JPWO2020127417A5

JPWO2020127417A5 -

Info

Publication number: JPWO2020127417A5
Application number: JP2021535101A
Authority: JP
Publication date: 2022-12-26

Description

実験は、配列番号２または配列番号６を有するラクトース結合アルファ－１，３－フコシルトランスフェラーゼ遺伝子を発現する突然変異体Ｅ．ｃｏｌｉ株は、バイオリアクタースケールでの流加回分発酵において、配列番号１８を含有する同様の株よりも高い３－ＦＬ純度を有するブロスを産生することを示す。
特定の実施形態では、例えば、以下が提供される：
（項目１）
α－１，３－フコシルラクトースを産生するための方法であって、
ａ）α－１，３－フコシルトランスフェラーゼ活性を有し、ラクトースをアクセプター基質として使用する能力を有するポリペプチドを提供するステップであって、前記ポリペプチドが、
ｉ）保存されたＧＤＰ－フコース結合ドメイン［Ｙ／Ｗ／Ｌ／Ｈ／Ｆ／Ｍ］Ｘ［Ｔ／Ｓ／Ｃ］［Ｅ／Ｑ／Ｄ／Ａ］［Ｋ／Ｒ］（配列番号３３）をコードするアミノ酸配列、
ｉｉ）保存された［Ｋ／Ｄ］［Ｌ／Ｋ／Ｍ］ＸＸＸ［Ｆ／Ｙ］ドメイン（配列番号３４）をコードするアミノ酸配列
を含み、
ｉｉｉ）加えて、ｉｉ）の前記ドメインがＤＭ［Ａ／Ｓ］ＶＳＦ（配列番号３６）に等しい場合、保存されたモチーフ［Ｎ／Ｈ］ＸＤＰＡＸＬＤ（配列番号３５）が、Ｎ末端領域に存在し、
式中、Ｘが、任意の別個のアミノ酸であってもよく、
前記ポリペプチドのＣ末端が、前記ＧＤＰ－フコース結合ドメインの第１のアミノ酸から開始して１００個未満またはそれに等しいアミノ酸を有する、ステップと、
ｂ）ステップａ）のα－１，３－フコシルトランスフェラーゼ活性を有する前記ポリペプチドを、ＧＤＰ－フコースをドナー基質として、およびラクトースをアクセプター基質として含む混合物と、前記ポリペプチドが、フコース残基の、前記ドナー基質から前記アクセプター基質への移行を触媒する条件下で接触させ、
それによって、α－１，３－フコシルラクトースを産生するステップと、
ｃ）必要に応じて、前記α－１，３－フコシルラクトースを分離するステップと
を含む方法。
（項目２）
前記ポリペプチドが、細胞非含有系で提供される、項目１に記載の方法。
（項目３）
前記ポリペプチドが、前記ポリペプチドをコードするポリヌクレオチドを含む細胞によって産生される、項目１に記載の方法。
（項目４）
前記ＧＤＰ－フコースおよび／またはラクトースが、前記ＧＤＰ－フコースおよび／またはラクトースを産生する細胞によって提供される、項目１または３に記載の方法。
（項目５）
ｉ）α－１，３－フコシルラクトースの産生のために遺伝子改変された細胞を提供するステップであって、前記細胞が、α－１，３－フコシルラクトース合成のための酵素をコードする少なくとも１つの核酸配列を含み、
前記細胞が、α－１，３－フコシルトランスフェラーゼ活性を有し、ラクトースをアクセプター基質として使用する能力を有する前記ポリペプチドの発現を含む、ステップと、
ｉｉ）前記細胞を、培地中でα－１，３－フコシルラクトースの産生を許容する条件下で培養するステップと、
ｉｉｉ）好ましくは、前記α－１，３－フコシルラクトースを培養から分離するステップと
を含む、項目１、３または４のいずれか一項に記載の方法。
（項目６）
ａ）α－１，３－フコシルトランスフェラーゼ活性を有し、ラクトースをアクセプター基質として使用する能力を有する前記ポリペプチドを発現する宿主細胞を提供するステップと、
ｂ）前記宿主細胞を、α－１，３－フコシルラクトースの産生を許容し、α－１，３－フコシルトランスフェラーゼ活性を有する前記ポリペプチドの発現を許容する好適な栄養条件下で、増殖させるステップと、
ｃ）α－１，３－フコシルトランスフェラーゼポリペプチドがフコース残基の、ドナー基質ＧＤＰ－フコースからアクセプター基質ラクトースへの移行を触媒するために、ＧＤＰ－フコースおよびラクトースをステップｂ）と同時に、またはそれに続いて提供し、それによって、α－１，３－フコシルラクトースを産生するステップと、
ｄ）必要に応じて、前記α－１，３－フコシルラクトースを前記宿主細胞またはその増殖培地から分離するステップと
を含む、項目３に記載の方法。
（項目７）
前記宿主細胞が、α－１，３－フコシルトランスフェラーゼ活性を有し、ラクトースをアクセプター基質として使用する能力を有する外因性ポリペプチドを発現するように形質転換またはトランスフェクトされる、項目５または６に記載の方法。
（項目８）
前記ＧＤＰ－フコースおよび／またはラクトースが、前記宿主細胞で同時発現される酵素によって、または前記宿主細胞の代謝によって提供されることを特徴とする、項目３から７のいずれか一項に記載の方法。
（項目９）
α－１，３－フコシルラクトースの精製をさらに含む、項目１から８のいずれか一項に記載の方法。
（項目１０）
前記ポリペプチドが、
ｉ）添付の配列表の配列番号６、２、４、８、１０、１２、１４、１６、２０、２２、２８、３０または３２のうちのいずれか１つ；
ｉｉ）配列番号２、２０または２２の全長アミノ酸配列との８７％またはそれよりも高い配列同一性を有するアミノ酸配列；
ｉｉｉ）配列番号６、８、１０、１２、１４、１６、２８、３０または３２のうちのいずれか１つの全長アミノ酸配列との８０％またはそれよりも高い配列同一性を有するアミノ酸配列；
ｉｖ）配列番号２、２０または２２で示されるアミノ酸配列の断片であって、少なくとも４５個のその連続アミノ酸を含む断片；
ｖ）配列番号６、８、１０、１２、１４、１６、２８、３０または３２のうちのいずれか１つで示されるアミノ酸配列の断片であって、少なくとも１０個のその連続アミノ酸を含み、ラクトース結合アルファ－１，３－フコシルトランスフェラーゼ活性を有する断片
からなる群から選択され、
必要に応じて、前記ポリペプチドが、Ｎ末端および／またはＣ末端アミノ酸伸長部によってさらに改変される、先行項目のいずれか一項に記載の方法。
（項目１１）
先行項目のいずれか一項に記載の３－フコシルラクトースの産生のための方法であって、以下のステップ：
ｉ）培養培地に、開始リアクター体積につき少なくとも５０、より好ましくは少なくとも７５、より好ましくは少なくとも１００、より好ましくは少なくとも１２０、より好ましくは少なくとも１５０グラムのラクトースを含むラクトース供給を、好ましくは連続様式で、好ましくは前記培養培地の最終体積が、前記ラクトース供給の添加前の前記培養培地の体積の３倍以下、好ましくは２倍以下、より好ましくは２倍未満であるように、添加するステップ；
ｉｉ）ラクトース供給を、連続様式で前記培養培地に１日、２日、３日、４日、５日の期間にわたり供給溶液の手段によって添加するステップ；
ｉｉｉ）ラクトース供給を、連続様式で前記培養培地に１日、２日、３日、４日、５日の期間にわたり供給溶液の手段によって添加するステップであって、前記ラクトース供給の溶液の濃度が、５０ｇ／Ｌ、好ましくは７５ｇ／Ｌ、より好ましくは１００ｇ／Ｌ、より好ましくは１２５ｇ／Ｌ、より好ましくは１５０ｇ／Ｌ、より好ましくは１７５ｇ／Ｌ、より好ましくは２００ｇ／Ｌ、より好ましくは２２５ｇ／Ｌ、より好ましくは２５０ｇ／Ｌ、より好ましくは２７５ｇ／Ｌ、より好ましくは３００ｇ／Ｌ、より好ましくは３２５ｇ／Ｌ、より好ましくは３５０ｇ／Ｌ、より好ましくは３７５ｇ／Ｌ、より好ましくは４００ｇ／Ｌ、より好ましくは４５０ｇ／Ｌ、より好ましくは５００ｇ／Ｌ、さらにより好ましくは５５０ｇ／Ｌ、最も好ましくは６００ｇ／Ｌであり、好ましくは前記溶液のｐＨが、３～７の間に設定され、好ましくは前記供給溶液の温度が、２０℃～８０℃の間で維持される、ステップ
のうちの少なくとも１つをさらに含み、
ｉｖ）前記方法が、前記培養培地の最終体積中の少なくとも５０ｇ／Ｌ、好ましくは少なくとも７５ｇ／Ｌ、より好ましくは少なくとも９０ｇ／Ｌ、より好ましくは少なくとも１００ｇ／Ｌ、より好ましくは少なくとも１２５ｇ／Ｌ、より好ましくは少なくとも１５０ｇ／Ｌ、より好ましくは少なくとも１７５ｇ／Ｌ、より好ましくは少なくとも２００ｇ／Ｌの３－フコシルラクトース濃度をもたらす、方法。
（項目１２）
α－１，３－フコシルラクトースの産生のために遺伝子改変された宿主細胞であって、α－１，３－フコシルラクトース合成に関与する酵素をコードする少なくとも１つの核酸配列を含み、α－１，３－フコシルトランスフェラーゼ活性を有し、ラクトースをアクセプター基質として使用する能力を有するポリペプチドの発現を含み、前記ポリペプチドが、
ｉ）保存されたＧＤＰ－フコース結合ドメイン［Ｙ／Ｗ／Ｌ／Ｈ／Ｆ／Ｍ］Ｘ［Ｔ／Ｓ／Ｃ］［Ｅ／Ｑ／Ｄ／Ａ］［Ｋ／Ｒ］（配列番号３３）をコードするアミノ酸配列、
ｉｉ）保存された［Ｋ／Ｄ］［Ｌ／Ｋ／Ｍ］ＸＸＸ［Ｆ／Ｙ］ドメイン（配列番号３４）をコードするアミノ酸配列
を含み、
ｉｉｉ）加えて、ｉｉ）の前記ドメインがＤＭ［Ａ／Ｓ］ＶＳＦ（配列番号３６）に等しい場合、保存されたモチーフ［Ｎ／Ｈ］ＸＤＰＡＸＬＤ（配列番号３５）が、Ｎ末端領域に存在し、
式中、Ｘが、任意の別個のアミノ酸であってもよく、
前記ポリペプチドのＣ末端が、前記ＧＤＰ－フコース結合ドメインの第１のアミノ酸から開始して１００個未満またはそれに等しいアミノ酸を有する、宿主細胞。
（項目１３）
ｉ）ラクトース結合アルファ－１，３－フコシルトランスフェラーゼ活性を有する前記ポリペプチドをコードするポリヌクレオチドを含む配列であって、前記宿主細胞にとって外来性の配列であり、前記宿主細胞のゲノムに組み込まれる配列を含むか、またはｉｉ）前記ポリペプチドをコードするポリヌクレオチドを含むベクターを含有し、前記ポリヌクレオチドが、前記ベクターで形質転換される宿主細胞によって認識される制御配列に作動可能に連結されている、項目１２に記載の細胞。
（項目１４）
前記ポリペプチドが、
ｉ）添付の配列表の配列番号６、２、４、８、１０、１２、１４、１６、２０、２２、２８、３０または３２のうちのいずれか１つ；
ｉｉ）配列番号２、２０または２２の全長アミノ酸配列との８７％またはそれよりも高い配列同一性を有するアミノ酸配列；
ｉｉｉ）配列番号６、８、１０、１２、１４、１６、２８、３０または３２のうちのいずれか１つの全長アミノ酸配列との８０％またはそれよりも高い配列同一性を有するアミノ酸配列；
ｉｖ）配列番号２、２０または２２で示されるアミノ酸配列の断片であって、少なくとも４５個のその連続アミノ酸を含む断片；
ｖ）配列番号６、８、１０、１２、１４、１６、２８、３０または３２のうちのいずれか１つで示されるアミノ酸配列の断片であって、少なくとも１０個のその連続アミノ酸を含み、ラクトース結合アルファ－１，３－フコシルトランスフェラーゼ活性を有する断片
からなる群から選択されるアミノ酸配列を含み、
必要に応じて、前記ポリペプチドが、Ｎ末端および／またはＣ末端アミノ酸伸長部によってさらに改変される、項目１２または１３に記載の細胞。
（項目１５）
前記細胞が、微生物、植物または動物細胞からなる群から選択され、好ましくは前記微生物が、細菌、真菌または酵母であり、好ましくは前記植物が、イネ、ワタ、ナタネ、ダイズ、トウモロコシまたはコーン植物であり、好ましくは前記動物が、昆虫、魚類、鳥類または非ヒト哺乳動物であり；好ましくは前記細胞が、Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ細胞である、項目３から１１のいずれか一項に記載の方法、または項目１２、１３もしくは１４のいずれか一項に記載の細胞。
（項目１６）
細菌の、好ましくはＥｓｃｈｅｒｉｃｈｉａｃｏｌｉ株の、より好ましくはＫ１２株であるＥｓｃｈｅｒｉｃｈｉａｃｏｌｉ株の細胞であり、さらにより好ましくは前記ＥｓｃｈｅｒｉｃｈｉａｃｏｌｉＫ１２株が、ＥｓｃｈｅｒｉｃｈｉａｃｏｌｉＭＧ１６５５であることを特徴とする、項目１２から１５のいずれか一項に記載の宿主細胞。
（項目１７）
酵母細胞であることを特徴とする、項目１２から１５のいずれか一項に記載の宿主細胞。
（項目１８）
ラクトース結合アルファ－１，３－フコシルトランスフェラーゼ（alpha-1,2-fucosyltransferase）活性を有する前記ポリペプチドをコードする前記ポリヌクレオチドが、各々の宿主細胞のコドン利用に適合される、項目１２から１７のいずれか一項に記載の宿主細胞。
（項目１９）
α－１，３－フコシルラクトースの産生のための方法であって、
ａ）項目１２から１８のいずれか一項に記載の細胞を提供するステップと、
ｂ）前記細胞を、培地中でα－１，３－フコシルトランスフェラーゼの産生を許容する条件下で培養するステップと、
ｃ）好ましくは、前記α－１，３－フコシルトランスフェラーゼを培養から分離するステップと
を含む方法。
（項目２０）
α－１，３－フコシルラクトースの産生のための、項目１２から１８のいずれか一項に記載の宿主細胞の使用。
（項目２１）
α－１，３－フコシルラクトースの産生のための、項目１または１１に記載の方法で説明されるポリペプチドの使用。
（項目２２）
ラクトース結合アルファ－１，３－フコシルトランスフェラーゼポリペプチドを異種性に発現する微生物であって、前記ポリペプチドが、
ｉ）保存されたＧＤＰ－フコース結合ドメイン［Ｙ／Ｗ／Ｌ／Ｈ／Ｆ／Ｍ］Ｘ［Ｔ／Ｓ／Ｃ］［Ｅ／Ｑ／Ｄ／Ａ］［Ｋ／Ｒ］（配列番号３３）をコードするアミノ酸配列、
ｉｉ）保存された［Ｋ／Ｄ］［Ｌ／Ｋ／Ｍ］ＸＸＸ［Ｆ／Ｙ］ドメイン（配列番号３４）をコードするアミノ酸配列
を含み、
ｉｉｉ）加えて、ｉｉ）の前記ドメインがＤＭ［Ａ／Ｓ］ＶＳＦ（配列番号３６）に等しい場合、保存されたモチーフ［Ｎ／Ｈ］ＸＤＰＡＸＬＤ（配列番号３５）が、Ｎ末端領域に存在し、
式中、Ｘが、任意の別個のアミノ酸であってもよく、
前記ポリペプチドのＣ末端が、前記ＧＤＰ－フコース結合ドメインの第１のアミノ酸から開始して１００個未満またはそれに等しいアミノ酸を有する、微生物。
（項目２３）
前記ポリペプチドが、
ｉ）添付の配列表の配列番号６、２、４、８、１０、１２、１４、１６、２０、２２、２８、３０または３２のうちのいずれか１つ；
ｉｉ）配列番号２、２０または２２の全長アミノ酸配列との８７％またはそれよりも高い配列同一性を有するアミノ酸配列；
ｉｉｉ）配列番号６、８、１０、１２、１４、１６、２８、３０または３２のうちのいずれか１つの全長アミノ酸配列との８０％またはそれよりも高い配列同一性を有するアミノ酸配列；
ｉｖ）配列番号２、２０または２２で示されるアミノ酸配列の断片であって、少なくとも４５個のその連続アミノ酸を含む断片；
ｖ）配列番号６、８、１０、１２、１４、１６、２８、３０または３２のうちのいずれか１つで示されるアミノ酸配列の断片であって、少なくとも１０個のその連続アミノ酸を含み、ラクトース結合アルファ－１，３－フコシルトランスフェラーゼ活性を有する断片
からなる群から選択されるアミノ酸配列を含み、
必要に応じて、前記ポリペプチドが、Ｎ末端および／またはＣ末端アミノ酸伸長部によってさらに改変される、項目２２に記載の微生物。
（項目２４）
アルファ－１，３－フコシルラクトースの産生のための、項目２２または２３に記載の微生物の使用。
（項目２５）
前記アルファ－１，３－フコシルラクトースを前記宿主細胞またはその増殖培地から分離するステップをさらに含む、項目１から１１、１５または１９のいずれか一項に記載の方法。
（項目２６）
前記分離が、以下のステップ：浄化、限外濾過、ナノ濾過、逆浸透法、精密濾過、活性炭もしくは炭素処理、接線流高性能濾過、接線流限外濾過、親和性クロマトグラフィー、イオン交換クロマトグラフィー、疎水性相互作用クロマトグラフィーおよび／またはゲル濾過、リガンド交換クロマトグラフィーのうちの少なくとも１つを含む、項目１から１１、１５、１９または２５のいずれか一項に記載の方法。
（項目２７）
アルファ－１，３－フコシルラクトースの精製をさらに含む、項目１から１１、１５、１９、２５または２６のいずれか一項に記載の方法。
（項目２８）
前記アルファ－１，３－フコシルラクトースの前記精製が、以下のステップ：活性炭または炭素の使用、炭の使用、ナノ濾過、限外濾過またはイオン交換、アルコールの使用、水性アルコール混合物の使用、結晶化、蒸発、沈殿、乾燥、噴霧乾燥または凍結乾燥のうちの少なくとも１つを含む、項目２７に記載の方法。
（項目２９）
前記ポリペプチドが、真菌、酵母、細菌、昆虫、動物および植物発現系で産生される、項目１から１１、１５、１９、２５から２８のいずれか一項に記載の方法。
（項目３０）
前記宿主細胞が、細菌の、好ましくはＥｓｃｈｅｒｉｃｈｉａｃｏｌｉ株の、より好ましくはＫ１２株であるＥｓｃｈｅｒｉｃｈｉａｃｏｌｉ株の細胞であり、さらにより好ましくは前記ＥｓｃｈｅｒｉｃｈｉａｃｏｌｉＫ１２株が、ＥｓｃｈｅｒｉｃｈｉａｃｏｌｉＭＧ１６５５である、項目２９に記載の方法。
（項目３１）
前記宿主細胞が、酵母細胞である、項目２９に記載の方法。
（項目３２）
前記培養培地中のラクトース濃度が、５０～１５０ｇ／Ｌの範囲に及ぶ、項目１から１１、１５、１９、２５から３１のいずれか一項に記載の方法。
（項目３３）
３－フコシルラクトースの最終濃度が、７０ｇ／Ｌ～２００ｇ／Ｌの範囲に及ぶ、項目１から１１、１５、１９、２５から３２のいずれか一項に記載の方法。
（項目３４）
前記産生が、発酵の終わりに、１：５未満のラクトース濃度の３－フコシルラクトース濃度に対する比をもたらす、項目１から１１、１５、１９、２５から３３のいずれか一項に記載の方法。
（項目３５）
前記産生が、発酵の終わりに、８０％またはそれよりも高い３－フコシルラクトース純度をもたらす、項目１から１１、１５、１９、２５から３４のいずれか一項に記載の方法。
（項目３６）
α－１，３－フコシルラクトースの産生のための方法であって、
ａ）α－１，３－フコシルトランスフェラーゼ活性を有し、ラクトースをアクセプター基質として使用する能力を有するポリペプチドを提供するステップと、
ｂ）ステップａ）のα－１，３－フコシルトランスフェラーゼ活性を有する前記ポリペプチドを、ＧＤＰ－フコースをドナー基質として、およびラクトースをアクセプター基質として含む混合物と、前記ポリペプチドが、フコース残基の、前記ドナー基質から前記アクセプター基質への移行を触媒する条件下で接触させ、
それによって、α－１，３－フコシルラクトースを産生するステップであって、
ｃ）触媒が、発酵の終わりに、１：５未満のラクトース濃度の３－フコシルラクトース濃度に対する比をもたらす、ステップと、
ｄ）必要に応じて、前記α－１，３－フコシルラクトースを分離するステップと
を含む方法。
（項目３７）
α－１，３－フコシルラクトースの産生のための方法であって、
ａ）α－１，３－フコシルトランスフェラーゼ活性を有し、ラクトースをアクセプター基質として使用する能力を有するポリペプチドを提供するステップと、
ｂ）ステップａ）のα－１，３－フコシルトランスフェラーゼ活性を有する前記ポリペプチドを、ＧＤＰ－フコースをドナー基質として、およびラクトースをアクセプター基質として含む混合物と、前記ポリペプチドが、フコース残基の、前記ドナー基質から前記アクセプター基質への移行を触媒する条件下で接触させ、
それによって、α－１，３－フコシルラクトースを産生するステップであって、
ｃ）触媒が、発酵の終わりに、８０％またはそれよりも高い３－フコシルラクトース純度をもたらす、ステップと、
ｄ）必要に応じて、前記α－１，３－フコシルラクトースを分離するステップと
を含む方法。
（項目３８）
３－フコシルラクトースの産生のための方法であって、以下：
ｉ）培養培地に、開始リアクター体積につき少なくとも５０、より好ましくは少なくとも７５、より好ましくは少なくとも１００、より好ましくは少なくとも１２０、より好ましくは少なくとも１５０グラムのラクトースを含むラクトース供給を、好ましくは連続様式で、好ましくは前記培養培地の最終体積が、前記ラクトース供給の添加前の前記培養培地の体積の３倍以下、好ましくは２倍以下、より好ましくは２倍未満であるように、添加するステップ；
ｉｉ）ラクトース供給を、連続様式で前記培養培地に１日、２日、３日、４日、５日の期間にわたり供給溶液の手段によって添加するステップ；
ｉｉｉ）ラクトース供給を、連続様式で前記培養培地に１日、２日、３日、４日、５日の期間にわたり供給溶液の手段によって添加するステップであって、ラクトース供給溶液の濃度が、５０ｇ／Ｌ、好ましくは７５ｇ／Ｌ、より好ましくは１００ｇ／Ｌ、より好ましくは１２５ｇ／Ｌ、より好ましくは１５０ｇ／Ｌ、より好ましくは１７５ｇ／Ｌ、より好ましくは２００ｇ／Ｌ、より好ましくは２２５ｇ／Ｌ、より好ましくは２５０ｇ／Ｌ、より好ましくは２７５ｇ／Ｌ、より好ましくは３００ｇ／Ｌ、より好ましくは３２５ｇ／Ｌ、より好ましくは３５０ｇ／Ｌ、より好ましくは３７５ｇ／Ｌ、より好ましくは４００ｇ／Ｌ、より好ましくは４５０ｇ／Ｌ、より好ましくは５００ｇ／Ｌ、さらにより好ましくは５５０ｇ／Ｌ、最も好ましくは６００ｇ／Ｌであり、好ましくは前記溶液のｐＨが、３～７の間に設定され、好ましくは前記供給溶液の温度が、２０℃～８０℃の間で維持される、ステップ
のうちの少なくとも１つを含み、
前記方法が、前記培養培地の最終体積中の少なくとも５０ｇ／Ｌ、好ましくは少なくとも７５ｇ／Ｌ、より好ましくは少なくとも９０ｇ／Ｌ、より好ましくは少なくとも１００ｇ／Ｌ、より好ましくは少なくとも１２５ｇ／Ｌ、より好ましくは少なくとも１５０ｇ／Ｌ、より好ましくは少なくとも１７５ｇ／Ｌ、より好ましくは少なくとも２００ｇ／Ｌの３－フコシルラクトース濃度、および好ましくは培養液の最終体積中の１：５未満、より好ましくは１：１０、さらにより好ましくは１：２０、最も好ましくは１：４０のラクトース濃度の３ＦＬ濃度に対する比をもたらす、方法。
（項目３９）
３－フコシルラクトースの産生のための方法であって、以下：
ｉ）培養培地に、開始リアクター体積につき少なくとも５０、より好ましくは少なくとも７５、より好ましくは少なくとも１００、より好ましくは少なくとも１２０、より好ましくは少なくとも１５０グラムのラクトースを含むラクトース供給を、好ましくは連続様式で、好ましくは前記培養培地の最終体積が、前記ラクトース供給の添加前の前記培養培地の体積の３倍以下、好ましくは２倍以下、より好ましくは２倍未満であるように、添加するステップ；
ｉｉ）ラクトース供給を、連続様式で前記培養培地に１日、２日、３日、４日、５日の期間にわたり供給溶液の手段によって添加するステップ；
ｉｉｉ）ラクトース供給を、連続様式で前記培養培地に１日、２日、３日、４日、５日の期間にわたり供給溶液の手段によって添加するステップであって、ラクトース供給溶液の濃度が、５０ｇ／Ｌ、好ましくは７５ｇ／Ｌ、より好ましくは１００ｇ／Ｌ、より好ましくは１２５ｇ／Ｌ、より好ましくは１５０ｇ／Ｌ、より好ましくは１７５ｇ／Ｌ、より好ましくは２００ｇ／Ｌ、より好ましくは２２５ｇ／Ｌ、より好ましくは２５０ｇ／Ｌ、より好ましくは２７５ｇ／Ｌ、より好ましくは３００ｇ／Ｌ、より好ましくは３２５ｇ／Ｌ、より好ましくは３５０ｇ／Ｌ、より好ましくは３７５ｇ／Ｌ、より好ましくは４００ｇ／Ｌ、より好ましくは４５０ｇ／Ｌ、より好ましくは５００ｇ／Ｌ、さらにより好ましくは５５０ｇ／Ｌ、最も好ましくは６００ｇ／Ｌであり、好ましくは前記溶液のｐＨが、３～７の間に設定され、好ましくは前記供給溶液の温度が、２０℃～８０℃の間で維持される、ステップ
のうちの少なくとも１つを含み、
前記方法が、前記培養培地の最終体積中の少なくとも５０ｇ／Ｌ、好ましくは少なくとも７５ｇ／Ｌ、より好ましくは少なくとも９０ｇ／Ｌ、より好ましくは少なくとも１００ｇ／Ｌ、より好ましくは少なくとも１２５ｇ／Ｌ、より好ましくは少なくとも１５０ｇ／Ｌ、より好ましくは少なくとも１７５ｇ／Ｌ、より好ましくは少なくとも２００ｇ／Ｌの３－フコシルラクトース濃度、および好ましくは培養液の最終体積中の８０％またはそれよりも高い３ＦＬ純度をもたらす、方法。 Experiments were performed on mutant E. cerevisiae expressing the lactose-binding alpha-1,3-fucosyltransferase gene having SEQ ID NO:2 or SEQ ID NO:6. The E. coli strain is shown to produce a broth with higher 3-FL purity than similar strains containing SEQ ID NO: 18 in fed-batch fermentation on bioreactor scale.
In certain embodiments, for example, the following are provided:
(Item 1)
A method for producing α-1,3-fucosyllactose, comprising:
a) providing a polypeptide having α-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate, said polypeptide comprising:
i) Conserved GDP-fucose binding domain [Y/W/L/H/F/M]X[T/S/C][E/Q/D/A][K/R] (SEQ ID NO: 33) an amino acid sequence that encodes a
ii) the amino acid sequence encoding the conserved [K/D][L/K/M]XXX[F/Y] domain (SEQ ID NO: 34)
including
iii) In addition, if said domain of ii) equals DM[A/S]VSF (SEQ ID NO: 36), the conserved motif [N/H]XDPAXLD (SEQ ID NO: 35) is present in the N-terminal region. ,
wherein X may be any distinct amino acid,
the C-terminus of said polypeptide has less than or equal to 100 amino acids starting from the first amino acid of said GDP-fucose binding domain;
b) a mixture comprising said polypeptide having α-1,3-fucosyltransferase activity of step a), GDP-fucose as a donor substrate and lactose as an acceptor substrate; contacting under conditions that catalyze the transfer from the donor substrate to the acceptor substrate;
thereby producing α-1,3-fucosyllactose;
c) optionally separating the α-1,3-fucosyllactose;
method including.
(Item 2)
A method according to item 1, wherein said polypeptide is provided in a cell-free system.
(Item 3)
2. The method of item 1, wherein said polypeptide is produced by a cell containing a polynucleotide encoding said polypeptide.
(Item 4)
4. Method according to item 1 or 3, wherein said GDP-fucose and/or lactose is provided by said GDP-fucose and/or lactose-producing cells.
(Item 5)
i) providing a cell genetically modified for the production of α-1,3-fucosyllactose, said cell encoding at least one enzyme for α-1,3-fucosyllactose synthesis comprising one nucleic acid sequence,
said cell expressing said polypeptide having alpha-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate;
ii) culturing said cells in a medium under conditions permissive for the production of α-1,3-fucosyllactose;
iii) preferably separating said α-1,3-fucosyllactose from the culture;
5. The method of any one of items 1, 3 or 4, comprising:
(Item 6)
a) providing a host cell expressing said polypeptide having α-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate;
b) growing said host cell under suitable nutritional conditions permissive for the production of α-1,3-fucosyllactose and for the expression of said polypeptide having α-1,3-fucosyltransferase activity; When,
c) GDP-fucose and lactose simultaneously or simultaneously with step b) for the α-1,3-fucosyltransferase polypeptide to catalyze the transfer of a fucose residue from the donor substrate GDP-fucose to the acceptor substrate lactose subsequently providing, thereby producing α-1,3-fucosyllactose;
d) optionally separating said α-1,3-fucosyllactose from said host cell or its growth medium;
4. The method of item 3, comprising
(Item 7)
Item 5 or 6, wherein said host cell is transformed or transfected to express an exogenous polypeptide having α-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate. described method.
(Item 8)
Method according to any one of items 3 to 7, characterized in that said GDP-fucose and/or lactose are provided by enzymes co-expressed in said host cell or by metabolism of said host cell. .
(Item 9)
9. The method according to any one of items 1 to 8, further comprising purification of α-1,3-fucosyllactose.
(Item 10)
said polypeptide is
i) any one of SEQ ID NOS: 6, 2, 4, 8, 10, 12, 14, 16, 20, 22, 28, 30 or 32 of the attached Sequence Listing;
ii) an amino acid sequence having 87% or greater sequence identity with the full-length amino acid sequence of SEQ ID NO: 2, 20 or 22;
iii) an amino acid sequence having 80% or greater sequence identity with the full-length amino acid sequence of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 28, 30 or 32;
iv) a fragment of the amino acid sequence set forth in SEQ ID NO: 2, 20 or 22, comprising at least 45 contiguous amino acids thereof;
v) a fragment of the amino acid sequence set forth in any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 28, 30 or 32, comprising at least 10 contiguous amino acids thereof and lactose Fragments with bound alpha-1,3-fucosyltransferase activity
is selected from the group consisting of
A method according to any one of the preceding items, wherein optionally said polypeptide is further modified by N-terminal and/or C-terminal amino acid extensions.
(Item 11)
A method for the production of 3-fucosyllactose according to any one of the preceding items, comprising the steps of:
i) to the culture medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per starting reactor volume, preferably in a continuous manner; adding preferably such that the final volume of said culture medium is no more than 3 times, preferably no more than 2 times, more preferably less than 2 times the volume of said culture medium prior to addition of said lactose feed;
ii) adding a lactose feed in a continuous manner to said culture medium over a period of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feed solution;
iii) adding a lactose feed in a continuous manner to said culture medium over a period of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feed solution, wherein the concentration of said lactose feed solution is , 50 g/L, preferably 75 g/L, more preferably 100 g/L, more preferably 125 g/L, more preferably 150 g/L, more preferably 175 g/L, more preferably 200 g/L, more preferably 225 g /L, more preferably 250 g/L, more preferably 275 g/L, more preferably 300 g/L, more preferably 325 g/L, more preferably 350 g/L, more preferably 375 g/L, more preferably 400 g/L L, more preferably 450 g/L, more preferably 500 g/L, even more preferably 550 g/L, most preferably 600 g/L, preferably the pH of said solution is set between 3 and 7, Preferably the temperature of said feed solution is maintained between 20°C and 80°C
further comprising at least one of
iv) said method reduces the final volume of said culture medium to at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L; More preferably, the method results in a 3-fucosyllactose concentration of at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L.
(Item 12)
A host cell genetically modified for the production of α-1,3-fucosyllactose, comprising at least one nucleic acid sequence encoding an enzyme involved in α-1,3-fucosyllactose synthesis, α-1 ,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate, said polypeptide comprising:
i) Conserved GDP-fucose binding domain [Y/W/L/H/F/M]X[T/S/C][E/Q/D/A][K/R] (SEQ ID NO: 33) an amino acid sequence that encodes a
ii) the amino acid sequence encoding the conserved [K/D][L/K/M]XXX[F/Y] domain (SEQ ID NO: 34)
including
iii) In addition, if said domain of ii) equals DM[A/S]VSF (SEQ ID NO: 36), the conserved motif [N/H]XDPAXLD (SEQ ID NO: 35) is present in the N-terminal region. ,
wherein X may be any distinct amino acid,
A host cell, wherein the C-terminus of said polypeptide has less than or equal to 100 amino acids starting from the first amino acid of said GDP-fucose binding domain.
(Item 13)
i) a sequence comprising a polynucleotide encoding said polypeptide having lactose-binding alpha-1,3-fucosyltransferase activity, said sequence being foreign to said host cell and integrated into said host cell's genome; or ii) a vector comprising a polynucleotide encoding said polypeptide, said polynucleotide being operably linked to regulatory sequences recognized by a host cell transformed with said vector , item 12.
(Item 14)
said polypeptide is
i) any one of SEQ ID NOS: 6, 2, 4, 8, 10, 12, 14, 16, 20, 22, 28, 30 or 32 of the attached Sequence Listing;
ii) an amino acid sequence having 87% or greater sequence identity with the full-length amino acid sequence of SEQ ID NO: 2, 20 or 22;
iii) an amino acid sequence having 80% or greater sequence identity with the full-length amino acid sequence of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 28, 30 or 32;
iv) a fragment of the amino acid sequence set forth in SEQ ID NO: 2, 20 or 22, comprising at least 45 contiguous amino acids thereof;
v) a fragment of the amino acid sequence set forth in any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 28, 30 or 32, comprising at least 10 contiguous amino acids thereof and lactose Fragments with bound alpha-1,3-fucosyltransferase activity
comprising an amino acid sequence selected from the group consisting of
14. A cell according to items 12 or 13, wherein optionally said polypeptide is further modified by N-terminal and/or C-terminal amino acid extensions.
(Item 15)
Said cells are selected from the group consisting of microorganisms, plant or animal cells, preferably said microorganisms are bacteria, fungi or yeast, preferably said plants are rice, cotton, rapeseed, soybean, maize or corn plants. A method according to any one of items 3 to 11, or item 12, wherein preferably said animal is an insect, fish, bird or non-human mammal; preferably said cell is an Escherichia coli cell , 13 or 14.
(Item 16)
from item 12, characterized in that it is a cell of an Escherichia coli strain of bacteria, preferably of an Escherichia coli strain, more preferably strain K12, even more preferably characterized in that said Escherichia coli K12 strain is Escherichia coli MG1655 16. The host cell according to any one of 15.
(Item 17)
16. Host cell according to any one of items 12 to 15, characterized in that it is a yeast cell.
(Item 18)
18. The method of items 12 to 17, wherein said polynucleotide encoding said polypeptide having lactose-binding alpha-1,2-fucosyltransferase activity is adapted to the codon usage of each host cell. A host cell according to any one of paragraphs.
(Item 19)
A method for the production of α-1,3-fucosyllactose comprising:
a) providing a cell according to any one of items 12-18;
b) culturing said cells in a medium under conditions permissive for the production of α-1,3-fucosyltransferase;
c) preferably isolating said α-1,3-fucosyltransferase from the culture;
method including.
(Item 20)
Use of a host cell according to any one of items 12-18 for the production of α-1,3-fucosyllactose.
(Item 21)
Use of a polypeptide as described in the method of items 1 or 11 for the production of α-1,3-fucosyllactose.
(Item 22)
A microorganism heterologously expressing a lactose-binding alpha-1,3-fucosyltransferase polypeptide, said polypeptide comprising:
i) Conserved GDP-fucose binding domain [Y/W/L/H/F/M]X[T/S/C][E/Q/D/A][K/R] (SEQ ID NO: 33) an amino acid sequence that encodes a
ii) the amino acid sequence encoding the conserved [K/D][L/K/M]XXX[F/Y] domain (SEQ ID NO: 34)
including
iii) In addition, if said domain of ii) equals DM[A/S]VSF (SEQ ID NO: 36), the conserved motif [N/H]XDPAXLD (SEQ ID NO: 35) is present in the N-terminal region. ,
wherein X may be any distinct amino acid,
A microorganism, wherein the C-terminus of said polypeptide has less than or equal to 100 amino acids starting from the first amino acid of said GDP-fucose binding domain.
(Item 23)
said polypeptide is
i) any one of SEQ ID NOS: 6, 2, 4, 8, 10, 12, 14, 16, 20, 22, 28, 30 or 32 of the attached Sequence Listing;
ii) an amino acid sequence having 87% or greater sequence identity with the full-length amino acid sequence of SEQ ID NO: 2, 20 or 22;
iii) an amino acid sequence having 80% or greater sequence identity with the full-length amino acid sequence of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 28, 30 or 32;
iv) a fragment of the amino acid sequence set forth in SEQ ID NO: 2, 20 or 22, comprising at least 45 contiguous amino acids thereof;
v) a fragment of the amino acid sequence set forth in any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 28, 30 or 32, comprising at least 10 contiguous amino acids thereof and lactose Fragments with bound alpha-1,3-fucosyltransferase activity
comprising an amino acid sequence selected from the group consisting of
23. Microorganism according to item 22, wherein said polypeptide is optionally further modified by N-terminal and/or C-terminal amino acid extensions.
(Item 24)
Use of a microorganism according to items 22 or 23 for the production of alpha-1,3-fucosyllactose.
(Item 25)
20. The method of any one of items 1 to 11, 15 or 19, further comprising separating said alpha-1,3-fucosyllactose from said host cell or its growth medium.
(Item 26)
Said separation comprises the following steps: clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated carbon or carbon treatment, tangential flow high performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography. , hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography.
(Item 27)
27. The method of any one of items 1 to 11, 15, 19, 25 or 26, further comprising purification of alpha-1,3-fucosyllactose.
(Item 28)
Said purification of said alpha-1,3-fucosyllactose comprises the following steps: use of activated carbon or carbon, use of charcoal, nanofiltration, ultrafiltration or ion exchange, use of alcohol, use of hydroalcoholic mixtures, crystallization. , evaporation, precipitation, drying, spray-drying or freeze-drying.
(Item 29)
29. The method of any one of items 1-11, 15, 19, 25-28, wherein said polypeptide is produced in fungal, yeast, bacterial, insect, animal and plant expression systems.
(Item 30)
30, wherein said host cell is a bacterial cell, preferably of an Escherichia coli strain, more preferably of an Escherichia coli strain that is K12 strain, even more preferably that said Escherichia coli K12 strain is Escherichia coli MG1655 described method.
(Item 31)
30. The method of item 29, wherein said host cell is a yeast cell.
(Item 32)
32. The method of any one of items 1-11, 15, 19, 25-31, wherein the lactose concentration in said culture medium ranges from 50 to 150 g/L.
(Item 33)
33. The method of any one of items 1-11, 15, 19, 25-32, wherein the final concentration of 3-fucosyllactose ranges from 70 g/L to 200 g/L.
(Item 34)
34. A method according to any one of items 1 to 11, 15, 19, 25 to 33, wherein said production results in a ratio of lactose concentration to 3-fucosyllactose concentration of less than 1:5 at the end of fermentation.
(Item 35)
35. The method of any one of items 1-11, 15, 19, 25-34, wherein said production results in a 3-fucosyllactose purity of 80% or higher at the end of fermentation.
(Item 36)
A method for the production of α-1,3-fucosyllactose comprising:
a) providing a polypeptide having α-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate;
b) a mixture comprising said polypeptide having α-1,3-fucosyltransferase activity of step a), GDP-fucose as a donor substrate and lactose as an acceptor substrate; contacting under conditions that catalyze the transfer from the donor substrate to the acceptor substrate;
thereby producing α-1,3-fucosyllactose,
c) the catalyst provides a ratio of lactose concentration to 3-fucosyllactose concentration of less than 1:5 at the end of fermentation;
d) optionally separating the α-1,3-fucosyllactose;
method including.
(Item 37)
A method for the production of α-1,3-fucosyllactose comprising:
a) providing a polypeptide having α-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate;
b) a mixture comprising said polypeptide having α-1,3-fucosyltransferase activity of step a), GDP-fucose as a donor substrate and lactose as an acceptor substrate; contacting under conditions that catalyze the transfer from the donor substrate to the acceptor substrate;
thereby producing α-1,3-fucosyllactose,
c) the catalyst provides a 3-fucosyllactose purity of 80% or higher at the end of the fermentation;
d) optionally separating the α-1,3-fucosyllactose;
method including.
(Item 38)
A method for the production of 3-fucosyllactose, comprising:
i) to the culture medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per starting reactor volume, preferably in a continuous manner; adding preferably such that the final volume of said culture medium is no more than 3 times, preferably no more than 2 times, more preferably less than 2 times the volume of said culture medium prior to addition of said lactose feed;
ii) adding a lactose feed in a continuous manner to said culture medium over a period of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feed solution;
iii) adding a lactose feed in a continuous manner to said culture medium over a period of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feed solution, wherein the concentration of the lactose feed solution is 50 g /L, preferably 75 g/L, more preferably 100 g/L, more preferably 125 g/L, more preferably 150 g/L, more preferably 175 g/L, more preferably 200 g/L, more preferably 225 g/L , more preferably 250 g/L, more preferably 275 g/L, more preferably 300 g/L, more preferably 325 g/L, more preferably 350 g/L, more preferably 375 g/L, more preferably 400 g/L, More preferably 450 g/L, more preferably 500 g/L, even more preferably 550 g/L, most preferably 600 g/L, preferably the pH of said solution is set between 3 and 7, preferably wherein the temperature of said feed solution is maintained between 20°C and 80°C
including at least one of
Said method comprises at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 125 g/L in the final volume of said culture medium. is a 3-fucosyllactose concentration of at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L, and preferably less than 1:5, more preferably 1:10 in the final volume of the culture medium, Even more preferably, the method results in a ratio of lactose concentration to 3FL concentration of 1:20, most preferably 1:40.
(Item 39)
A method for the production of 3-fucosyllactose, comprising:
i) to the culture medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per starting reactor volume, preferably in a continuous manner; adding preferably such that the final volume of said culture medium is no more than 3 times, preferably no more than 2 times, more preferably less than 2 times the volume of said culture medium prior to addition of said lactose feed;
ii) adding a lactose feed in a continuous manner to said culture medium over a period of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feed solution;
iii) adding a lactose feed in a continuous manner to said culture medium over a period of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feed solution, wherein the concentration of the lactose feed solution is 50 g /L, preferably 75 g/L, more preferably 100 g/L, more preferably 125 g/L, more preferably 150 g/L, more preferably 175 g/L, more preferably 200 g/L, more preferably 225 g/L , more preferably 250 g/L, more preferably 275 g/L, more preferably 300 g/L, more preferably 325 g/L, more preferably 350 g/L, more preferably 375 g/L, more preferably 400 g/L, More preferably 450 g/L, more preferably 500 g/L, even more preferably 550 g/L, most preferably 600 g/L, preferably the pH of said solution is set between 3 and 7, preferably wherein the temperature of said feed solution is maintained between 20°C and 80°C
including at least one of
Said method comprises at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L, more preferably at least 125 g/L in the final volume of said culture medium. provides a 3-fucosyllactose concentration of at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L, and preferably a 3FL purity of 80% or greater in the final volume of the culture, Method.

Claims

A method for producing α-1,3-fucosyllactose, comprising:
a) a polypeptide having α-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate , produced in a cell-free system or by a cell containing a polynucleotide encoding said polypeptide wherein the polypeptide is
i) Conserved GDP-fucose binding domain [Y/W/L/H/F/M]X[T/S/C][E/Q/D/A][K/R] (SEQ ID NO: 33) an amino acid sequence that encodes a
ii) comprising an amino acid sequence encoding a conserved [K/D][L/K/M]XXX[F/Y] domain (SEQ ID NO: 34);
iii) In addition, if said domain of ii) equals DM[A/S]VSF (SEQ ID NO: 36), the conserved motif [N/H]XDPAXLD (SEQ ID NO: 35) is present in the N-terminal region. ,
wherein X may be any distinct amino acid,
the C-terminus of said polypeptide has less than or equal to 100 amino acids starting from the first amino acid of said GDP-fucose binding domain ;
said polypeptide is
i) any one of SEQ ID NOS: 6, 2, 4, 8, 10, 12, 14, 16, 20, 22, 28, 30 or 32 of the attached Sequence Listing;
ii) an amino acid sequence having 87% or greater sequence identity with the full-length amino acid sequence of SEQ ID NO: 2, 20 or 22;
iii) an amino acid sequence having 80% or greater sequence identity with the full-length amino acid sequence of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 28, 30 or 32;
iv) a fragment of the amino acid sequence set forth in SEQ ID NO: 2, 20 or 22, comprising at least 45 contiguous amino acids thereof;
v) a fragment of the amino acid sequence set forth in any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 28, 30 or 32, comprising at least 10 contiguous amino acids thereof and lactose Fragments with bound alpha-1,3-fucosyltransferase activity
is selected from the group consisting of
optionally, said polypeptide is further modified by N-terminal and/or C-terminal amino acid extensions ;
b) a mixture comprising said polypeptide having α-1,3-fucosyltransferase activity of step a), GDP-fucose as a donor substrate and lactose as an acceptor substrate; contacting under conditions that catalyze the transfer from the donor substrate to the acceptor substrate;
thereby producing α-1,3-fucosyllactose;
c) optionally separating the α-1,3-fucosyllactose ;
including
d) optionally further comprising purification of α-1,3-fucosyllactose,
Preferably, said GDP-fucose and/or lactose is provided by said GDP-fucose and/or lactose-producing cells .

i) providing a cell genetically modified for the production of α-1,3-fucosyllactose, said cell encoding at least one enzyme for α-1,3-fucosyllactose synthesis comprising one nucleic acid sequence,
said cell expressing said polypeptide having alpha-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate;
ii) culturing said cells in a medium under conditions permissive for the production of α-1,3-fucosyllactose;
iii) preferably separating said α-1,3-fucosyllactose from the culture ,
iv) The method according to claim 1 , further comprising purification of α-1,3-fucosyllactose, if desired .

a) providing a host cell expressing said polypeptide having α-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate;
b) growing said host cell under suitable nutritional conditions permissive for the production of α-1,3-fucosyllactose and for the expression of said polypeptide having α-1,3-fucosyltransferase activity; When,
c) GDP-fucose and lactose simultaneously or simultaneously with step b) for the α-1,3-fucosyltransferase polypeptide to catalyze the transfer of a fucose residue from the donor substrate GDP-fucose to the acceptor substrate lactose subsequently providing, thereby producing α-1,3-fucosyllactose;
d) optionally separating said α-1,3-fucosyllactose from said host cell or its growth medium ;
e) The method of claim 1 , further comprising purification of α-1,3-fucosyllactose, if desired .

Claim 2 or 3 , wherein said host cell is transformed or transfected to express an exogenous polypeptide having alpha-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate. The method described in .

5. The method according to any one of claims 1 to 4 , characterized in that said GDP-fucose and/or lactose are provided by enzymes co-expressed in said host cell or by metabolism of said host cell. Method.

A method for the production of α- 1,3-fucosyllactose according to any one of the preceding claims, comprising the steps of:
i) to the culture medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per starting reactor volume, preferably in a continuous manner; adding preferably such that the final volume of said culture medium is no more than 3 times, preferably no more than 2 times, more preferably less than 2 times the volume of said culture medium prior to addition of said lactose feed;
ii) adding a lactose feed in a continuous manner to said culture medium over a period of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feed solution;
iii) adding a lactose feed in a continuous manner to said culture medium over a period of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feed solution, wherein the concentration of said lactose feed solution is , 50 g/L, preferably 75 g/L, more preferably 100 g/L, more preferably 125 g/L, more preferably 150 g/L, more preferably 175 g/L, more preferably 200 g/L, more preferably 225 g /L, more preferably 250 g/L, more preferably 275 g/L, more preferably 300 g/L, more preferably 325 g/L, more preferably 350 g/L, more preferably 375 g/L, more preferably 400 g/L L, more preferably 450 g/L, more preferably 500 g/L, even more preferably 550 g/L, most preferably 600 g/L, preferably the pH of said solution is set between 3 and 7, preferably further comprising at least one of the steps wherein the temperature of said feed solution is maintained between 20°C and 80°C,
iv) said method reduces the final volume of said culture medium to at least 50 g/L, preferably at least 75 g/L, more preferably at least 90 g/L, more preferably at least 100 g/L, more preferably at least 125 g/L; More preferably, the method results in an α- 1,3-fucosyllactose concentration of at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L.

A host cell genetically modified for the production of α-1,3-fucosyllactose, comprising at least one nucleic acid sequence encoding an enzyme involved in α-1,3-fucosyllactose synthesis, α-1 ,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate, said polypeptide comprising:
i) Conserved GDP-fucose binding domain [Y/W/L/H/F/M]X[T/S/C][E/Q/D/A][K/R] (SEQ ID NO: 33) an amino acid sequence that encodes a
ii) comprising an amino acid sequence encoding a conserved [K/D][L/K/M]XXX[F/Y] domain (SEQ ID NO: 34);
iii) In addition, if said domain of ii) equals DM[A/S]VSF (SEQ ID NO: 36), the conserved motif [N/H]XDPAXLD (SEQ ID NO: 35) is present in the N-terminal region. ,
wherein X may be any distinct amino acid,
the C-terminus of said polypeptide has less than or equal to 100 amino acids starting from the first amino acid of said GDP-fucose binding domain ;
said polypeptide is
i) any one of SEQ ID NOS: 6, 2, 4, 8, 10, 12, 14, 16, 20, 22, 28, 30 or 32 of the attached Sequence Listing;
ii) an amino acid sequence having 87% or greater sequence identity with the full-length amino acid sequence of SEQ ID NO: 2, 20 or 22;
iii) an amino acid sequence having 80% or greater sequence identity with the full-length amino acid sequence of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 28, 30 or 32;
iv) a fragment of the amino acid sequence set forth in SEQ ID NO: 2, 20 or 22, comprising at least 45 contiguous amino acids thereof;
v) a fragment of the amino acid sequence set forth in any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 28, 30 or 32, comprising at least 10 contiguous amino acids thereof and lactose Fragments with bound alpha-1,3-fucosyltransferase activity
comprising an amino acid sequence selected from the group consisting of
Optionally, a host cell wherein said polypeptide is further modified by N-terminal and/or C-terminal amino acid extensions .

i) a sequence comprising a polynucleotide encoding said polypeptide having lactose-binding alpha-1,3-fucosyltransferase activity, said sequence being foreign to said host cell and integrated into said host cell's genome; or ii) a vector comprising a polynucleotide encoding said polypeptide, said polynucleotide being operably linked to regulatory sequences recognized by a host cell transformed with said vector 8. The cell of claim 7 .

Said cells are selected from the group consisting of microorganisms, plant or animal cells, preferably said microorganisms are bacteria, fungi or yeast, preferably said plants are rice, cotton, rapeseed, soybean, maize or corn plants. preferably said animal is an insect, fish, bird or non-human mammal; preferably said cell is an Escherichia coli cell , more preferably a cell of an Escherichia coli strain which is the K12 strain, and further 9. A host cell according to any one of claims 7 or 8 , wherein more preferably said Escherichia coli K12 strain is Escherichia coli MG1655; preferably said cell is a yeast cell.

A method for the production of α-1,3-fucosyllactose comprising:
a) providing a cell according to any one of claims 7 to 9 ;
b) culturing said cells in a medium under conditions permissive for the production of α-1,3-fucosyltransferase;
c) preferably isolating said α-1,3-fucosyltransferase from the culture.

Use of the host cell according to any one of claims 7-9 for the production of α-1,3- fucosyllactose .

Use of a polypeptide as described in the method of claims 1 or 6 for the production of α-1,3-fucosyllactose.

A microorganism heterologously expressing a lactose-binding alpha-1,3-fucosyltransferase polypeptide, said polypeptide comprising:
i) Conserved GDP-fucose binding domain [Y/W/L/H/F/M]X[T/S/C][E/Q/D/A][K/R] (SEQ ID NO: 33) an amino acid sequence that encodes a
ii) comprising an amino acid sequence encoding a conserved [K/D][L/K/M]XXX[F/Y] domain (SEQ ID NO: 34);
iii) In addition, if said domain of ii) equals DM[A/S]VSF (SEQ ID NO: 36), the conserved motif [N/H]XDPAXLD (SEQ ID NO: 35) is present in the N-terminal region. ,
wherein X may be any distinct amino acid,
the C-terminus of said polypeptide has less than or equal to 100 amino acids starting from the first amino acid of said GDP-fucose binding domain ;
Preferably, said polypeptide is
i) any one of SEQ ID NOS: 6, 2, 4, 8, 10, 12, 14, 16, 20, 22, 28, 30 or 32 of the attached Sequence Listing;
ii) an amino acid sequence having 87% or greater sequence identity with the full-length amino acid sequence of SEQ ID NO: 2, 20 or 22;
iii) an amino acid sequence having 80% or greater sequence identity with the full-length amino acid sequence of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 28, 30 or 32;
iv) a fragment of the amino acid sequence set forth in SEQ ID NO: 2, 20 or 22, comprising at least 45 contiguous amino acids thereof;
v) a fragment of the amino acid sequence set forth in any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 28, 30 or 32, comprising at least 10 contiguous amino acids thereof and lactose Fragments with bound alpha-1,3-fucosyltransferase activity
comprising an amino acid sequence selected from the group consisting of
A microorganism , wherein said polypeptide is optionally further modified by an N-terminal and/or C-terminal amino acid extension .

Use of the microorganism according to claim 13 for the production of alpha-1,3-fucosyllactose.

further comprising the step of separating said alpha-1,3-fucosyllactose from said host cell or its growth medium , preferably said separation comprising the steps of: clarification, ultrafiltration, nanofiltration, reverse osmosis, at least of microfiltration, activated carbon or carbon treatment, tangential flow high performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography 11. The method of any one of claims 1-6 or 10 , comprising one .

Further comprising purification of alpha-1,3-fucosyllactose, preferably said purification of said alpha-1,3-fucosyllactose comprises the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, 16. The method of claim 1 to 6 , 10 or 15 comprising at least one of extrafiltration or ion exchange, use of alcohol, use of hydroalcoholic mixtures, crystallization, evaporation, precipitation, drying, spray drying or freeze drying . A method according to any one of paragraphs.

Said polypeptide is produced in fungal, yeast, bacterial, insect, animal and plant expression systems, preferably said host cell is bacterial, preferably Escherichia coli strain, more preferably K12 strain. strain, even more preferably said Escherichia coli K12 strain is Escherichia coli MG1655 , preferably said host cell is a yeast cell . The method according to item 1.

1. The lactose concentration in said culture medium ranges from 50 to 150 g/L and/or the final concentration of α-1,3-fucosyllactose ranges from 70 g/L to 200 g/L. 18. The method of any one of 6 , 10, 15-17 .

said production results in a ratio of lactose concentration to α- 1,3-fucosyllactose concentration of less than 1:5 at the end of fermentation and/or said production results in 80% or more at the end of fermentation 19. The method of any one of claims 1 to 6 , 10, 15 to 18 , resulting in α-1,3-fucosyllactose purity .

A method for the production of α-1,3-fucosyllactose comprising:
a) providing a polypeptide having α-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate , said polypeptide comprising:
i) Conserved GDP-fucose binding domain [Y/W/L/H/F/M]X[T/S/C][E/Q/D/A][K/R] (SEQ ID NO: 33) an amino acid sequence that encodes a
ii) the amino acid sequence encoding the conserved [K/D][L/K/M]XXX[F/Y] domain (SEQ ID NO: 34)
including
iii) In addition, if said domain of ii) equals DM[A/S]VSF (SEQ ID NO: 36), the conserved motif [N/H]XDPAXLD (SEQ ID NO: 35) is present in the N-terminal region. ,
wherein X may be any distinct amino acid,
the C-terminus of said polypeptide has less than or equal to 100 amino acids starting from the first amino acid of said GDP-fucose binding domain;
said polypeptide is
i) any one of SEQ ID NOS: 6, 2, 4, 8, 10, 12, 14, 16, 20, 22, 28, 30 or 32 of the attached Sequence Listing;
ii) an amino acid sequence having 87% or greater sequence identity with the full-length amino acid sequence of SEQ ID NO: 2, 20 or 22;
iii) an amino acid sequence having 80% or greater sequence identity with the full-length amino acid sequence of any one of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 28, 30 or 32;
iv) a fragment of the amino acid sequence set forth in SEQ ID NO: 2, 20 or 22, comprising at least 45 contiguous amino acids thereof;
v) a fragment of the amino acid sequence set forth in any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 28, 30 or 32, comprising at least 10 contiguous amino acids thereof and lactose Fragments with bound alpha-1,3-fucosyltransferase activity
is selected from the group consisting of
optionally, said polypeptide is further modified by N-terminal and/or C-terminal amino acid extensions ;
b) a mixture comprising said polypeptide having α-1,3-fucosyltransferase activity of step a), GDP-fucose as a donor substrate and lactose as an acceptor substrate; contacting under conditions that catalyze the transfer from the donor substrate to the acceptor substrate;
thereby producing α-1,3-fucosyllactose,
c) the catalyst has a ratio of lactose concentration to α -1,3-fucosyllactose concentration of less than 1:5 at the end of fermentation and/or an α-1,3 - a step that results in fucosyllactose purity ;
d) optionally separating said α-1,3-fucosyllactose.