入することが、形質転換、形質導入または電気穿孔によって実施される、請求項14から23までのいずれか一項に記載の方法。
- 前記生体分子が、低分子、アミノ酸、ヌクレオチド、有機酸、またはアルコールである、請求項14から24までのいずれか一項に記載の方法。
- 前記アミノ酸が、リシン、グルタミン酸、チロシン、フェニルアラニン、トリプトファン、アスパラギン酸、アスパラギン、トレオニン、イソロイシン、またはメチオニンである、請求項25に記載の方法。
- 前記有機酸が、スクシネート、ラクテートまたはピルベートである、請求項25に記載の方法。
- 前記アルコールが、エタノールまたはイソブタノールである、請求項25に記載の方法。
- 細菌ヘモグロビン遺伝子のライブラリーであって、細菌ヘモグロビン遺伝子の該ライブラリー内の各細菌ヘモグロビン遺伝子が、配列番号1、配列番号2、配列番号3、配列番号4、配列番号5、配列番号6、配列番号7、および配列番号8から選択されるヌクレオチド配列を含むプロモーターに機能的に連結している、ライブラリー。
- 細菌ヘモグロビン遺伝子の前記ライブラリーが配列番号12、配列番号10、配列番号11、配列番号9、配列番号13、配列番号14、配列番号15、配列番号16、配列番号17、配列番号18、配列番号19、および配列番号20から選択される1種または複数種の細菌ヘモグロビン遺伝子を含む、請求項29に記載のライブラリー。
- 細菌ヘモグロビン遺伝子の前記ライブラリーが配列番号26、配列番号24、配列番号25、配列番号23、配列番号27、配列番号28、配列番号29、配列番号30、配列番号31、配列番号32、配列番号33および配列番号34から選択される1種または複数種のポリペプチド配列をコードする1種または複数種の細菌ヘモグロビン遺伝子を含む、請求項29に記載のライブラリー。
- 細菌ヘモグロビン遺伝子の前記ライブラリーが、表2に列挙された微生物の株、種、または亜種に由来する1種または複数種の細菌ヘモグロビン遺伝子を含む、請求項29に記載のライブラリー。
- 前記ライブラリー内の前記細菌ヘモグロビン遺伝子のそれぞれが、細菌フラボヘモグロビン遺伝子である、請求項29に記載のライブラリー。
- 細菌フラボヘモグロビン遺伝子の前記ライブラリーが、表2に列挙された微生物の株、種、または亜種に由来する1種または複数種の細菌フラボヘモグロビン遺伝子を含む、請求項33に記載のライブラリー。
- 細菌ヘモグロビン遺伝子の前記ライブラリー内の前記細菌ヘモグロビン遺伝子の少なくとも1種が、細菌フラボヘモグロビン遺伝子である、請求項29に記載のライブラリー。
- 細菌ヘモグロビン遺伝子の前記ライブラリーが、表2に列挙された微生物の株、種、または亜種に由来する1種または複数種の細菌ヘモグロビン遺伝子および表2に列挙された微生物の株、種、または亜種に由来する1種または複数種の細菌フラボヘモグロビン遺伝子を含む、請求項35に記載のライブラリー。
- 生体分子を産生させる方法であって、請求項29から36までのいずれか一項に記載のライブラリーに由来する細菌ヘモグロビン遺伝子を宿主細胞に導入するステップと、該宿主細胞を該生体分子の産生に適した条件下で培養するステップとを含む方法。
- 前記生体分子が、低分子、アミノ酸、ヌクレオチド、有機酸、またはアルコールである、請求項37に記載の方法。
- 前記アミノ酸が、リシン、グルタミン酸、チロシン、フェニルアラニン、トリプトファン、アスパラギン酸、アスパラギン、トレオニン、イソロイシン、またはメチオニンである、請求項38に記載の方法。
- 前記有機酸が、スクシネート、ラクテートまたはピルベートである、請求項38に記載の方法。
- 前記アルコールが、エタノールまたはイソブタノールである、請求項38に記載の方法。
- 前記宿主細胞が、Corynebacterium属に属する、請求項37から41までのいずれか一項に記載の方法。
- 前記宿主細胞が、Corynebacterium glutamicumである、請求項37から42までのいずれか一項に記載の方法。
- 前記導入するステップが、形質転換、形質導入または電気穿孔によって実施される、請求項37から43までのいずれか一項に記載の方法。
- 配列番号12、配列番号10、配列番号11、配列番号9、配列番号13、配列番号14、配列番号15、配列番号16、配列番号17、配列番号18、配列番号19および配列番号20から選択される配列を有するコドン最適化ポリヌクレオチドを含む、単離された、合成または組換えポリヌクレオチドであって、宿主細胞における発現のためにコドン最適化されている、単離された、合成または組換えポリヌクレオチド。
- 前記宿主細胞が、E.coliおよび/またはC.glutamicumである、請求項45に記載の単離された、合成または組換えポリヌクレオチド。
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KR102345899B1 (ko) | 2021-12-31 |
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WO2018005655A2 (en) | 2018-01-04 |
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