JP5836271B2 - グリセロールを含まない発酵によるエタノール生成 - Google Patents
グリセロールを含まない発酵によるエタノール生成 Download PDFInfo
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- JP5836271B2 JP5836271B2 JP2012521593A JP2012521593A JP5836271B2 JP 5836271 B2 JP5836271 B2 JP 5836271B2 JP 2012521593 A JP2012521593 A JP 2012521593A JP 2012521593 A JP2012521593 A JP 2012521593A JP 5836271 B2 JP5836271 B2 JP 5836271B2
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Description
アセチルコエンザイムA+NADH+H+<−>アセトアルデヒド+NAD++コエンザイムA。
菌株の構築および維持
使用したサッカロマイセス・セレビシエ株(表1)は、CEN.PKファミリー由来であり、遺伝学および生理学を組み合わせた研究に好適なバックグランドとして以前確認された(ファン・ダイケン(van Dijken)ら(2000年)エンザイム・アンド・マイクロバイアル・テクノロジー第26巻:p.706−714)。
attBl配列およびattB2配列をそれぞれ含むプライマー対mhpF−FW(5’−GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAGTAAGCGTAAAGTCGCCATTATCGG−3’[配列番号3])およびmhpF−RV(5’−GGGGACCACTTTGTACAAGAAAGCTGGGTGTTCATGCCGCTTCTCCTGCCTTGC−3’、[配列番号4])を用いて、E.コリmhpF遺伝子(EMBL受託番号Y09555.7)を、E.コリK12菌株JM109ゲノムDNAからPCR増幅した。製造者の仕様に従いPhusion(登録商標)ホット・スタート・ハイフィデリティーDNAポリメラーゼ(Hot Start High−Fidelity DNA Polymerase)(フィンザイム・オイ(Finnzymes Oy)、エスポー(Espoo)、フィンランド)を用いて、以下の条件のBiometra TGradientサーモサイクラー(バイオメトラ(Biometra)、ゲッティンゲン(Goettingen)、ドイツ)でポリメラーゼ連鎖反応(PCR)を行った:変性98℃で10秒、アニーリング30秒、伸長72℃で25サイクル。1011bpのPCR産物を、Gateway(登録商標)クローニングテクノロジー(cloning technology)(インビトロジェン、カールズバッド、カリフォルニア、米国)を用いてクローニングした。BP反応によりプラスミドpDONR221を用いてエントリークローンを作製し、プラスミドpUD64と命名した。このエントリークローン、および多コピープラスミドpAG426GPD−ccdB(アドジーン(Addgene)、ケンブリッジ、マサチューセッツ州、米国)から、LR反応を利用して酵母発現プラスミドpUDE43を構築した。Z−Competent(商標)E.トランスフォーメーションキット(Transformation Kit)(ザイモリサーチ・コーポレーション(Zymoresearch Corporation)、オレンジ、米国)に従い、コンピテントなE.コリK12菌株JM109への組換え反応産物の形質転換を行い、アンピシリン(100mg l−1)あるいはカナマイシン(50mg.l−1)のどちらかを含むLB培地に蒔いた。酵母形質転換は、バークら(メソッズ・イン・イースト・ジェネティックス(2000年)、コールド・スプリング・ハーバー・ラボラトリー・プレス、プレーンビュー、ニューヨーク)に従い実施した。酵母発現プラスミドによる形質転換後、細胞を合成培地に蒔いた。多コピープラスミドpUDE43の挿入が成功したかを、クローニング用のプライマー対を用いてPCR診断により確認した。
合成培地(46)を用いて30℃で振盪フラスコ培養を行った。培地のpHは、滅菌の前に2MのKOHで6.0に調整した。500mL振盪フラスコ中に20g l−1のグルコースを含む100mlの培地に、(1mlの)凍結した保存培養物を接種して、前培養液を調製した。Innova(登録商標)インキュベーターシェーカー(200rpm、ニューブランズウィックサイエンティフィック(New Brunswick Scientific)、ニュージャージー州、米国)を用いた30℃で24時間のインキュベーション後、培養物をバイオリアクターに移した。
予め秤量したニトロセルロースフィルター(細孔サイズ0.45μm;ゲルマンラボラトリー(Gelman Laboratory)、アナーバー(Ann Arbor)、米国)により所定の時間間隔で培養サンプル(10ml)を濾過した。培地の除去後、フィルターを脱イオン水で洗浄し、電子レンジ(ボッシュ(Bosch)、シュトゥットガルト(Stuttgart)、ドイツ)で350W、20min乾燥させ、秤量した。2反復測定のばらつきは1%未満であった。さらに、Novaspec(登録商標)II分光光度計を用いて波長660nmの光学密度の測定値により培養増殖もモニターした。
排気ガスを冷却器(condensor)(2℃)で冷却し、パーマピュアドライヤー(Permapure dryer)タイプMD−110−48P−4(パーマピュア、トムズリバー(Toms River)、米国)で乾燥させた。酸素および二酸化炭素の濃度をNGA 2000アナライザー(ローズマウント・アナリティカル(Rosemount Analytical)、オービル(Orrville)、米国)で判定した。排気ガス流速および二酸化炭素生成速度を、以前記載されたように判定した(3)。これらのバイオマス特有の速度を算出する際、培養サンプルの除去により起こる体積変化の補正を行った。
バイオラッド(Biorad)HPX 87Hカラム(バイオラッド、ハーキュリーズ(Hercules)、米国)を含むウォーターズアライアンス(Waters Alliance)2690HPLC(ウォーターズ、ミルフォード(Milford)、米国)を用いたHPLC解析で、培養サンプルの遠心分離により得られた上清を、グルコース、酢酸、コハク酸、乳酸、グリセロールおよびエタノールについて解析した。カラムは、0.5g l−1のH2SO4を用いて60℃、0.6ml min−1の流速で溶出した。検出は、ウォーターズ2410屈折率検出器、およびウォーターズ2487 UV検出器により行った。さらに、酵素的測定キット(アールバイオファーム(R−Biopharm)AG、ダルムシュタット(Darmstadt)、ドイツ)を用いて最初と最後のグリセロール濃度を判定した。窒素ガスをスパージするバイオレアクターでの培養の過程で、オフガスによりエタノールのかなりの部分が失われる。これを補正するため、無菌合成培地を用いて同一条件下、異なる有効容積で運転したバイオレアクターでエタノール蒸発のカイネティクスを解析した。得られた容積依存性のエタノール蒸発定数(この構成の場合、リットル単位の容積で割った商0.0080に相当し、h−1で表す)を用いて、培養上清におけるエタノール濃度のHPLC測定値を補正し、サンプリングにより起こる容積の変化を考慮した。
以前記載されたように指数関数的に増殖する嫌気回分培養から、NAD+依存性アセトアルデヒドデヒドロゲナーゼ(アセチル化)活性アッセイのための細胞抽出物を調製した(アボット(Abbott)ら、アプライド・アンド・エンバイロンメンタル・マイクロバイオロジー、第75巻:p.2320−2325)。340nmでNADHの酸化をモニタリングすることにより、NAD+依存性アセトアルデヒドデヒドロゲナーゼ(アセチル化)活性を30℃で測定した。反応混合物(全容積1ml)は、50mMのリン酸カリウム緩衝液(pH7.5)、15mMのNADHおよび細胞抽出物を含んでいた。0.5mMのアセチルコエンザイムAを添加して反応を開始した。グリセロール3−リン酸デヒドロゲナーゼ(EC1.1.1.8)活性の判定では、リン酸塩緩衝液をトリエタノールアミン緩衝液(10mM、pH5)(5、19)と交換したこと以外は上記のように細胞抽出物を調製した。グリセロール−3−リン酸デヒドロゲナーゼ活性は、以前記載されたように細胞抽出物を用いて30℃でアッセイした(ブロムバーグ(Blomberg)およびアドラー(Adler)(1989年)、ジャーナル・オブ・バクテリオロジー第171巻:p.1087−1092。反応速度は、添加した細胞抽出物の量に比例した。タンパク質濃度は、ウシ血清アルブミンを標準物質としてローリー(Lowry)法により判定した(ローリーet al(1951年)ジャーナル・オブ・バイオロジカル・ケミストリー第193巻:p.265−275)。
嫌気回分培養における増殖および産物形成
原栄養性の基準菌株S.セレビシエIME076(GPD1 GPD2)の培養物に2.0g l−1の酢酸を補充した場合、比増殖速度(0.32h−1)は、酢酸の非存在下で増殖させた培養物で報告された比増殖速度(0.34h−1)、カイパー(Kuyper)ら(2005年)FEMSイーストリサーチ第5巻:p.399−409と同一であった。酢酸の添加によりバイオマスの収率は若干低下し、その結果、酢酸の非存在下で増殖した培養物と比較してグルコースのグリセロールの収率も低下した(図2、表3)。
本研究は、化学量論的には、S.セレビシエの嫌気的増殖のレドックスシンクとしてのグリセロールの役割を、アセテートのエタノールへのNADH依存性還元における線形経路で十分に置き換えられるという原理の証拠となる。これは、リグノセルロース加水分解物など酢酸を含む供給原料からの大規模エタノール生成の興味深い視点を与える。
嫌気回分培養における増殖および生成物の形成
gpd1Δ gpd2ΔS.セレビシエ株におけるシュードモナス・エスピーCF600dmpF遺伝子の発現の結果は、同じ菌株におけるE.コリmhpF遺伝子の発現で得られた結果と類似していた。嫌気性回分発酵では、菌株IMZ132(gpd1Δ gpd2ΔmhpF)と類似して、2.0g l−1の酢酸を含む培地の補充に伴いdmpF遺伝子の機能発現は指数増殖し、比増殖速度は0.11h−1であった(図1)。菌株IMZ130(gpd1Δ gpd2ΔdmpF)の回分時間(55h)は、菌株IMZ132(40h)よりやや長かった。培養中、グルコースの初濃度20g l−1は完全に消化される一方、グリセロールの形成が観察された。同時に、アセテートは初濃度2.1g l−1から最終濃度1.6g l−1に消化された。エタノールが主な有機生成物であり、グルコースのエタノール収率は0.48g g−1(エタノール蒸発で補正した収率)を示した。同じ条件下で増殖させた基準菌株の培養で観察されたのと同様に、少量のスクシネートおよびラクテートが生成された。
配列表
SEQ ID NO: 1
E. coli mhpF gene Acetaldehyde dehydrogenase, acylating
ATGAGTAAGCGTAAAGTCGCCATTATCGGTTCTGGCAACATTGGTACCGATCTGATGATTAAAATTTTGCGTCACGGTCAGCATCTGGAGATGGCGGTGATGGTTGGCATTGATCCTCAGTCCGACGGTCTGGCGCGCGCCAGACGTATGGGCGTCGCCACCACCCATGAAGGGGTGATCGGACTGATGAACATGCCTGAATTTGCTGATATCGACATTGTATTTGATGCGACCAGCGCCGGTGCTCATGTGAAAAACGATGCCGCTTTACGCGAAGCGAAACCGGATATTCGCTTAATTGACCTGACGCCTGCTGCCATCGGCCCTTACTGCGTGCCGGTGGTTAACCTCGAGGCGAACGTCGATCAACTGAACGTCAACATGGTCACCTGCGGCGGCCAGGCCACCATTCCAATGGTGGCGGCAGTTTCACGCGTGGCGCGTGTTCATTACGCCGAAATTATCGCTTCTATCGCCAGTAAATCTGCCGGACCTGGCACGCGTGCCAATATCGATGAATTTACGGAAACCACTTCCCGAGCCATTGAAGTGGTGGGCGGCGCGGCAAAAGGGAAGGCGATTATTGTGCTTAACCCAGCA
GAGCCACCGTTGATGATGCGTGACACGGTGTATGTATTGAGCGACGAAGCTTCACAAGATGATATCGAAGCCTCAATCAATGAAATGGCTGAGGCGGTGCAGGCTTACGTACCGGGTTATCGCCTGAAACAGCGCGTGCAGTTTGAAGTTATCCCGCAGGATAAACCGGTCAATTTACCGGGCGTGGGGCAATTCTCCGGACTGAAAACAGCGGTCTGGCTGGAAGTCGAAGGCGCAGCGCATTATCTGCCTGCCTATGCGGGCAACCTCGACATTATGACTTCCAGTGCGCTGGCGACAGCGGAAAAAATGGCCCAGTCACTGGCGCGCAAGGCAGGAGAAGCGGCATGA
SEQ ID NO: 2
E. coli Acetaldehyde dehydrogenase OS=Escherichia coli (strain K12) GN=mhpF PE=1 SV=1
MSKRKVAIIGSGNIGTDLMIKILRHGQHLEMAVMVGIDPQSDGLARARRMGVATTHEGVIGLMNMPEFADIDIVFDATSAGAHVKNDAALREAKPDIRLIDLTPAAIGPYCVPVVNLEANVDQLNVNMVTCGGQATIPMVAAVSRVARVHYAEIIASIASKSAGPGTRANIDEFTETTSRAIEVVGGAAKGKAIIVLNPAEPPLMMRDTVYVLSDEASQDDIEASINEMAEAVQAYVPGYRLKQRVQFEVIPQDKPVNLPGVGQFSGLKTAVWLEVEGAAHYLPAYAGNLDIMTSSALATAEKMAQSLARKAGEAA
SEQ ID NO: 3
primer mhpF-FW
GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAGTAAGCGTAAAGTCGCCATTATCGG
SEQ ID NO: 4
primer mhpF-RV GGGGACCACTTTGTACAAGAAAGCTGGGTGTTCATGCCGCTTCTCCTGCCTTGC
SEQ ID NO: 5 GPD1/YDL022W fw gene disruption primer
TTGTACACCCCCCCCCTCCACAAACACAAATATTGATAATATAAAcagctgaagcttcgtacgc
SEQ ID NO: 6 GPD1/YDL022W rv gene disruption primer
AATCTAATCTTCATGTAGATCTAATTCTTCAATCATGTCCGGCGgcataggccactagtggatctg
SEQ ID NO: 7 GPD2/YOL059W fw gene disruption primer
TCAATTCTCTTTCCCTTTCCTTTTCCTTCGCTCCCCTTCCTTATC ccaggctgaagcttcgtacg
SEQ ID NO: 8 GPD2/YOL059W rv gene disruption primer
GTGTCTATTCGTCATCGATGTCTAGCTCTTCAATCATCTCCGGTAGgcataggccactagtggatc
SEQ ID NO: 9 GPD1/YDL022W fw verification primer
CCCACCCACACCACCAATAC
SEQ ID NO: 10 GPD1/YDL022Wrv verification primer
CGGACGCCAGATGCTAGAAG
SEQ ID NO: 11 GPD2/YOL059W fw verification primer
GTTCAGCAGCTCTTCTCTAC
SEQ ID NO: 12 GPD2/YOL059W rv verification primer
CCAAATGCGACATGAGTCAC
SEQ ID NO: 13 GPD1/YDL022W fw disruption cassette specific verification primer
CGCACGTCAAGACTGTCAAG
SEQ ID NO: 14 GPD1/YDL022W rv disruption cassette specific verification primer
TCGTATGTGAATGCTGGTCG
SEQ ID NO: 15 GPD2/YOL059W fw disruption cassette specific verification primer
CGCACGTCAAGACTGTCAAG
SEQ ID NO: 16 GPD2/YOL059W rv disruption cassette specific verification primer
TCGTATGTGAATGCTGGTCG
SEQ ID NO: 17 S. cerevisiae ACS 1 gene
ATGTCGCCCTCTGCCGTACAATCATCAAAACTAGAAGAACAGTCAAGTGAAATTGACAAG
TTGAAAGCAAAAATGTCCCAGTCTGCCGCCACTGCGCAGCAGAAGAAGGAACATGAGTAT
GAACATTTGACTTCGGTCAAGATCGTGCCACAACGGCCCATCTCAGATAGACTGCAGCCC
GCAATTGCTACCCACTATTCTCCACACTTGGACGGGTTGCAGGACTATCAGCGCTTGCAC
AAGGAGTCTATTGAAGACCCTGCTAAGTTCTTCGGTTCTAAAGCTACCCAATTTTTAAAC
TGGTCTAAGCCATTCGATAAGGTGTTCATCCCAGACCCTAAAACGGGCAGGCCCTCCTTC
CAGAACAATGCATGGTTCCTCAACGGCCAATTAAACGCCTGTTACAACTGTGTTGACAGA
CATGCCTTGAAGACTCCTAACAAGAAAGCCATTATTTTCGAAGGTGACGAGCCTGGCCAA
GGCTATTCCATTACCTACAAGGAACTACTTGAAGAAGTTTGTCAAGTGGCACAAGTGCTG
ACTTACTCTATGGGCGTTCGCAAGGGCGATACTGTTGCCGTGTACATGCCTATGGTCCCA
GAAGCAATCATAACCTTGTTGGCCATTTCCCGTATCGGTGCCATTCACTCCGTAGTCTTT
GCCGGGTTTTCTTCCAACTCCTTGAGAGATCGTATCAACGATGGGGACTCTAAAGTTGTC
ATCACTACAGATGAATCCAACAGAGGTGGTAAAGTCATTGAGACTAAAAGAATTGTTGAT
GACGCGCTAAGAGAGACCCCAGGCGTGAGACACGTCTTGGTTTATAGAAAGACCAACAAT
CCATCTGTTGCTTTCCATGCCCCCAGAGATTTGGATTGGGCAACAGAAAAGAAGAAATAC
AAGACCTACTATCCATGCACACCCGTTGATTCTGAGGATCCATTATTCTTGTTGTATACG
TCTGGTTCTACTGGTGCCCCCAAGGGTGTTCAACATTCTACCGCAGGTTACTTGCTGGGA
GCTTTGTTGACCATGCGCTACACTTTTGACACTCACCAAGAAGACGTTTTCTTCACAGCT
GGAGACATTGGCTGGATTACAGGCCACACTTATGTGGTTTATGGTCCCTTACTATATGGT
TGTGCCACTTTGGTCTTTGAAGGGACTCCTGCGTACCCAAATTACTCCCGTTATTGGGAT
ATTATTGATGAACACAAAGTCACCCAATTTTATGTTGCGCCAACTGCTTTGCGTTTGTTG
AAAAGAGCTGGTGATTCCTACATCGAAAATCATTCCTTAAAATCTTTGCGTTGCTTGGGT
TCGGTCGGTGAGCCAATTGCTGCTGAAGTTTGGGAGTGGTACTCTGAAAAAATAGGTAAA
AATGAAATCCCCATTGTAGACACCTACTGGCAAACAGAATCTGGTTCGCATCTGGTCACC
CCGCTGGCTGGTGGTGTTACACCAATGAAACCGGGTTCTGCCTCATTCCCCTTCTTCGGT
ATTGATGCAGTTGTTCTTGACCCTAACACTGGTGAAGAACTTAACACCAGCCACGCAGAG
GGTGTCCTTGCCGTCAAAGCTGCATGGCCATCATTTGCAAGAACTATTTGGAAAAATCAT
GATAGGTATCTAGACACTTATTTGAACCCTTACCCTGGCTACTATTTCACTGGTGATGGT
GCTGCAAAGGATAAGGATGGTTATATCTGGATTTTGGGTCGTGTAGACGATGTGGTGAAC
GTCTCTGGTCACCGTCTGTCTACCGCTGAAATTGAGGCTGCTATTATCGAAGATCCAATT
GTGGCCGAGTGTGCTGTTGTCGGATTCAACGATGACTTGACTGGTCAAGCAGTTGCTGCA
TTTGTGGTGTTGAAAAACAAATCTAGTTGGTCCACCGCAACAGATGATGAATTACAAGAT
ATCAAGAAGCATTTGGTCTTTACTGTTAGAAAAGACATCGGGCCATTTGCCGCACCAAAA
TTGATCATTTTAGTGGATGACTTGCCCAAGACAAGATCCGGCAAAATTATGAGACGTATT
TTAAGAAAAATCCTAGCAGGAGAAAGTGACCAACTAGGCGACGTTTCTACATTGTCAAAC
CCTGGCATTGTTAGACATCTAATTGATTCGGTCAAGTTGTAA
SEQ ID NO: 18 S. cerevisiae ACS 2 gene
ATGACAATCAAGGAACATAAAGTAGTTTATGAAGCTCACAACGTAAAGGCTCTTAAGGCT
CCTCAACATTTTTACAACAGCCAACCCGGCAAGGGTTACGTTACTGATATGCAACATTAT
CAAGAAATGTATCAACAATCTATCAATGAGCCAGAAAAATTCTTTGATAAGATGGCTAAG
GAATACTTGCATTGGGATGCTCCATACACCAAAGTTCAATCTGGTTCATTGAACAATGGT
GATGTTGCATGGTTTTTGAACGGTAAATTGAATGCATCATACAATTGTGTTGACAGACAT
GCCTTTGCTAATCCCGACAAGCCAGCTTTGATCTATGAAGCTGATGACGAATCCGACAAC
AAAATCATCACATTTGGTGAATTACTCAGAAAAGTTTCCCAAATCGCTGGTGTCTTAAAA
AGCTGGGGCGTTAAGAAAGGTGACACAGTGGCTATCTATTTGCCAATGATTCCAGAAGCG
GTCATTGCTATGTTGGCTGTGGCTCGTATTGGTGCTATTCACTCTGTTGTCTTTGCTGGG
TTCTCCGCTGGTTCGTTGAAAGATCGTGTCGTTGACGCTAATTCTAAAGTGGTCATCACT
TGTGATGAAGGTAAAAGAGGTGGTAAGACCATCAACACTAAAAAAATTGTTGACGAAGGT
TTGAACGGAGTCGATTTGGTTTCCCGTATCTTGGTTTTCCAAAGAACTGGTACTGAAGGT
ATTCCAATGAAGGCCGGTAGAGATTACTGGTGGCATGAGGAGGCCGCTAAGCAGAGAACT
TACCTACCTCCTGTTTCATGTGACGCTGAAGATCCTCTATTTTTATTATACACTTCCGGT
TCCACTGGTTCTCCAAAGGGTGTCGTTCACACTACAGGTGGTTATTTATTAGGTGCCGCT
TTAACAACTAGATACGTTTTTGATATTCACCCAGAAGATGTTCTCTTCACTGCCGGTGAC
GTCGGCTGGATCACGGGTCACACCTATGCTCTATATGGTCCATTAACCTTGGGTACCGCC
TCAATAATTTTCGAATCCACTCCTGCCTACCCAGATTATGGTAGATATTGGAGAATTATC
CAACGTCACAAGGCTACCCATTTCTATGTGGCTCCAACTGCTTTAAGATTAATCAAACGT
GTAGGTGAAGCCGAAATTGCCAAATATGACACTTCCTCATTACGTGTCTTGGGTTCCGTC
GGTGAACCAATCTCTCCAGACTTATGGGAATGGTATCATGAAAAAGTGGGTAACAAAAAC
TGTGTCATTTGTGACACTATGTGGCAAACAGAGTCTGGTTCTCATTTAATTGCTCCTTTG
GCAGGTGCTGTCCCAACAAAACCTGGTTCTGCTACCGTGCCATTCTTTGGTATTAACGCT
TGTATCATTGACCCTGTTACAGGTGTGGAATTAGAAGGTAATGATGTCGAAGGTGTCCTT
GCCGTTAAATCACCATGGCCATCAATGGCTAGATCTGTTTGGAACCACCACGACCGTTAC
ATGGATACTTACTTGAAACCTTATCCTGGTCACTATTTCACAGGTGATGGTGCTGGTAGA
GATCATGATGGTTACTACTGGATCAGGGGTAGAGTTGACGACGTTGTAAATGTTTCCGGT
CATAGATTATCCACATCAGAAATTGAAGCATCTATCTCAAATCACGAAAACGTCTCGGAA
GCTGCTGTTGTCGGTATTCCAGATGAATTGACCGGTCAAACCGTCGTTGCATATGTTTCC
CTAAAAGATGGTTATCTACAAAACAACGCTACTGAAGGTGATGCAGAACACATCACACCA
GATAATTTACGTAGAGAATTGATCTTACAAGTTAGGGGTGAGATTGGTCCTTTCGCCTCA
CCAAAAACCATTATTCTAGTTAGAGATCTACCAAGAACAAGGTCAGGAAAGATTATGAGA
AGAGTTCTAAGAAAGGTTGCTTCTAACGAAGCCGAACAGCTAGGTGACCTAACTACTTTG
GCCAACCCAGAAGTTGTACCTGCCATCATTTCTGCTGTAGAGAACCAATTTTTCTCTCAA
AAAAAGAAATAA
SEQ ID NO: 19 S. cerevisiae ADH1
ATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCACGGTAAGTTGGAA
TACAAAGATATTCCAGTTCCAAAGCCAAAGGCCAACGAATTGTTGATCAACGTTAAATAC
TCTGGTGTCTGTCACACTGACTTGCACGCTTGGCACGGTGACTGGCCATTGCCAGTTAAG
CTACCATTAGTCGGTGGTCACGAAGGTGCCGGTGTCGTTGTCGGCATGGGTGAAAACGTT
AAGGGCTGGAAGATCGGTGACTACGCCGGTATCAAATGGTTGAACGGTTCTTGTATGGCC
TGTGAATACTGTGAATTGGGTAACGAATCCAACTGTCCTCACGCTGACTTGTCTGGTTAC
ACCCACGACGGTTCTTTCCAACAATACGCTACCGCTGACGCTGTTCAAGCCGCTCACATT
CCTCAAGGTACCGACTTGGCCCAAGTCGCCCCCATCTTGTGTGCTGGTATCACCGTCTAC
AAGGCTTTGAAGTCTGCTAACTTGATGGCCGGTCACTGGGTTGCTATCTCCGGTGCTGCT
GGTGGTCTAGGTTCTTTGGCTGTTCAATACGCCAAGGCTATGGGTTACAGAGTCTTGGGT
ATTGACGGTGGTGAAGGTAAGGAAGAATTATTCAGATCCATCGGTGGTGAAGTCTTCATT
GACTTCACTAAGGAAAAGGACATTGTCGGTGCTGTTCTAAAGGCCACTGACGGTGGTGCT
CACGGTGTCATCAACGTTTCCGTTTCCGAAGCCGCTATTGAAGCTTCTACCAGATACGTT
AGAGCTAACGGTACCACCGTTTTGGTCGGTATGCCAGCTGGTGCCAAGTGTTGTTCTGAT
GTCTTCAACCAAGTCGTCAAGTCCATCTCTATTGTTGGTTCTTACGTCGGTAACAGAGCT
GACACCAGAGAAGCTTTGGACTTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCAAGGTT
GTCGGCTTGTCTACCTTGCCAGAAATTTACGAAAAGATGGAAAAGGGTCAAATCGTTGGT
AGATACGTTGTTGACACTTCTAAATAA
SEQ ID NO: 20 S. cerevisiae ADH2
ATGTCTATTCCAGAAACTCAAAAAGCCATTATCTTCTACGAATCCAACGGCAAGTTGGAG
CATAAGGATATCCCAGTTCCAAAGCCAAAGCCCAACGAATTGTTAATCAACGTCAAGTAC
TCTGGTGTCTGCCACACCGATTTGCACGCTTGGCATGGTGACTGGCCATTGCCAACTAAG
TTACCATTAGTTGGTGGTCACGAAGGTGCCGGTGTCGTTGTCGGCATGGGTGAAAACGTT
AAGGGCTGGAAGATCGGTGACTACGCCGGTATCAAATGGTTGAACGGTTCTTGTATGGCC
TGTGAATACTGTGAATTGGGTAACGAATCCAACTGTCCTCACGCTGACTTGTCTGGTTAC
ACCCACGACGGTTCTTTCCAAGAATACGCTACCGCTGACGCTGTTCAAGCCGCTCACATT
CCTCAAGGTACTGACTTGGCTGAAGTCGCGCCAATCTTGTGTGCTGGTATCACCGTATAC
AAGGCTTTGAAGTCTGCCAACTTGAGAGCAGGCCACTGGGCGGCCATTTCTGGTGCTGCT
GGTGGTCTAGGTTCTTTGGCTGTTCAATATGCTAAGGCGATGGGTTACAGAGTCTTAGGT
ATTGATGGTGGTCCAGGAAAGGAAGAATTGTTTACCTCGCTCGGTGGTGAAGTATTCATC
GACTTCACCAAAGAGAAGGACATTGTTAGCGCAGTCGTTAAGGCTACCAACGGCGGTGCC
CACGGTATCATCAATGTTTCCGTTTCCGAAGCCGCTATCGAAGCTTCTACCAGATACTGT
AGGGCGAACGGTACTGTTGTCTTGGTTGGTTTGCCAGCCGGTGCAAAGTGCTCCTCTGAT
GTCTTCAACCACGTTGTCAAGTCTATCTCCATTGTCGGCTCTTACGTGGGGAACAGAGCT
GATACCAGAGAAGCCTTAGATTTCTTTGCCAGAGGTCTAGTCAAGTCTCCAATAAAGGTA
GTTGGCTTATCCAGTTTACCAGAAATTTACGAAAAGATGGAGAAGGGCCAAATTGCTGGT
AGATACGTTGTTGACACTTCTAAATAA
SEQ ID NO: 21 S. cerevisiae ADH3
ATGTTGAGAACGTCAACATTGTTCACCAGGCGTGTCCAACCAAGCCTATTTTCTAGAAAC
ATTCTTAGATTGCAATCCACAGCTGCAATCCCTAAGACTCAAAAAGGTGTCATCTTTTAT
GAGAATAAGGGGAAGCTGCATTACAAAGATATCCCTGTCCCCGAGCCTAAGCCAAATGAA
ATTTTAATCAACGTTAAATATTCTGGTGTATGTCACACCGATTTACATGCTTGGCACGGC
GATTGGCCATTACCTGTTAAACTACCATTAGTAGGTGGTCATGAAGGTGCTGGTGTAGTT
GTCAAACTAGGTTCCAATGTCAAGGGCTGGAAAGTCGGTGATTTAGCAGGTATCAAATGG
CTGAACGGTTCTTGTATGACATGCGAATTCTGTGAATCAGGTCATGAATCAAATTGTCCA
GATGCTGATTTATCTGGTTACACTCATGATGGTTCTTTCCAACAATTTGCGACCGCTGAT
GCTATTCAAGCCGCCAAAATTCAACAGGGTACCGACTTGGCCGAAGTAGCCCCAATATTA
TGTGCTGGTGTTACTGTATATAAAGCACTAAAAGAGGCAGACTTGAAAGCTGGTGACTGG
GTTGCCATCTCTGGTGCTGCAGGTGGCTTGGGTTCCTTGGCCGTTCAATATGCAACTGCG
ATGGGTTACAGAGTTCTAGGTATTGATGCAGGTGAGGAAAAGGAAAAACTTTTCAAGAAA
TTGGGGGGTGAAGTATTCATCGACTTTACTAAAACAAAGAATATGGTTTCTGACATTCAA
GAAGCTACCAAAGGTGGCCCTCATGGTGTCATTAACGTTTCCGTTTCTGAAGCCGCTATT
TCTCTATCTACGGAATATGTTAGACCATGTGGTACCGTCGTTTTGGTTGGTTTGCCCGCT
AACGCCTACGTTAAATCAGAGGTATTCTCTCATGTGGTGAAGTCCATCAATATCAAGGGT
TCTTATGTTGGTAACAGAGCTGATACGAGAGAAGCCTTAGACTTCTTTAGCAGAGGTTTG
ATCAAATCACCAATCAAAATTGTTGGATTATCTGAATTACCAAAGGTTTATGACTTGATG
GAAAAGGGCAAGATTTTGGGTAGATACGTCGTCGATACTAGTAAATAA
SEQ ID NO: 22 S. cerevisiae ADH4
ATGTCTTCCGTTACTGGGTTTTACATTCCACCAATCTCTTTCTTTGGTGAAGGTGCTTTA
GAAGAAACCGCTGATTACATCAAAAACAAGGATTACAAAAAGGCTTTGATCGTTACTGAT
CCTGGTATTGCAGCTATTGGTCTCTCCGGTAGAGTCCAAAAGATGTTGGAAGAACGTGAC
TTAAACGTTGCTATCTATGACAAAACTCAACCAAACCCAAATATTGCCAATGTCACAGCT
GGTTTGAAGGTTTTGAAGGAACAAAACTCTGAAATTGTTGTTTCCATTGGTGGTGGTTCT
GCTCACGACAATGCTAAGGCCATTGCTTTATTGGCTACTAACGGTGGGGAAATCGGAGAC
TATGAAGGTGTCAATCAATCTAAGAAGGCTGCTTTACCACTATTTGCCATCAACACTACT
GCTGGTACTGCTTCCGAAATGACCAGATTCACTATTATCTCTAATGAAGAAAAGAAAATC
AAGATGGCTATCATTGACAACAACGTCACTCCAGCTGTTGCTGTCAACGATCCATCTACC
ATGTTTGGTTTGCCACCTGCTTTGACTGCTGCTACTGGTCTAGATGCTTTGACTCACTGT
ATCGAAGCTTATGTTTCCACCGCCTCTAACCCAATCACCGATGCCTGTGCTTTGAAGGGT
ATTGATTTGATCAATGAAAGCTTAGTCGCTGCATACAAAGACGGTAAAGACAAGAAGGCC
AGAACTGACATGTGTTACGCTGAATACTTGGCAGGTATGGCTTTCAACAATGCTTCTCTA
GGTTATGTTCATGCCCTTGCTCATCAACTTGGTGGTTTCTACCACTTGCCTCATGGTGTT
TGTAACGCTGTCTTGTTGCCTCATGTTCAAGAGGCCAACATGCAATGTCCAAAGGCCAAG
AAGAGATTAGGTGAAATTGCTTTGCATTTCGGTGCTTCTCAAGAAGATCCAGAAGAAACC
ATCAAGGCTTTGCACGTTTTAAACAGAACCATGAACATTCCAAGAAACTTGAAAGAATTA
GGTGTTAAAACCGAAGATTTTGAAATTTTGGCTGAACACGCCATGCATGATGCCTGCCAT
TTGACTAACCCAGTTCAATTCACCAAAGAACAAGTGGTTGCCATTATCAAGAAAGCCTAT
GAATATTAA
SEQ ID NO: 23 S. cerevisiae ADH5
ATGCCTTCGCAAGTCATTCCTGAAAAACAAAAGGCTATTGTCTTTTATGAGACAGATGGA
AAATTGGAATATAAAGACGTCACAGTTCCGGAACCTAAGCCTAACGAAATTTTAGTCCAC
GTTAAATATTCTGGTGTTTGTCATAGTGACTTGCACGCGTGGCACGGTGATTGGCCATTT
CAATTGAAATTTCCATTAATCGGTGGTCACGAAGGTGCTGGTGTTGTTGTTAAGTTGGGA
TCTAACGTTAAGGGCTGGAAAGTCGGTGATTTTGCAGGTATAAAATGGTTGAATGGGACT
TGCATGTCCTGTGAATATTGTGAAGTAGGTAATGAATCTCAATGTCCTTATTTGGATGGT
ACTGGCTTCACACATGATGGTACTTTTCAAGAATACGCAACTGCCGATGCCGTTCAAGCT
GCCCATATTCCACCAAACGTCAATCTTGCTGAAGTTGCCCCAATCTTGTGTGCAGGTATC
ACTGTTTATAAGGCGTTGAAAAGAGCCAATGTGATACCAGGCCAATGGGTCACTATATCC
GGTGCATGCGGTGGCTTGGGTTCTCTGGCAATCCAATACGCCCTTGCTATGGGTTACAGG
GTCATTGGTATCGATGGTGGTAATGCCAAGCGAAAGTTATTTGAACAATTAGGCGGAGAA
ATATTCATCGATTTCACGGAAGAAAAAGACATTGTTGGTGCTATAATAAAGGCCACTAAT
GGCGGTTCTCATGGAGTTATTAATGTGTCTGTTTCTGAAGCAGCTATCGAGGCTTCTACG
AGGTATTGTAGGCCCAATGGTACTGTCGTCCTGGTTGGTATGCCAGCTCATGCTTACTGC
AATTCCGATGTTTTCAATCAAGTTGTAAAATCAATCTCCATCGTTGGATCTTGTGTTGGA
AATAGAGCTGATACAAGGGAGGCTTTAGATTTCTTCGCCAGAGGTTTGATCAAATCTCCG
ATCCACTTAGCTGGCCTATCGGATGTTCCTGAAATTTTTGCAAAGATGGAGAAGGGTGAA
ATTGTTGGTAGATATGTTGTTGAGACTTCTAAATGA
SEQ ID NO 24: S. cerevisiae GPD1
ATGTCTGCTGCTGCTGATAGATTAAACTTAACTTCCGGCCACTTGAATGCTGGTAGAAAG
AGAAGTTCCTCTTCTGTTTCTTTGAAGGCTGCCGAAAAGCCTTTCAAGGTTACTGTGATT
GGATCTGGTAACTGGGGTACTACTATTGCCAAGGTGGTTGCCGAAAATTGTAAGGGATAC
CCAGAAGTTTTCGCTCCAATAGTACAAATGTGGGTGTTCGAAGAAGAGATCAATGGTGAA
AAATTGACTGAAATCATAAATACTAGACATCAAAACGTGAAATACTTGCCTGGCATCACT
CTACCCGACAATTTGGTTGCTAATCCAGACTTGATTGATTCAGTCAAGGATGTCGACATC
ATCGTTTTCAACATTCCACATCAATTTTTGCCCCGTATCTGTAGCCAATTGAAAGGTCAT
GTTGATTCACACGTCAGAGCTATCTCCTGTCTAAAGGGTTTTGAAGTTGGTGCTAAAGGT
GTCCAATTGCTATCCTCTTACATCACTGAGGAACTAGGTATTCAATGTGGTGCTCTATCT
GGTGCTAACATTGCCACCGAAGTCGCTCAAGAACACTGGTCTGAAACAACAGTTGCTTAC
CACATTCCAAAGGATTTCAGAGGCGAGGGCAAGGACGTCGACCATAAGGTTCTAAAGGCC
TTGTTCCACAGACCTTACTTCCACGTTAGTGTCATCGAAGATGTTGCTGGTATCTCCATC
TGTGGTGCTTTGAAGAACGTTGTTGCCTTAGGTTGTGGTTTCGTCGAAGGTCTAGGCTGG
GGTAACAACGCTTCTGCTGCCATCCAAAGAGTCGGTTTGGGTGAGATCATCAGATTCGGT
CAAATGTTTTTCCCAGAATCTAGAGAAGAAACATACTACCAAGAGTCTGCTGGTGTTGCT
GATTTGATCACCACCTGCGCTGGTGGTAGAAACGTCAAGGTTGCTAGGCTAATGGCTACT
TCTGGTAAGGACGCCTGGGAATGTGAAAAGGAGTTGTTGAATGGCCAATCCGCTCAAGGT
TTAATTACCTGCAAAGAAGTTCACGAATGGTTGGAAACATGTGGCTCTGTCGAAGACTTC
CCATTATTTGAAGCCGTATACCAAATCGTTTACAACAACTACCCAATGAAGAACCTGCCG
GACATGATTGAAGAATTAGATCTACATGAAGATTAG
SEQ ID NO: 25: S. cerevisiae GPD2
ATGCTTGCTGTCAGAAGATTAACAAGATACACATTCCTTAAGCGAACGCATCCGGTGTTA
TATACTCGTCGTGCATATAAAATTTTGCCTTCAAGATCTACTTTCCTAAGAAGATCATTA
TTACAAACACAACTGCACTCAAAGATGACTGCTCATACTAATATCAAACAGCACAAACAC
TGTCATGAGGACCATCCTATCAGAAGATCGGACTCTGCCGTGTCAATTGTACATTTGAAA
CGTGCGCCCTTCAAGGTTACAGTGATTGGTTCTGGTAACTGGGGGACCACCATCGCCAAA
GTCATTGCGGAAAACACAGAATTGCATTCCCATATCTTCGAGCCAGAGGTGAGAATGTGG
GTTTTTGATGAAAAGATCGGCGACGAAAATCTGACGGATATCATAAATACAAGACACCAG
AACGTTAAATATCTACCCAATATTGACCTGCCCCATAATCTAGTGGCCGATCCTGATCTT
TTACACTCCATCAAGGGTGCTGACATCCTTGTTTTCAACATCCCTCATCAATTTTTACCA
AACATAGTCAAACAATTGCAAGGCCACGTGGCCCCTCATGTAAGGGCCATCTCGTGTCTA
AAAGGGTTCGAGTTGGGCTCCAAGGGTGTGCAATTGCTATCCTCCTATGTTACTGATGAG
TTAGGAATCCAATGTGGCGCACTATCTGGTGCAAACTTGGCACCGGAAGTGGCCAAGGAG
CATTGGTCCGAAACCACCGTGGCTTACCAACTACCAAAGGATTATCAAGGTGATGGCAAG
GATGTAGATCATAAGATTTTGAAATTGCTGTTCCACAGACCTTACTTCCACGTCAATGTC
ATCGATGATGTTGCTGGTATATCCATTGCCGGTGCCTTGAAGAACGTCGTGGCACTTGCA
TGTGGTTTCGTAGAAGGTATGGGATGGGGTAACAATGCCTCCGCAGCCATTCAAAGGCTG
GGTTTAGGTGAAATTATCAAGTTCGGTAGAATGTTTTTCCCAGAATCCAAAGTCGAGACC
TACTATCAAGAATCCGCTGGTGTTGCAGATCTGATCACCACCTGCTCAGGCGGTAGAAAC
GTCAAGGTTGCCACATACATGGCCAAGACCGGTAAGTCAGCCTTGGAAGCAGAAAAGGAA
TTGCTTAACGGTCAATCCGCCCAAGGGATAATCACATGCAGAGAAGTTCACGAGTGGCTA
CAAACATGTGAGTTGACCCAAGAATTCCCATTATTCGAGGCAGTCTACCAGATAGTCTAC
AACAACGTCCGCATGGAAGACCTACCGGAGATGATTGAAGAGCTAGACATCGATGACGAA
TAG
SEQ ID NO 26: S. cerevisiae GPP1
ATGCCTTTGACCACAAAACCTTTATCTTTGAAAATCAACGCCGCTCTATTCGATGTTGAC
GGTACCATCATCATCTCTCAACCAGCCATTGCTGCTTTCTGGAGAGATTTCGGTAAAGAC
AAGCCTTACTTCGATGCCGAACACGTTATTCACATCTCTCACGGTTGGAGAACTTACGAT
GCCATTGCCAAGTTCGCTCCAGACTTTGCTGATGAAGAATACGTTAACAAGCTAGAAGGT
GAAATCCCAGAAAAGTACGGTGAACACTCCATCGAAGTTCCAGGTGCTGTCAAGTTGTGT
AATGCTTTGAACGCCTTGCCAAAGGAAAAATGGGCTGTCGCCACCTCTGGTACCCGTGAC
ATGGCCAAGAAATGGTTCGACATTTTGAAGATCAAGAGACCAGAATACTTCATCACCGCC
AATGATGTCAAGCAAGGTAAGCCTCACCCAGAACCATACTTAAAGGGTAGAAACGGTTTG
GGTTTCCCAATTAATGAACAAGACCCATCCAAATCTAAGGTTGTTGTCTTTGAAGACGCA
CCAGCTGGTATTGCTGCTGGTAAGGCTGCTGGCTGTAAAATCGTTGGTATTGCTACCACT
TTCGATTTGGACTTCTTGAAGGAAAAGGGTTGTGACATCATTGTCAAGAACCACGAATCT
ATCAGAGTCGGTGAATACAACGCTGAAACCGATGAAGTCGAATTGATCTTTGATGACTAC
TTATACGCTAAGGATGACTTGTTGAAATGGTAA
SEQ ID NO: 27 S. cerevisiae GPP2
ATGGGATTGACTACTAAACCTCTATCTTTGAAAGTTAACGCCGCTTTGTTCGACGTCGAC
GGTACCATTATCATCTCTCAACCAGCCATTGCTGCATTCTGGAGGGATTTCGGTAAGGAC
AAACCTTATTTCGATGCTGAACACGTTATCCAAGTCTCGCATGGTTGGAGAACGTTTGAT
GCCATTGCTAAGTTCGCTCCAGACTTTGCCAATGAAGAGTATGTTAACAAATTAGAAGCT
GAAATTCCGGTCAAGTACGGTGAAAAATCCATTGAAGTCCCAGGTGCAGTTAAGCTGTGC
AACGCTTTGAACGCTCTACCAAAAGAGAAATGGGCTGTGGCAACTTCCGGTACCCGTGAT
ATGGCACAAAAATGGTTCGAGCATCTGGGAATCAGGAGACCAAAGTACTTCATTACCGCT
AATGATGTCAAACAGGGTAAGCCTCATCCAGAACCATATCTGAAGGGCAGGAATGGCTTA
GGATATCCGATCAATGAGCAAGACCCTTCCAAATCTAAGGTAGTAGTATTTGAAGACGCT
CCAGCAGGTATTGCCGCCGGAAAAGCCGCCGGTTGTAAGATCATTGGTATTGCCACTACT
TTCGACTTGGACTTCCTAAAGGAAAAAGGCTGTGACATCATTGTCAAAAACCACGAATCC
ATCAGAGTTGGCGGCTACAATGCCGAAACAGACGAAGTTGAATTCATTTTTGACGACTAC
TTATATGCTAAGGACGATCTGTTGAAATGGTAA
SEQ ID NO: 28 dmpF ORF synthetic codon optimized for Saccharomyces cerevisiae. Reference sequence: Pseudomonas sp. CF600
EMBL Accession number: X60835.1
ATGAATCAGAAACTGAAAGTAGCTATCATAGGTTCCGGTAATATCGGAAC
AGACCTGATGATTAAGGTACTGCGTAATGCAAAGTACTTAGAAATGGGCG
CGATGGTCGGTATCGATGCAGCCTCTGATGGACTGGCCAGAGCTCAAAGA
ATGGGCGTTACGACTACTTATGCAGGTGTAGAAGGGCTAATCAAGCTTCC
TGAATTTGCAGACATAGATTTCGTCTTCGATGCTACATCTGCATCAGCCC
ACGTTCAGAACGAGGCTTTATTAAGACAAGCTAAACCTGGTATTAGATTG
ATCGACCTTACCCCGGCGGCAATCGGTCCTTACTGTGTCCCCGTAGTTAA
TCTCGAGGAACATTTGGGCAAGTTGAACGTTAACATGGTTACTTGCGGTG
GCCAAGCTACTATTCCGATGGTCGCAGCTGTCTCACGTGTAGCCAAAGTC
CATTATGCTGAGATTGTTGCTTCTATTTCAAGCAAGAGTGCCGGACCTGG
AACCAGAGCCAATATAGATGAATTCACTGAGACAACCAGTAAAGCCATAG
AAGTTATTGGTGGTGCTGCAAAGGGTAAAGCTATAATTATTATGAACCCA
GCTGAACCACCATTGATTATGAGGGATACGGTGTATGTGCTTTCCGCCGC
CGCTGATCAAGCCGCTGTCGCAGCTTCTGTGGCTGAAATGGTTCAAGCGG
TTCAAGCATACGTGCCAGGCTATAGGTTAAAACAACAGGTTCAGTTTGAC
GTGATTCCCGAGTCCGCGCCACTAAACATCCCCGGTTTGGGGAGATTCAG
CGGGTTGAAAACAAGTGTGTTCCTAGAAGTAGAAGGTGCTGCTCATTATT
TGCCAGCATACGCAGGAAACTTAGATATTATGACTTCCGCAGCGTTAGCT
ACAGCCGAACGTATGGCGCAATCAATGTTGAATGCATAG
SEQ ID NO: 29 dmpF ORF
MNQKLKVAIIGSGNIGTDLMIKVLRNAKYLEMGAMVGIDAASDGLARAQR
MGVTTTYAGVEGLIKLPEFADIDFVFDATSASAHVQNEALLRQAKPGIRL
IDLTPAAIGPYCVPVVNLEEHLGKLNVNMVTCGGQATIPMVAAVSRVAKV
HYAEIVASISSKSAGPGTRANIDEFTETTSKAIEVIGGAAKGKAIIIMNP
AEPPLIMRDTVYVLSAAADQAAVAASVAEMVQAVQAYVPGYRLKQQVQFD
VIPESAPLNIPGLGRFSGLKTSVFLEVEGAAHYLPAYAGNLDIMTSAALA
TAERMAQSMLNA
SEQ ID NO: 30 primer dmpF-FW
CATTGATTGCGCCATACG
SEQ ID NO: 31 primer dmpF-RV
CCGGTAATATCGGAACAGAC
SEQ ID NO: 32 Wild type sequence of dmpF gene from Pseudomonas sp. CF600. EMBL Accession number: X60835.1
ATGAACCAGAAACTCAAAGTCGCGATCATCGGTTCGGGCAATATCGGCACCGACCTGATGATCAAGGTGCTGCGCAACGCCAAGTACCTGGAAATGGGCGCCATGGTCGGCATCGACGCCGCCTCCGACGGCCTGGCCCGCGCCCAGCGCATGGGCGTGACGACCACCTATGCCGGCGTCGAAGGGCTGATCAAGCTGCCCGAATTCGCCGACATCGATTTCGTCTTCGACGCCACCTCGGCCAGTGCCCACGTGCAGAACGAGGCGCTGCTGCGCCAGGCCAAACCTGGCATCCGCCTGATCGACCTGACCCCGGCGGCCATCGGCCCGTACTGCGTGCCGGTGGTCAATCTGGAGGAGCACCTCGGCAAGCTCAACGTCAACATGGTTACCTGCGGCGGCCAGGCGACCATCCCGATGGTCGCCGCGGTCTCCCGTGTGGCCAAGGTCCATTACGCCGAGATCGTCGCCTCGATCAGCAGCAAGTCGGCCGGACCCGGCACCCGCGCCAACATCGACGAGTTCACCGAGACCACCAGCAAAGCCATCGAAGTGATCGGTGGTGCGGCCAAGGGCAAGGCGATCATCATCATGAACCCGGCTGAGCCGCCGCTGATCATGCGCGACACCGTGTATGTGCTGTCCGCCGCCGCCGATCAGGCCGCCGTCGCGGCCTCGGTGGCGGAAATGGTTCAGGCGGTGCAGGCCTACGTGCCCGGCTATCGCCTGAAGCAGCAGGTGCAGTTCGACGTGATCCCCGAGTCCGCGCCGCTGAACATCCCCGGTCTCGGCCGGTTCAGCGGGTTGAAGACCTCGGTGTTCCTCGAAGTCGAAGGCGCCGCCCATTACCTGCCGGCCTACGCCGGCAACCTCGACATCATGACCTCCGCCGCGCTGGCTACCGCCGAGCGTATGGCGCAGTCGATGTTGAACGCCTGA
Claims (14)
- 組換え酵母細胞であって、NAD + 依存性アセチル化アセトアルデヒドデヒドロゲナーゼ(EC1.2.1.10)活性を有するタンパク質をコードする1つまたは複数の組換え核酸配列を含み、NADH依存性グリセロール合成に必要な酵素活性を欠損しているか、あるいは、その対応する野生型酵母細胞と比較してNADH依存性グリセロール合成に対する酵素活性が低下している、酵母細胞。
- 前記細胞は、配列番号2もしくは配列番号29で表されるNAD + 依存性アセチル化アセトアルデヒドデヒドロゲナーゼ、または配列番号2もしくは配列番号29の機能的ホモログをコードする1つまたは複数の異種核酸配列を含み、前記ホモログは配列番号2もしくは配列番号29との配列同一性が少なくとも90%である、請求項1に記載の細胞。
- 前記細胞は、前記デヒドロゲナーゼをコードする核酸配列であって、配列番号1、配列番号28もしくは配列番号32に記載の配列または配列番号1、配列番号28もしくは配列番号32の機能的ホモログを含む、核酸配列を含み、前記ホモログは配列番号1、配列番号28もしくは配列番号32との配列同一性が少なくとも90%である、請求項2に記載の細胞。
- 前記細胞はNAD + 依存性グリセロール3−リン酸デヒドロゲナーゼ活性を含まないか、もしくは、NAD + 依存性グリセロール3−リン酸デヒドロゲナーゼ活性がその対応する野生型細胞と比較して低下しており、および/または、前記細胞はグリセロールリン酸ホスファターゼ活性を含まないか、もしくは、グリセロールリン酸ホスファターゼ活性がその対応する野生型細胞と比較して低下している、請求項1〜3のいずれか一項に記載の細胞。
- 前記細胞のゲノムは、GPD1、GPD2、GPP1およびGPP2の群から選択される少なくとも1つの遺伝子に突然変異を含み、前記突然変異はノックアウト突然変異でもよく、前記ノックアウト突然変異は前記細胞に対応する野生型酵母遺伝子に対する、前記遺伝子の少なくとも1つの完全な欠失でもよい、請求項4に記載の細胞。
- 前記細胞はNAD + 依存性グリセロール3−リン酸デヒドロゲナーゼ(EC1.1.1.8)をコードする遺伝子を含まない、請求項4または5に記載の細胞。
- アセチルコエンザイムAシンテターゼ活性(EC6.2.1.1)を有するタンパク質をコードする1つまたは複数の核酸配列、およびNAD + 依存性アルコールデヒドロゲナーゼ活性(EC1.1.1.1)を有するタンパク質をコードする1つまたは複数の核酸配列を含む、請求項1〜6にいずれか一項に記載の細胞。
- 前記細胞はサッカロマイセス科から選択される酵母細胞である、請求項1〜7のいずれか一項に記載の細胞。
- 前記細胞は2009年7月16日にセントラルビューロー・フォー・シュメルカルチャーズに寄託された寄託番号CBS125049のサッカロマイセス・セレビシエ株の細胞である、請求項1〜8のいずれか一項に記載の細胞。
- エタノールの調製のための、請求項1〜9のいずれか一項に記載の細胞の使用。
- アセテートと、グルコース、フルクトース、スクロース、マルトース、キシロース、アラビノース、ガラクトースおよびマンノースの群から選択される発酵性糖質とからエタノールを調製することを含み、前記調製は酵母細胞を用いて嫌気条件下で行われ、前記細胞はアセチルコエンザイムAシンテターゼ活性、およびNAD + 依存性アセチル化アセトアルデヒドデヒドロゲナーゼ活性を含み、前記細胞は、NADH依存性グリセロール合成に必要な酵素活性を欠損している細胞、およびその対応する野生型酵母細胞と比較してNADH依存性グリセロール合成に対する酵素活性が低下している細胞の群から選択される細胞である、エタノールを調製する方法。
- 前記調製は発酵培地内で行われ、前記発酵培地は前記アセテートおよび前記発酵性糖質を含み、前記発酵性糖質に対する前記アセテートのモル比が0.7以下である、請求項11に記載の方法。
- 前記発酵性糖質の少なくとも一部および前記アセテートの少なくとも一部はリグノセルロース、セルロース、ヘミセルロースおよびペクチンの群から選択される多糖を加水分解することにより得られる、請求項11または12に記載の方法。
- リグノセルロース系バイオマスは加水分解され、それにより前記発酵性糖質およびアセテートを得る、請求項13に記載の方法。
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EP09166360A EP2277989A1 (en) | 2009-07-24 | 2009-07-24 | Fermentative glycerol-free ethanol production |
EP09166360.9 | 2009-07-24 | ||
PCT/NL2010/050475 WO2011010923A1 (en) | 2009-07-24 | 2010-07-23 | Fermentative glycerol-free ethanol production |
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JP (1) | JP5836271B2 (ja) |
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AU (1) | AU2010275085A1 (ja) |
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CA (1) | CA2768897C (ja) |
DK (1) | DK2456851T3 (ja) |
EA (1) | EA201270187A1 (ja) |
ES (1) | ES2706653T3 (ja) |
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UA (1) | UA107467C2 (ja) |
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