JP2018533378A - キメラ微生物ハイブリッドを製造する方法 - Google Patents
キメラ微生物ハイブリッドを製造する方法 Download PDFInfo
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- JP2018533378A JP2018533378A JP2018545116A JP2018545116A JP2018533378A JP 2018533378 A JP2018533378 A JP 2018533378A JP 2018545116 A JP2018545116 A JP 2018545116A JP 2018545116 A JP2018545116 A JP 2018545116A JP 2018533378 A JP2018533378 A JP 2018533378A
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Classifications
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- C—CHEMISTRY; METALLURGY
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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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
- 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
- C12N1/16—Yeasts; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- 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
- 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
- C12N1/20—Bacteria; Culture media therefor
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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Abstract
Description
本出願は、2015年11月17日に提出した米国仮出願第62/256,625号、および2016年7月15日に提出した米国出願第15/212,068号に基づく優先権を主張し、その両方が、参照によりそれらの全体で本明細書に組み込まれる。
本発明は、遺伝子操作なしで2つの微生物種の間で染色体DNAを導入する方法、特に、ベクターを使用せずに水平遺伝子導入により表現型を加えるか、または削除するために使用され得る方法に関する。この方法から生じる株は、両方の親由来のDNAの組み合わせを発現することができるキメラ微生物ハイブリッドである。
細菌および真菌は、様々な商業的応用を有し、アルコール飲料、食品、駆除剤、食品添加物、バイオ燃料、プロバイオティクス、および医薬の製造において使用される。一般には天然で遭遇しない表現型形質の組み合わせを有する微生物が、生物学的治療のような、幾つかの応用に頻繁に必要である。野生型微生物株は、生存および複製のため進化的発生を示す。しかしながら、商業的応用は、産業プロセスを最適化し、かつ微生物の天然の環境内では大抵有利ではない量で産物を産生する形質の混在した株を大抵必要とする。株の変更ならびに改良方法を開発して、新規商品を創出し、および/または収益性を増大させた。従来の株の変更は、3つの異なるアプローチにより支配され、後述の通り、それぞれが、それ独自の制限を伴う。
ベクターを使用せずに、水平遺伝子導入を通じてキメラ微生物ハイブリッドを製造する方法が、本明細書において提供される。これらの方法は、一連の環境圧および選択ツールを使用して、微生物種に渡る遺伝子導入のため、天然の接合、形質転換、または別の機序を使用して指向進化を行う。多くの刊行物は、天然に生じる生物のゲノム配列データを使用して、例えば、形質転換、形質導入、接合、摩擦による、または幾つかの他の遺伝子導入現象による、天然での多様な種の間での外遺伝子導入の仮説を支持している。しかしながら、この現象は、ベクターまたはバクテリオファージを使用しない、異なる種に渡る実験室設定においては生じないことが報告されている。理論により結び付けられることを望んではいないが、本明細書において開示される方法は、多様な微生物種に渡る遺伝子導入が、環境が、複製がもはや可能でないようなレベルのストレスに至ったときに関与する最後の頼みの綱の生存機序であり得ることを証明する。
「キメラ生物」または「キメラ」は、2つもしくはそれ以上の微生物種由来の機能遺伝子を含有する単細胞生物を指す。
キメラ微生物ハイブリッドを製造する方法が提供される。以下の工程の幾つかまたは全てが行われてもよい。
キメラ微生物ハイブリッドは、本明細書において開示される方法から生じる株であり、ハイブリッド表現型および遺伝子型を有する。それらは、ベクターを使用しない、インビトロの外側遺伝子導入の結果である。キメラ微生物は、両方の親宿主と親ドナー由来のDNAを保持し、発現する。
キメラ微生物ハイブリッドは、多くの商業および産業応用を有する。新規商品を創出するため、もしくは現在の商業的微生物の収益性を改善するために形質を加えるか、または差し引くことは、広範な効力のある使用、ならびにアルコール製造、発酵食品、除草剤製造、真菌剤製造、殺菌剤製造、駆除剤製造、食品添加物および保存剤、バイオ燃料および生化学の製造、プロバイオティクス、生きた生物学的治療、薬効のある食べ物、ならびに医薬を含むが、これらに限定されない、かかる産業における商品の製造を含む。
エタノール前ストレス要因でのラクトバチルス・プランタルムアミノ酸栄養要求性除去
この実験における所望の親宿主は、ラクトバチルス・プランタルムであった。取得させるべき表現型は、親ドナーであるクロストリジウム・ベイジェリンキが提供した。遺伝子導入の所望の形質は、アミノ酸原栄養性、またはタンパク質もしくはアミノ酸を含有しない最小培地上での生存であり、クロストリジウム親は、最小培地上で35〜37℃にて嫌気的に成長することができる。ラクトバチルス親は、タンパク質または加えられたアミノ酸を含まない培地上で成長することができない。
原栄養体ハイブリッドをもたらす遺伝子導入の頻度は、200mMエタノール前ストレス要因条件について1×10-8であった。適用した前ストレス要因を含まない条件から生じたコロニーはなかった。生じたキメラ微生物ハイブリッドC-1の分析を、実施例2由来の9つのキメラと共に並行して行った。以下の実施例2は、より詳細な結果を提供する。
UV前ストレス要因でのラクトバチルス・プランタルムアミノ酸栄養要求性除去
所望の親宿主は、ラクトバチルス・プランタルムであった。導入すべき所望の表現型は、親ドナーであるクロストリジウム・ベイジェリンキが提供した。遺伝子導入の所望の形質は、アミノ酸原栄養性、またはタンパク質もアミノ酸も含有しない最小培地プレート上での生存であった。
原栄養体ハイブリッドをもたらす遺伝子導入の頻度は、UV前ストレス要因のみのラクトバチルスについて1×10-8であった。これは、200mMエタノールについて実施例1において観察したのと同じ頻度であった。二重UV前ストレス要因条件における上出来の原栄養体ハイブリッドは、5×10-8の増大した頻度を有していた。最終的に、いずれかの親についての前ストレス要因も含まない条件は、クロストリジウム・ベイジェリンキドナー細胞当たり3×10-8の頻度を有していた。計10種の株を分析し、キメラ微生物ハイブリッドであることを見出した。
サッカロマイセス・セレビシエ・バー・ボウラディソルビトール代謝付加
この実験における所望の親宿主は、サッカロマイセス・セレビシエ・バー・ボウラディであった。獲得すべき表現型は、親ドナーメチニコビア・リューカフィー(Metschnikowia reukaffi)野生花蜜酵母が提供した。遺伝子導入の所望の形質は、ソルビトール代謝、または唯一の炭水化物供給源としてソルビトールを含むタンパク質リッチな培地上での生存であった。花蜜酵母ドナーは、この同じ、ソルビトールを含有するタンパク質リッチな培地上で23〜34℃にて好気的に成長することができる。サッカロマイセス親は、唯一の炭素供給源としてソルビトールを含む最小培地上で成長することができないが、それは、炭素供給源としてでんぷんまたはグルコースを含む最小培地上で成長することができる。
ソルビトールを代謝するハイブリッドをもたらす遺伝子導入の頻度は、前ストレス要因なしについて1.7×10-5であり、UV前ストレス要因を使用して2×10-5であった。最小培地ならびにクロストリジウム・ディフィシル毒素AおよびBの追加選択は、コロニー数を10倍低減した。これは、細菌の遺伝子導入において見られるものより顕著に高い。酵母細胞間での遺伝子導入現象であったと信じられているものは、図9Aにおける遺伝子導入顕微鏡写真を、図9Bにおける通常の出芽と比較して、図9A〜9Bにおいて示される通り、顕微鏡下で見られた。11種のキメラ細菌ハイブリッドを分析した。
グラム陰性ドナーを介したビフィドバクテリウム・フィーカレ(B. faecale)アミノ酸栄養要求性除去
所望の親宿主は、ビフィドバクテリウム・フィーカレであった。導入すべき所望の表現型は、親ドナーグリモンテラ・セネガレンシスが提供した。遺伝子導入についての所望の形質は、アミノ酸原栄養性、またはタンパク質もアミノ酸も含有しない最小培地プレート上での生存であった。
原栄養体ハイブリッドをもたらす遺伝子導入の頻度は、従前の細菌の遺伝子導入においてよりずっと高かった。頻度率1.1×10-6について、計2,300コロニーが存在した。これらのうち3種のキメラ微生物ハイブリッドのみを詳細に分析し、それらを、プレート上でのそれらの異なる形態について選択した。
Claims (20)
- ベクターを使用しない、2つの異なる微生物の親株の間での染色体の水平遺伝子導入方法であって、第1と第2の親株の細胞を、固形成長培地上または液体成長培地に一緒に組み合わせることを含み、前記成長培地、および/または前記成長培地の周囲もしくはそれと接触する環境が、それぞれの親株についての少なくとも1つの別々のストレス要因を含有し、前記第1と第2の親微生物ハイブリッド株の遺伝子ハイブリッドであり、前記第1の親株から前記第2の親株に導入された遺伝子材料由来の少なくとも1つの表現型形質を発現する、微生物ハイブリッド株が製造される、方法。
- 前記第1と第2の親株が、異なる微生物種である、請求項1に記載の方法。
- 前記微生物ハイブリッドが、両方の親株から与えられた遺伝子材料由来の表現型形質を発現する、請求項1に記載の方法。
- それぞれのストレス要因が、成長を約80%〜約100%阻害するのに十分である濃度またはレベルにて存在する、請求項1に記載の方法。
- 前記微生物の親株が、細菌または真菌である、請求項1に記載の方法。
- 前記微生物ハイブリッド株が、1つまたは複数の病態の生物学的治療である、請求項1に記載の方法。
- 前記微生物ハイブリッド株が、それがもたらされた前記微生物の親株と比較し、1つもしくは複数の産業応用に対する増大または低下した適応性を有する、請求項1に記載の方法。
- 前記微生物ハイブリッド株が、それがもたらされた前記微生物の親株と比較し、新規商品を産生する、請求項1に記載の方法。
- 前記微生物ハイブリッド株が、プロバイオティック特性を有する、請求項1に記載の方法。
- 前記微生物ハイブリッド株の少なくとも1つの親が、安全(GRAS)と一般に認められる、請求項1に記載の方法。
- 前記微生物ハイブリッドが、ラクトバチルス、ビフィドバクテリウム、ストレプトコッカス、クロストリジウム、マイコプラズマ、バチルス、スタフィロコッカス、ラクトコッカス、ロイコノストック、ペディオコッカス、エンテロコッカス、エンテロバクテリアッセ、エシェリキア、シュードモナス、バクテリオデス(Bacteroidetes)、またはアクチノバクテリアの細菌種である親株由来のDNAを含有する、請求項1に記載の方法。
- 前記微生物ハイブリッドが、サッカロマイセス、シゾサッコロミセス(Schizosacchoromyces)、シェフェロマイセス、ザイゴサッカロミセス、ヤロウイア、ピキア、デッケラ、クリベロマイセス、カンジダ、メチニコビア、またはトルラスポラの真菌種である親株由来のDNAを含有する、請求項1に記載の方法。
- 前記ラクトバチルス種が、ラクトバチルス・プランタルム、ラクトバチルス・デルブリュッキ、ラクトバチルス・アシドフィルス、ラクトバチルス・ブレビス、ラクトバチルス・カゼイ、ラクトバチルス・パラカゼイ(L. paracasei)、ラクトバチルス・サンフランシスエンシス、ラクトバチルス・ラムノサス、ラクトバチルス・ヘルベティカス、ラクトバチルス・カルバータス、ラクトバチルス・サケイ、ラクトバチルス・ブフネリ、ラクトバチルス・ファーメンタム、またはラクトバチルス・ロイテリである、請求項11に記載の方法。
- 前記ビフィドバクテリウム種が、ビフィドバクテリウム・アニマリス(B. animalis)、ビフィドバクテリウム・アステロイデス(B. asteroids)、ビフィドバクテリウム・ビフィドゥム(B bifidum)、ビフィドバクテリウム・ブレベ(B. breve)、ビフィドバクテリウム・デンチクム(B. denticum)、ビフィドバクテリウム・フィーカレ、ビフィドバクテリウム・インファンティス(B. infantis)、ビフィドバクテリウム・ロングム(B. longum)、ビフィドバクテリウム・メリシカム(B. merycicum)、ビフィドバクテリウム・ルミナンティウム(B. ruminantium)、ビフィドバクテリウム・サーマシドフィルム(B. thermacidophilum)、またはビフィドバクテリウム・ツルミエンス(B. tsurumiense)である、請求項11に記載の方法。
- 前記サッカロマイセス種が、サッカロマイセス・セレビシエ・バー・ボウラディ、サッカロマイセス・セレビシエ(S. cerevisiae)、サッカロマイセス・パストリアヌス(S. pastorianus)、サッカロマイセス・フラギリス(S. fragilis)またはサッカロマイセス・バヤヌス(S. bayanus)である、請求項12に記載の方法。
- 請求項1に記載の方法により製造される、微生物ハイブリッド株。
- 請求項1に記載の方法により製造される、生物学的治療用微生物株。
- 請求項1に記載の方法により製造される、プロバイオティック微生物株。
- 商品を産生する、請求項1に記載の方法により製造される、微生物株。
- 組換えDNA技術を使用せず、2つの異なる微生物の親株または種からもたらされる天然に生じない微生物ハイブリッド。
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CN104293716A (zh) * | 2014-10-08 | 2015-01-21 | 湖南民康生物技术研究所 | 一种用大型真菌菌液(质)制备高效益生菌制剂的方法 |
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2017
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2018
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CN108351354B (zh) | 2021-06-04 |
AU2016355409A1 (en) | 2018-05-17 |
US9765358B2 (en) | 2017-09-19 |
WO2017087429A1 (en) | 2017-05-26 |
EP3377901A4 (en) | 2019-04-24 |
CN108351354A (zh) | 2018-07-31 |
JP2022064330A (ja) | 2022-04-25 |
EP3377901A1 (en) | 2018-09-26 |
US20180087071A1 (en) | 2018-03-29 |
US20190367946A1 (en) | 2019-12-05 |
CA3002823A1 (en) | 2017-05-26 |
US20170137844A1 (en) | 2017-05-18 |
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