JP2011518564A5 - - Google Patents

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JP2011518564A5
JP2011518564A5 JP2011506460A JP2011506460A JP2011518564A5 JP 2011518564 A5 JP2011518564 A5 JP 2011518564A5 JP 2011506460 A JP2011506460 A JP 2011506460A JP 2011506460 A JP2011506460 A JP 2011506460A JP 2011518564 A5 JP2011518564 A5 JP 2011518564A5
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host cell
polypeptide
amino acid
isoprene
variant
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JP2011506460A
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JP2011518564A (ja
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Priority claimed from PCT/US2009/041581 external-priority patent/WO2009132220A2/en
Publication of JP2011518564A publication Critical patent/JP2011518564A/ja
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Claims (22)

  1. プロモータに作動可能に結合されるとともに切断されたイソプレンシンターゼ変異体をコードする非相同ポリヌクレオチド配列を備える単離された宿主細胞であって、前記切断されたイソプレンシンターゼ変異体がSEQ ID NO: 120に示す配列を有するポプライソプレンシンターゼにおいて対応する位置の1以上のアミノ酸残基に1以上のアミノ酸置換を有し、前記アミノ酸置換がV10M, F12S, T15A, E18G, V58I, V58F, L70Q, L70R, L70V, L70T, T71P, V79L, E89D, G94A, S119F, F120L, G127R, E175V, T212I, S257A, R262G, A266G, F280L, N297K, F305L, L319M, E323K, A328T, D342E, A359T, K366N, E368D, L374M, S396T, V418S, K438N, H440R, T442A, I449V, A469S, K500R, K505Q, G507S, S509N, F511Y, 及びN532Kから成る群から選択され、前記変異体は置換を有していないイソプレンシンターゼ変異体よりもジメチルアリルピロホスフェート (DMAPP)をイソプレンへより効率的に転換することができる、単離された宿主細胞。
  2. 少なくとも1のアミノ酸置換がL70R置換である、請求項１に記載の宿主細胞。
  3. 前記変異体が、G127R/F511Y, L70Q/G94A/R262G/F305L, F12S/T15A/E18G/N297K, S396T/T442I, V10M/E323K, F120L/A266G, K438N/K500R, V79L/S509N, E175V/S257A/E368D/A469S, T71P/L374M, F280L/H440R, E89D/H440R, V58F/A328T/N532K, S119F/D342E/I449V, 及び K366N/G507Sから成る群から選択される1以上のアミノ酸置換を有する、請求項１に記載の宿主細胞。
  4. 前記ポリヌクレオチド配列がプラスミド中に含まれている、請求項１に記載の宿主細胞。
  5. 前記ポリヌクレオチド配列が宿主細胞中の染色体に組み込まれている、請求項４に記載の宿主細胞。
  6. 前記宿主細胞が、グラム陽性バクテリア細胞、グラム陰性バクテリア細胞、糸状菌類細胞、及びイースト細胞から成る群から選択される、請求項１に記載の方法。
  7. 前記宿主細胞が、エシェリキア(Escherichia)属 (E. coli)、パントエア(Panteoa) 属 (P. シトレア(citrea) )、バシラス(Bacillus) 属(B. スブチリス(subtilis))、ヤロウイア(Yarrowia) 属(Y. リポリティカ(lipolytica) )、及びトリコデルマ(Trichoderma) 属(T. リーゼイ(reesei)) から成る群から選択される、請求項１に記載の方法。
  8. 前記宿主細胞が、グルコース、グリセロール、グリセリン、ジヒドロキシアセトン、酵母エキス、バイオマス、糖蜜、ショ糖、及びオイルからなる群より選択される炭素源を含有する培地を用いて培養される、請求項１に記載の宿主細胞。
  9. 前記宿主細胞がさらに、イソペンテニルジホスフェートイソメラーゼ（IDI）ポリペプチドをコードする非相同または天然の核酸、および／または、l-デオキシ-D-キシルロース-5-ホスフェートシンターゼ（DXS）ポリペプチドをコードする非相同または天然の核酸を備え、任意に天然の1-デオキシ-D-キシルロース5-ホスフェート（DXP）経路と組み合わされている、請求項１に記載の宿主細胞。
  10. 前記宿主細胞がさらに、IDIポリペプチド及びDXSポリペプチドをコードする1以上の核酸を備える、請求項１に記載の宿主細胞。
  11. 宿主細胞がイソプレンシンターゼ変異体、IDIポリペプチド及びDXSポリペプチドをコードする1のベクターを備える、請求項１に記載の宿主細胞。
  12. 宿主細胞がさらに、サッカロミセス・セレビシア(Saccharomyces cerevisia) 由来のメバロン酸（MVA）経路ポリペプチド、及びエンテロコッカス・ファエカリス(Enterococcus faecalis)由来のMVA経路ポリペプチドから成る群から選択されるMVA経路ポリペプチドをコードする非相同核酸を備える、請求項１１に記載の宿主細胞。
  13. 宿主細胞がさらにMVA経路ポリペプチド及びDXSポリペプチドをコードする1以上の核酸を備え、1のベクターがイソプレンシンターゼ変異体、MVA経路ポリペプチド及びDXSポリペプチドをコードする、請求項１に記載の宿主細胞。
  14. 宿主細胞がさらにDXSポリペプチド、IDIポリペプチド、または1以上の残りのDXP経路ポリペプチド、及びMVA経路ポリペプチドをコードする1以上の核酸を備える、請求項１３に記載の宿主細胞。
  15. イソプレンを産出する方法であって、(a)イソプレン産出に適した培養条件下で請求項１に記載の宿主細胞を培養する工程と、(b)イソプレンを産出する工程とが含まれる方法。
  16. さらに、(c)イソプレンを回収する工程が含まれる、請求項１５に記載の方法。
  17. さらに、(d)イソプレンを重合する工程が含まれる、請求項１６に記載の方法。
  18. 前記変異体がSEQ ID NO: 122で定義される配列を有するMEA変異体である、請求項１の宿主細胞。
  19. 前記MEA変異体がL70Rのアミノ酸置換を含む、請求項１８に記載の宿主細胞。
  20. 前記１つ以上のアミノ酸置換がG507Sである、請求項１の宿主細胞
  21. 前記MEA変異体がG507Sのアミノ酸置換を含む、請求項１８に記載の宿主細胞。
  22. さらに、MVA経路ポリペプチドをコードする１つ以上の非相同核酸が含まれている、請求項１の宿主細胞。
JP2011506460A 2008-04-23 2009-04-23 改良された微生物によるイソプレン産出用のイソプレンシンターゼ変異体 Pending JP2011518564A (ja)

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US12533608P 2008-04-23 2008-04-23
US61/125,336 2008-04-23
PCT/US2009/041581 WO2009132220A2 (en) 2008-04-23 2009-04-23 Isoprene synthase variants for improved microbial production of isoprene

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US (3) US8173410B2 (ja)
EP (1) EP2279251A2 (ja)
JP (2) JP2011518564A (ja)
KR (1) KR20110020234A (ja)
CN (1) CN102171335A (ja)
AU (1) AU2009240505B2 (ja)
BR (1) BRPI0911667A2 (ja)
CA (1) CA2722440A1 (ja)
MX (1) MX2010011706A (ja)
MY (1) MY153871A (ja)
RU (2) RU2516343C2 (ja)
SG (2) SG165884A1 (ja)
WO (1) WO2009132220A2 (ja)
ZA (2) ZA201007564B (ja)

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