JP2017502682A - 二酸化炭素を使用するように改変された酵母 - Google Patents
二酸化炭素を使用するように改変された酵母 Download PDFInfo
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Abstract
Description
a)適切なプロモータの転写制御下において、バクテリアのフォームIのRuBisCO酵素のRbcLサブユニットをコードする配列を包含する発現カセットと、
b)適切なプロモータの転写制御下において、前記RuBisCO酵素のRbcSサブユニットをコードする配列を包含する発現カセットと、
c)適切なプロモータの転写制御下において、前記RuBisCO酵素の特異的シャペロンRbcXをコードする配列を包含する発現カセットと、
d)適切なプロモータの転写制御下において、バクテリアシャペロンGroESをコードする配列を包含する発現カセットと、
e)適切なプロモータの転写制御下において、バクテリアシャペロンGroELをコードする配列を包含する発現カセットと、を含むことを特徴とする。
[実施例1:サッカロマイセス・セレビシエ(SACCHAROMYCES CEREVISIAE)酵母におけるシネココッカス・エロンガタス(SYNECHOCOCCUS ELONGATUS)RuBisCO複合体の発現と会合]
シネココッカス・エロンガタスpCC6301からのRuBisCOのRbcS及びRbcLサブユニットと特定のシャペロンRbcXとをコードし、酵母における発現に最適化された合成遺伝子を調製し、プラスミドpBSII(Genecust社)にクローニングした。また、コード配列の3′末端にHAタグを追加した変異体も構築した。
シネココッカス・エロンガタス(Syn)、ロドバクター・スフェロイデス(Rhodobacter sphaeroides、Rsph)、ロドシュードモナス・パルストリス(Rhodopseudomonas palustris、Rpal)、スピナシア・オレラセア(Spinacia oleracea、Sole)、ユーグレナ・グラシリス(Euglena gracilis、Egra)である5つの異なる由来のPRKをコードし、酵母における発現に最適化され、そしてC末端HAタグを有するか又は有しない、合成遺伝子を調製した。これら合成遺伝子の配列(HAタグ無し)は、添付の配列表において、それぞれ配列番号4〜8の番号のもとに示される。
・SynPRKでは抑制条件においてすら非常に高く(50%の誘導レベル)、この活性は細胞内ATPのレベルの重大な降下が付随する毒性を伴い、
・最小培地におけるEgraとSoleとのPRKについては検出可能であるがより弱く、
・RspHとRpaiとのPRKに用いられた条件下では検出不可能であり、
・SynPRKについては、培地次第で、リブロース−1,5−2リン酸蓄積レベルが富栄養培地より栄養の乏しい培地においてはるかに高い、
ということが見受けられる。
図4に記載されるように、人工的RuBisCO複合体の機能性は、完全又は部分的エンジニアリングを含む酵母抽出物からの合成基質(リブロース2リン酸)におけるRuBisCO活性をテストすることと、それが触媒する反応産生物、つまり3−グリセロリン酸の出現を評価することによって、インビトロにおいて示された。
本実施例は、以下の表7〜表9に記載の構成と形質転換株とを用いておこなわれた。
備考
1.pCM185は市販のプラスミド(ATCC87659)である。
CEN−PK3とCEN−PK4株の可溶性タンパク質の抽出のため、用いられるプラスミドの選択マーカに適した(ロイシン、ウラシル、及びトリプトファン非含有培地)市販のCSM培地(MP Biomedicals社)を含んで6.7g/l硫酸アンモニウム、20g/lグルコース、寒天用20g/l寒天で補完された、YNB(窒素源非含有酵母)培地において、細胞を、振動させながら周囲空気下30℃で成長させる。対数期終了の一世代前に4℃で冷却することで、培養を停止する。培養物を遠心分離し、スフェロプラストを、高張性ソルビトール培地(1.2Mソルビトール)においてザイモリエース・サイトへリカーゼ混合物で細胞壁の酵素消化をすることで調製する。スフェロプラストを、1mMのPMSF及びEDTA(プロテアーゼ阻害剤)の存在下で高張性ソルビトール培地において洗浄し、等張性培地(0.6Mソルビトール)において反復ピペッティングと弱い音波処理とで破砕する。大きな破片を除くために低速(200gで5分間)で、そして中程度のサイズの破片とミトコンドリアとを収集するために中速(1500gで10分間)で遠心分離した後、上清を収集する。
上述(図4)された同位体取込み実験は、標識3−グリセロリン酸の定量化によって表され、これは、13C標識バイカーボネート分子から炭素を固定することによりリブロース二リン酸から標識3−グリセロリン酸を産生するRuBisCO複合体の能力を示す。
炭酸脱水酵素は、溶媒和二酸化炭素へのバイカーボネートの相互変換を触媒することによって既知の反応補助因子である。この実施例では、そうした反応の予期される挙動を確認する。興味深いことに、インビトロにおける活性テストは、上述されたRuBisCO活性テストの反応量に10μg/mlの最終濃度のウシ炭酸脱水酵素を添加することで、RuBisCO複合体の能力を3〜4倍増加させることを示す(図10)。複合体周囲のCO2濃度の最適化により、再構成された活性の最小値を示す前述のテストで観察された活性が顕著に増加されることが提示される。その他の因子もインビボにおいて寄与し得り、故に測定値は単なる最小値である。
<4.1.嫌気培養はエタノール産生の増加を示す>
前培養を化学的合成培地において調製した。1mlのストック管(−80℃)を解凍後、これを用いて、(20g/lグルコースで補完された0.1g/lギ酸を含有する)10mlの培地を含むペニシリンボトル(100ml)に植え付けをおこない、18時間30℃で、120rpmにおいてインキュベートした。PRK遺伝子の存在下で見受けられる毒性の問題を避けるため、前培養物を、嫌気状態において(事前に窒素を流したボトル)、ドキシサイクリン(2μ/ml)の存在下で調製した。
実験プロトコル
前培養を化学的合成培地において調製した。1mlのストック管(−80℃)を解凍後、これを用いて、(20g/lグルコースで補完された0.1g/lギ酸を含有する)10mlの培地を含むペニシリンボトル(100ml)に植え付けをおこない、30℃で18時間、120rpmにおいてインキュベートした。PRK遺伝子の存在下で見受けられる毒性の問題を避けるため、前培養物を、嫌気状態において(事前に窒素を流したボトル)、ドキシサイクリン(2μ/ml)の存在下で調製した。
酵母において細胞の外部から内部へのCO2輸送は自然なプロセスではなく、S.エロンガタスにおける特殊化されたアクアポリンなどのトランスポータの同時発現によりこれをおこなうことが可能な補足的エンジニアリングが待たれることから、酵母デヒドロゲナーゼによって二酸化炭素へと酸化可能であるギ酸が、細胞内二酸化炭素源として用いられた。この二酸化炭素は、RuBisCO複合体を介して有機材料に再取込みされる可能性がある。つまり、13C標識ホルマートの存在下において、バイオマスに同位体を取込むことが期待される。しかし、酵母に他の自然なアナプレロティック反応(CO2を固定可能)があることから、これらの条件下において、RuBisCO複合体の非存在下においてすら13C取込みから顕著なバックグラウンドノイズ(標識の3〜4%)が観察され、観察される同位体取込みにおけるRuBisCOの寄与がはっきりと分からない理由が説明される。代謝経路の解析は、この第1の実験に使用された条件がインビボにおけるRuBisCO活性の同位体測定に実際には適さないことを示す。しかしながら、炭素源としてホルマートではなくグルコースを用いて培地に添加されるときに標識バイカーボネートのインビボにおける取込みがなかったことは、実際にはCO2(又はバイカーボネート/カーボネート)輸送の問題を理由とするものであり、代謝の問題のためではないということが、この実験により確認できたということが言及される必要がある。
ギ酸における成長を評価するため、ギ酸(0.45g/l)とグルコース(0.55g/l)とにおける好気培養を用いて、完全カーボイーストエンジニアリング又は単離要素を含む株を表現型的に特性化した。ギ酸は酵母においてCO2に代謝されることができ、ホルマートデヒドロゲナーゼによってパワーが減少(H2)するが、酵母は唯一の炭素源としてのギ酸において成長することができない。
この研究の課題は、RuBisCO(及びシャペロン)とホスホリブロキナーゼとの機能的同時発現による、酵母におけるカルビン回路の導入が、インビボにおけるエンジニアリングの機能性と合致する方向に内部代謝プロファイルを大きく改変するということを示すことである。この代謝プロファイルは、完全又は部分のみのエンジニアリングを保持する株の培養と、HPLC(逆相イオン対クロマトグラフィ)に連結された質量分析によるリン酸メタボロームの比較分析との後に評価された。
Claims (13)
- a)適切なプロモータの転写制御下において、バクテリアのフォームIRuBisCO酵素のRbcLサブユニットをコードする配列を包含する発現カセットと、
b)適切なプロモータの転写制御下において、前記RuBisCO酵素のRbcSサブユニットをコードする配列を包含する発現カセットと、
c)適切なプロモータの転写制御下において、前記RuBisCO酵素の特異的シャペロンRbcXをコードする配列を包含する発現カセットと、
d)適切なプロモータの転写制御下において、一般的なバクテリアシャペロンGroESをコードする配列を包含する発現カセットと、
e)適切なプロモータの転写制御下において、一般的なバクテリアシャペロンGroELをコードする配列を包含する発現カセットと、を含む、形質転換酵母細胞。 - 前記シャペロンRbcX、前記GroES、及び前記GroELは、少なくとも2つの異なる生物種に由来する、請求項1に記載の酵母細胞。
- 前記シャペロンRbcXはシアノバクテリアシャペロンである、請求項1又は請求項2に記載の酵母細胞。
- 前記一般的なシャペロンGroESとGroELとのうちの少なくとも1つは、シアノバクテリアにもRuBisCO複合体を発現する他のバクテリアにも由来しない、請求項1〜3のいずれか1項に記載の酵母細胞。
- 請求項1のc)、d)、及びe)に記載の3つの前記発現カセットは、遺伝子情報の連続的ブロックを形成する、請求項1〜4のいずれか1項に記載の酵母細胞。
- 請求項1のc)、d)、及びe)に記載の前記発現カセットは、単一のエピソーム遺伝要素によって保持される、請求項1〜5のいずれか1項に記載の酵母細胞。
- 前記酵母はサッカロマイセス・セレビシエ(Saccharomyces cerevisiae)種に属する、請求項1〜6のいずれか1項に記載の酵母細胞。
- 前記バクテリアのフォームIRuBisCO酵素はシアノバクテリアのRuBisCO酵素である、請求項1〜7のいずれか1項に記載の酵母細胞。
- 前記シアノバクテリアはシネココッカス(Synechococcus)属に属する、請求項1〜8のいずれか1項に記載の酵母細胞。
- 適切なプロモータの転写制御下において、ホスホリブロキナーゼ(PRK)をコードする配列を包含する発現カセットをさらに含む、請求項1〜9のいずれか1項に記載の酵母細胞。
- 前記PRKはクラスIIPRKである、請求項10に記載の酵母細胞。
- 前記クラスIIPRKは、スピナシア・オレラセア(Spinacia oleracea)、ユーグレナ・グラシリス(Euglena gracilis)、又はシネココッカス・エロンガタス(Synechococcus elongatus)のPRKから選択される、請求項11に記載の酵母細胞。
- 前記PRKをコードする配列の転写を制御する前記プロモータは誘導型プロモータである、請求項10〜12のいずれか1項に記載の酵母細胞。
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