TW200827448A - Real time monitoring of microbial enzymatic pathways - Google Patents
Real time monitoring of microbial enzymatic pathways Download PDFInfo
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- TW200827448A TW200827448A TW096134723A TW96134723A TW200827448A TW 200827448 A TW200827448 A TW 200827448A TW 096134723 A TW096134723 A TW 096134723A TW 96134723 A TW96134723 A TW 96134723A TW 200827448 A TW200827448 A TW 200827448A
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Description
200827448 九、發明說明: 【先前技術】 電子沿生物系統中酵素途徑之流動受大量因素控制。此 等因素包括(例如)途徑中各點處受質之濃度及酵素轉化產 物之正反饋及負反饋。詳言之,某些目標產物可對細胞具 有毋f生且進而充當其本身產生之負調節劑。舉例而言,對 於諸如乙醇及丁醇之某些醇而言此點確信無疑。 Γ
生物體中醱酵或合成途徑之諸如醇之某些產物具有商業 貝值田由微生物產生時該等化合物藉由培養微生物大量 產生n當細胞自指數生長發展為靜態平衡且當有毒 產物之累積抑制所需目標產物產生時,所需目標產物之產 生速率隨時“改變,首㈣加且隨後降低。 夺。養物維持在目標產生在較長時間内仍維持較高之狀 f中係有益的,由此增加具有商業價值之產物的總體產 【發明内容】 在L樣中,本發明提供包含轉錄調節核苦酸序列之重 組核酸分子,該魅^ $ ^料列與編碼自足發光報導 體的核苷酸序列操作性連 喟筋值、““ 連接其中該轉錄調節核苷酸序列 调即傳遞細胞中醱酵或人 因之^ μ 控之目標產物產生信號的基 因之表現。在本發明之一 Λ ^ Μ ## ^ ^ 也中,轉錄調節核苷酸序列 為、、、田滅轉錄,周郎核苷酸序 盆 調節編碼該途徑中料之其中該轉_節核㈣序列 變與目標產物產生之且報導體表現之改 生之改變正相關。或者,在本發明之另一 124909.doc 200827448 :施’報導體表現的改變與目標產物產生 =在本發明之-實施財,報導體之表現隨著目^ 加或減少。在本發明之另-實施例二: 一、現酼著目標產物產生之減少增加或減少。
C 中,目標產物為終錢。在本發明 一奋/例中,終產物為丙酮、乙醇或丁醇。在本發明 之κ轭例中,目標產物為酸中間物。在本發明之一與 施例中’酸中間物為乙酸、丁酸或乳酸。 Λ 本發月之實施例中,途徑為厭氧性途徑。在本發明 之另一實施例中’途徑為醱酵途徑。在本發明之另—實施 例中,途彳!為選自諸新生、糖酵解、恩峰道夫途^ (Entner-Doudoroff pathway)或非氧化戊醣磷酸途徑之受質 利用远徑。在本發明之另一實施例中,細菌將己醣、戊醣 或胺基酸轉化為酸或醇。 在本發明之一實施例中,基因編碼自乙醯<〇人變化為丁 醇之途徑或彼途徑之分枝中之酵素。在本發明之另一實施 例中’基因編碼丁醇脫氫酶、丁醛脫氫酶、乙醇脫氫酶、 酸駿脫氫酶、乙醯乙酸脫羧酶、丁酸激酶、磷酸丁醯基轉 移酶、磷酸轉乙醯基酶、乙酸激酶、醯基c〇 A轉移酶、乳 酸脫氫酶或丁基CoA轉移酶。在本發明之另一實施例中, 轉錄調節核苷酸序列來自梭菌(Clostridium)、大腸桿菌(£. 、運動醱酵單胞菌(Z· mo⑹以)或釀酒酵母 cerevisiae) 〇 在本發明之一實施例中,自足發光報導體為發光報導 124909.doc 200827448 體。在本發明之另一實施例中,發光報導體包含螢光素 酶。在本發明之另一實施例中,螢光素酶係來自鞘翅目 (Coleoptera)、發光桿菌(Photorhabdus)、5瓜菌(vibrio) ' 高 氏蟲(Gaussia)、雙翅目(Diptera)、海腎(Renilia)。在本發 明之另一實施例中,自足發光報導體包含螢光報導體。在 • 本發明之另一實施例中,螢光報導體包含綠色螢光蛋白 • ("GFP")。在本發明之另一實施例中,自足發光報導體包 含磷光報導體。 c 在一恶樣中’本發明提供包含在已誘導或抑制合成或醱 酵途徑以便影響該途徑目標產物之濃度時指示的自足報導 體構築體之細胞。 在另一態樣中,本發明提供包含包括與編碼自足發光報 導體的核苷酸序列操作性連接之轉錄調節核苷酸序列之重 組核酸分子之細胞,其中該轉錄調節核苷酸序列調節傳遞 細胞中醋酵或合成途徑之目標產物產生信號的基因之表 I; 現。在本發明之一實施例中,細胞為細菌細胞。在本發明 之另一實施例中,細胞為梭菌、大腸桿菌、運動醱酵單胞 • _或酿酒酵母。在本發明之一實施例中,細胞中該途徑之 • 目標產物為終產物。在本發明之另一實施例中,細胞中該 途徑之終產物為丁醇。在本發明之一實施例中,該基因編 碼丁醇脫氫酶、丁醛脫氫酶、乙醇脫氫酶、酸醛脫氫酶、 乙醯乙酸脫緩酶、丁酸激酶、鱗酸丁醢基轉移酶、磷酸轉 乙醢基酶、乙酸激酶、醯基c〇A轉移酶、乳酸脫氫酶或丁 基CoA轉移酶。在本發明之另一實施例中,細胞含有一個 124909.doc 200827448 包含與編碼自足發光報導體之核苷酸序列操作性連接的轉 錄調節核苷酸序列之基因,其中該轉錄調節核苷酸序列調 節丁駿脫氫酶之表現,且其另外含有另一與編碼自足發光 報導體之核苷酸序列操作性連接的轉錄調節核苷酸序列之 基因’其中該轉錄調節核苷酸序列調節丁醇脫氫酶之表 * 現。 - 在一態樣中,本發明提供包含產生商業上有價值之量的 ζ) 合成或酸酵途徑目標產物及發光報導體的細胞之培養物。 在另一態樣中,本發明提供一種方法,其包含:(a)培養 包3包括與編碼自足發光報導體之核苷酸序列操作性連接 的轉錄調節核苷酸序列之重組核酸分子之細胞,其中該轉 錄調節核苷酸序列調節傳遞細胞中醱酵或合成途徑之目標 產物產生信號的基因之表現,由此報導體之光線發射傳遞 目標產物產生信號;(b)量測自培養物中報導體發出的光; 及(C)基於由該發射光傳遞信號之產生而改變培養條件以調 (/ 整目標產物之產生。 在本發明之一實施例中,發光報導體為自足發光報導 , 體。在本發明之另一實施例中,目標產物為終產物。在本 ^ 發明之另一實施例中,目標產物為酸中間物。在本發明之 一實施例中,即時進行發射光之量測。在本發明之另一實 施例中,發射光隨增加目標產物之產生而增加或降低。在 本發明之另一實施例中,發射光隨降低目標產物之產生而 增加或降低。在本發明之一實施例中,在包含窗口之培養 容器中培養細胞且經由該口窗量測光。在本發明之另一實 124909.doc 200827448 施例中,在培養物中包含至少一個可感測發射光且直接或 遠端將信號傳遞至偵侧器的光敏元件之培養容器中培養細 胞。在本發明之一實施例中,在包含使培養液連續流經感 屑J η亥液々IL中的發射光之光敏元件之設備的培養容器中培養 細胞。在本發明之另一實施例中,若目標產生降低,則改 - 變培養條件以復活產生,此類作用包含移除目標產物、添 • 加營養物、稀釋培養物或移除細胞。 0 在一態樣中,本發明提供一種方法,其包含:(a)在產生 目私產物之培養條件下培養重組細胞,其中該細胞包含產 生光基信號之報導體構築體,該光基信號之強度指示目標 產物之產生量;(b)在複數個不同時刻隨時間連續監測培養 物中信號之強度以指示彼等時刻目標產物之產生水準;及 (0回應於目標產物產生之變化改變培養條件以將目標產物 產生設定在所需水準。 在另一態樣中,本發明提供一種由包含以下各物之軟體 1/ 監測且控制之培養物:(a)接收關於細胞或細胞培養物狀態 之 > 訊的代碼;(b)轉定培養條件是否應且應如何改變以優 • 化目標產生之代碼;(c)及傳送對改變培養條件之指示之代 碼。在本發明之一實施例中,該代碼確定細胞或細胞培養 物之狀態。 在一態樣中’本發明提供一種系統,其包含(a)用於培養 細胞之容器;(b)用於偵測容器中細胞培養物之光的光子偵 測器;及(C)回應於偵側器所偵測到之光而改變培養條件之 電腦控制裝置。在本發明之一實施例中,該系統進一步包 124909.doc 200827448 a將光子轉化為電子且將電子轉化為光子之設備。在本發 明之另一實施例中,該系統進一步包含包括至少一個窗口 或至少一個位於培養物中可直接或遠端發送信號至偵側器 之光敏元件的酸酵室,或包含對培養物取樣之連續流侦側 器,由此培養液流經量測光之偵側器/感應器。在本發明 之一實施例中,該系統進一步包含回應於來自電腦指示目 • #產物之產生量的信號而自容器移除目標產物之電腦控制 f 1 裝置。 在另一悲樣中,本發明提供大體上由丁醇構成且含有微 量來自莧菜(amaranth)或甜高粱(sweet s〇rghum)或兩者之 組分且大體上不含石油副產品的組合物。 在一態樣中,本發明提供一種商業方法,其包含在至少 一家生產經生物工程化之製造生物燃料之細胞的第一公司 與-家從事於煉油之第二公司之間建立合資企業;運作該 合貧企業,其中該第一公司提供對產生生物燃料之生物工 ^ ㈣菌株的所有權之許可,該第二公司在合資企業贊助針 對生物燃料產生之研究與開發,且該第二公司購買由合資 , 企業生產之生物燃料。 參考文獻之併入 本#明曰中所提及之所有公開案及專利中請案皆以引用 的方式併入本文中,其引用的程度如各個別公開案或專利 申請案經特定且單獨指示以引人的方式併入般。 【實施方式】 1·引言 124909.doc 200827448 本毛明提供用於提高來自生物體具有商業價值之產品的 、’心產里、尤其來自微生物培養物之產量的方法及物質。藉 由向生物體提供即時指示使得產生所需產物之生化途徑之 狀t的報導體系統來實現該方法。從業者使用此資訊來改 變^養條件’使料時資訊來使該途徑,,保持,,於所需目標 • 產生狀態。此可包括增加產生速率及隨時間維持產生速 率因此例如若報導體系統指示產物產生之速率降低, f)貝“文業者可修改培養條件以藉由(例%)添加受質或營養 物稀釋i口養物、移除細胞、移除有毒產物或改變諸如授 2速率、大氣壓或溫度之環境條件來增加產生。此過程可 藉由電腦運行系統來執行,該系統包括接收及處理關於培 養物狀態之資訊、執行確定培養條件是否需要且如何改變 以改變目標之產生速率且向裝置發送指令之算法的電腦代 碼;及執行該指令以改變培養條件之裝置。 生化途徑之狀態由催化受f之反應朝向或偏離目標產生 ° t酵素的產生水準來反映。可獲得酵素產生之絕對速率及 彼速率之變化的適用資訊。舉例而言,催化前驅物轉化成 •目‘之酵素的產生水準較高指示產物正以較高水準產生。 酵素之產生水準隨時間增加亦指示目標之產生增加。相 反’酵素產生水準較低或酵素產生速率降低分別指示目標 產生水準較低或目標產生速率降低。另一方面,使受質轉 向偏離目標產生之酵素的產生速率較高或產生速率增加指 示目標產生較低或降低。次最佳產生水準提供干涉該過程 以將條件改變成彼料於目標產生增加之條件之根據。 124909.doc 11 2UU827448 本發明之報導體構築體在無需直 下提供量測信號酵素之產生水 置娜酵素活性之情況 中,將調節該系統中信號酵素7表的方法。在此等構築體 與報導基因偶合以便該調節:轉錄調節核苦酸序列 此,報導°周即報道基因之表現。因 報導體之表現量反映該系 本發明之-態樣為㈣μ 就酵素之表現量。 途”持在…ί 件以保持培養物平衡以將
二:==準。其部分包括在進行同時量測啟 改變二速地報導量測值以容許在培養條件顯著 用於培養條件以調節途徑活性。因此,監測且調 即培養條件係即時發生。翱道 ^ &生報導基因經選擇以產生可即時量 7之報導體信號。對於此目的尤其適用之報導體類別為發 之類別。誶言之’本發明涵蓋發光蛋白質、螢光素酶。 先可易於以電子儀器量測且電子信號可易於讀取。 本發明涵蓋制此等方法以監測合成或_途徑之任何 勿之產生然'而’該方法在由微生物產生適用作燃料之 溶劑中存在特定用途。詳言之,本發明涵蓋使用本發明之 方法調節丁醇(―種高價值生物燃料)在丙酮丁醇梭菌(C. _糾吟〜謂)、拜氏梭菌(c〜如”·滅")、腓尼基梭菌 (Pumceum)或醣丁 酸梭卤(c sacchar〇6ufwcum)中之產 生。 2·產生所關注之目標之酵素途徑 2·1·途徑、產物及信號傳遞酵素 本發明適用於監測及調節所關注之化合物由生化途徑通 吊但不排外地在活體内之產生。生化途徑為一種生物化合 124909.doc -12- 200827448 物轉化為另-種生物化合物之酵素或其他反應序列。本發 明尤其涵蓋監測及調節醱酵或合成生化途徑。本發明可; 於原核及真核系統。生化途徑"目標產物"為由生物體或活 體外系統產生之化合物,其中該產物為欲由該途徑產生之 所需化合物。目標產物可為途徑"終產物"。途徑終產物為 自生物體或活體外系統產生之化合物,其中由於不存在將 - 該化合物轉化成另一化合物之可用酵素,因此可能無化合 Γ 物之進一步轉化。舉例而言,在微生物中無進一步酵素轉 化係可能的,此係由於基因組中不存在編碼該酵素之基 因。梭菌中終產物之實例包括溶劑:丙酮、丁醇及乙醇: 目標產物亦可為生化途徑中間物,其中該化合物之進一 步轉化係可能的。在梭菌中,途徑中間物包括,,酸中間物,。 當梭菌處於酸產生培養階段時’酸中間物乙酸及丁酸在培 養基中累積。稍後在溶劑產生階段中,此等酸中間物將^ 再吸收且用以合成溶劑。當在鐵限制及高ρΗ值條件下培養 〇 梭菌時,另一酸中間物乳酸在培養基中累積。 據稱表現提供關於系統中目標產物產生之資訊的酵素傳 • 遞產物產生"信號”且在本文中亦稱為"信號酵素”。在為途 徑終產物之目標產物的情況下,將途徑之中間物轉化成另 一中間物或終產物本身之任何酵素可為信號酵素。一般而 言,與彼等沿途徑進一步上行之酵素相比,為途徑之最終 酵素之酵素為終產物產生之更佳信號酵素。舉例而言,在 丙酮丁醇梭菌中,催化丁經還原成丁醇(步驟R,圖^之脫 氫酶代表適用信號酵素,此係由於其表現直接指示丁醇產 124909.doc -13- 200827448 生速率。因此,來自與此啟動子操作性連接之報導體的信 號之降低指示應改變培養條件以增加丁醇產生速率。
U 於在最後之反應中轉化以產生終產物之中間物極少至無 轉換之途徑中,催化途徑中最後之中間物(距終產物2個步 驟)產生之酵素亦充當優良信號酵素。舉例而言,當丙gjg 丁醇梭菌係處於溶劑產生階段時,由於酵素產生之所有丁 醛將隨後轉化成丁酵,因此丁醛脫氫酶(步驟Q,圖丨)將充 當理想信號酵素。因此,丁醛脫氫酶合成之速率將直接傳 遞丁醇產生速率之信號。 類似地,若目標產物為生化途徑之中間物,則催化中間 物產生之酵素亦為優良信號酵素。例如,在丙酮丁醇梭菌 中,乙酸激酶或丁酸激酶成為理想信號酵素,此係由於其 合成速率將分別指示酸中間物乙酸及丁酸之產生速率(步 驟Η及N,圖1)。若用以製造目標中間物之中間物無轉換, 則催化此等反應之酵素(沿生化途徑上行2個步驟)亦為優良 信號酵素。例如在丙酮丁醇㈣中,鱗酸轉乙酿基酶及麟 I丁醯基轉移酶將分別成分用於監測乙酸及丁酸產生之優 良信號酵素(步驟G及Μ,圖1)。 另外’使中間物再循環以使得此等化合物變得為所關注 之_或合成途徑可用之酵素亦為信號酵素。例如在丙嗣 丁醇梭菌中,乙醯7r 八 C〇A •乙酸/丁酸·· C〇A轉移酶複
口物使乙酸及丁酸分另丨丨A 刀別再循核成乙醯-CoA及丁醯-CoA(# " 圖1)。將乙醯乙醯-CoA · 7 ^ 合 coA.乙酸/丁酸:c〇A轉移酶複 亞早位用作信號酵素將指示酸中間物之再猶環 124909.doc -14- 200827448 速率。信號之出現亦將指示自酸中間物累積之酸產生階段 向酸中間物經微生物再吸收且隨後轉化成溶劑的培養物之 溶劑產生階段之轉移。因此,來自此酵素之信號的增加將 指示對於持續產生目標而言,培養條件不必改變。 相反,使中間物轉向偏離目標途徑之酵素亦可用作信號 - 酵素,此係由於信號之出現及信號強度之任何後續增加指 - *目標產物之產生速率降低,由此指示可能需要採取校正 (] 措施。例如在丙酮丁醇梭菌中,若酸中間物為所需目標, 則來自丁醛脫氫酶(步驟Q,圖”之信號之出現將指示培養 物轉入溶劑產生階段,由此酸中間物之累積由於其經再吸 收以用於;谷劑產生而停止且實際上降低。 2·2·使用分枝點酵素作為信號酵素 使用佔據醱酵途徑上分枝點緊接其上方或下方出現之位 置使文質離開途徑之酵素將不會如進一步沿所需醱酵途徑 向前之酵素般提供培養物狀態之資訊,除非該生物體已經 〇 工程化以取消或下調競爭途徑上酵素之表現。例如在丙: 丁醇梭菌中,若編碼競爭途徑上諸如乙醛脫氫酶之酵素之 基因經下調或缺失,由此容許更多乙醯_C0A可用於丁醇產 生而非乙醇產生,則使用乙醯-C0A乙醯基轉移酶(步驟h 圖1)將提供更多丁醇產生之資訊。 2.3使用信號酵素量測培養物之生活力 報導體可置於代謝途徑之較高位置處,其儘管不傳遞特 定產物產生之信號,但可用於依據碳及電子流提供關於培 養物總體狀態之資訊且因此提供關於生物體健康狀態之資 124909.doc •15· 200827448 訊。例如,在丙酮丁醇播 吁役国中,使用甘油醛-3_磷酸脫氫酶 (/驟A目υ作為^號酵素將不會如使用丁酸途徑更下游 氫酶之酵素(步驟Q,圖〇般提供關於丁醇產生 之簡明資訊。然而,使用 1更用如甘油醛-3-磷酸脫氫酶之酵素將 傳遞培養物之總體代謝產+ > & ^ 代谢旱之^號,其可隨後用作控制向培 養物中饋送介質之速率之方法。類似地,硫解酶(乙酸輔 酶A乙醯基轉移酶;步驟1,圖1)亦可用以提供關於培養物 之總體狀態的資訊。 2·4醱酵途徑 醱酵途徑為在厭氧條件下進行之代謝途徑,其中有機分 子而非氧充當終端電子受體,如好氧條件下所發生之氧化 磷酸化。糖酵解為細菌(丙酮丁醇梭菌及大腸桿菌)及酵母 中廣泛分布之醱酵途徑之實例。在糖酵解期間,細胞將諸 如葡萄糖之單糖轉化成丙酮酸,同時淨產生ΑΤρ及 NADH。至少95%之丙酮酸在再生NAD+(此為持續糖酵解 〇 及ATP產生之根本要求)之短途徑中消耗。此等NAD+再生 系統之廢物或終產物稱為醱酵產物。依賴於生物體及培養 條件’丙酮酸最終轉化為諸如有機酸(甲酸、乙酸、乳 酸、丙酮酸、丁酸、丁二酸、二羧酸、己二酸及胺基酸) 及中性溶劑(乙醇、丁醇、丙酮' 1,3-丙二醇、2,3-丙二 醇、乙酸、丁駿、2,3-丁二醇)之終產物。 TIGR 之綜合微生物資源(Comprehensive Microbial Resource,CMR)基於以下醱酵終產物在其圖譜中列出9種 類型之醱酵途徑:同塑乳酸(乳酸);異型乳酸(乳酸)、乙 124909.doc -16 - 200827448 醇酸、丙酸、混合酸(甲酸及乙酸)、丁二醇、丁酸、胺基 酸及產甲烧作用。本發明之方法可用於上文所述之任一酸 酵途徑。本發明描述之醱酵途徑可天然存在或經工程化。 浴劑為一類由微生物產生之具有特殊商業價值之終產 物。此等溶劑包括(例如)醇(乙醇、丁醇、丙醇、異丙醇、 1,3丙一醇、2,3-丙二醇、2,3-丁二醇、甘油)、酮(丙酮)及
U 醛(乙醛、丁醛)。圖1說明丙酮丁醇梭菌中溶劑丙酮、丁醇 及乙醇之產生。 2.5梭菌中之溶劑產生 在1912至1914年之時期内,當Weizmann尋找可用於製造 丁二浠或異戊二烯且由此提供合成橡膠之發展市場之丁醇 或異戊醇之醱酵來源時,其首先鑑別出細菌丙酮丁醇梭菌 (Jones D· T·及 Woods,D· R. Acetone-butanol fermentation revisited· Microbio· Rev. 50:484·524,1986)。丙酮丁醇梭 菌以約3:6:1之比率同時產生溶劑丙酮、丁醇及乙醇 (ABE) 〇在醱酵期間丙_ 丁醇梭菌亦產生氯及二氧化碳。 已知丁醇產生型梭菌之不同物種且其不同之處主要在於 其產生之溶劑之類型及比率。拜氏梭菌(同義詞丁醇梭菌 (C. 以約與丙酮丁醇梭菌相同之比率產生溶 劑,且在拜氏梭菌之一些菌株中,產生異丙醇替代丙酮 (George, Η. Α·等人。Acetone,isopropanol, and butanol production by Clostridium beijernickii (syn. Clostridium butylicum) and Clostridium. Aurantibutyricum. Appl. Environ· Microbiol· 45:1160-1163,1983)。 丁酸梭菌為經 124909.doc •17- 200827448 由遺傳及生理特性自醣分解工業菌種鑑別出之梭菌屬物種 之提議名(Keis,S·等人。Emended descriptions of Clostridium acetobutylicum,and Clostridium beijerinckii and descriptions of Clostridium saccharoperbutylacetonicum sp. nov. and Clostridium saccharobutylicum sp. nov. Inti. J. System. Evol. Microbio. 51:2095-2103,2001)。除丁 醇外,金黃丁 酸梭菌(C. awra 還產生丙酮與異丙醇(George, Η· A.,同上文)。破傷風桿菌(C iekHomorp/zwm)產生幾乎 等莫耳量之丁醇及乙醇,但未產生其他溶劑(Gottwald,M· 等人。Formation of n-butanol from D-glucose by strains of ’’Clostridium tetanomorphum” group. Appl. Environ. Microbio. 48:573-576, 1984)。 丙酮丁醇梭菌之分批培養物中之溶劑產生經由2個階段 進行。在指數生長期發生之稱為酸產生階段之第一階段, 丙酮丁醇梭菌產生氫、二氧化碳、乙酸及丁酸。培養基中 酸之累積降低pH值。當培養物中丁酸之未解離濃度達到約 9 mM時,過渡至該第二或溶劑產生階段(Hiisemann,Μ· H· W.及 Ε· T. Papoutsakis. Solventogenesis in Clostridium acetobutylicum fermentations related to carboxylic acid and proton concentrations. Biotechnol. Bioeng.3 2:843-852, 1988)。當丙酮丁酵梭菌達到早期生長停滯期時此階段開 始(Davies,R.及 Stephenson M. Studies on the acetone-butyl alcohol fermentation. I. Nutritional and other factors involved in the preparation of active suspensions of 124909.doc -18 - 200827448
Clostridium acetobutylicum. Biochem· J. 35:1320-133 1, 1941)。此處,伴隨自再吸收之酸及持續消耗之碳水化合 物合成丙酮、丁醇及乙醇,培養物之pH值提高。氫及二氧 化碳持續產生。 當丙酮丁醇梭菌生長於分批培養物中時,視稀釋比率及 介質組合物而定可產生不同比例之酸及溶劑(美國專利第 5,063,156號)。乙酸或丙酸之添加並不影響溶劑產生之起 始,但將增加所產生溶劑之總濃度(Hiisemann,Μ· H. W.及 E. T. Papoutsakis. Solventogenesis in Clostridium acetobutylicum fermentations related to carboxylic acid and proton concentrations· Biotechnol. Bioeng.32:843-852,1988) o 藉由以CO氣體喷射培養物亦可改變溶劑產量。此因隨 後不可用作丙酮產生之後續受質之丁酸之吸收導致丁酸產 生途徑逆轉,(Hartmanis,M. G. N.等人。Uptake and activation of acetate and butyrate in Clostridium acetobutylicum. Appl. Microbiol. Biotechnol. 20:66-71,1984) 〇 改變醱酵溫度亦可影響丁醇及溶劑產量。在以三種不同 溶劑產生菌株進行之分批醱酵實驗中,溶劑產量在30°C及 33°C下相當恆定地保持在約31%,但在37°C下降低至23-25%(McCutchan,W. N.,及 Hickey,R. J. The butanol-acetone fermentations. Ind. Fement. 1:347-388,1954)。更 近期研究中以丙酮丁醇梭菌NCIB 852獲得類似結果,其中 儘管當溫度增加時醱酵時間降低,但發現溶劑產量自25°C 下之 29% 降低至 40°C 下之 24%(McNeil Β·及 Kristiansen,B·, 124909.doc •19- 200827448
Effect of temperature upon growth rate and solvent production in batch cultures of Clostridium acetobutylicum. Biotech Lett· 7:499-502,1985)。溶劑產量之降低似乎反映 丙酮產生降低,而丁醇之產量未受影響。 在連續培養物中,丙酮丁醇梭菌可維持在3種不同穩定 代謝狀態。當在中性pH值下於葡萄糖上生長時產生酸,當 在低pH值下於葡萄糖上生長時產生溶劑,且當在中性pH 值高NAD(P)H可用性條件下生長時產生醇(Girbal,L.等 人。Regulation of metabolic shifts in Clostridium acetobutylicum ATCC824, FEMS Microbiol. Rev. 17:287-297,1995) 〇 以生 長限制量之磷酸或硫酸,但添加大量氮及碳源將使酸產生 培養物轉向pH值降低、乙酸及/或丁酸濃度降低之溶劑產 生階段(Bahl,H. Andersch,W,及 Gottschalk G. Continuous production of acetone and butanol by Clostridium acetobutylicum in a two-stage phosphate limited chemostat. Eur. J. Appl. Microbiol. Biotechnol. 15:201-205,1982 ; Bahl,H.,及 Gottschalk G·,Parameters affecting solvent production by Clostridium acetobutylicum in continuous culture,第 215-223 頁。於 Wang D. I· C.及 Scott. C. D. (編),生物技術及生物工程第14次年會(Biotechnology and bioengineering Symposium no. 14),燃料及化學品生物技 術第 6次年會(Sixth Symposium on Biotechnology for Fuels and Chemicals),John Wiley & Sons,Inc.,New York,1984 中)〇 124909.doc -20- 200827448 Ο u 溶劑產生之生理信號誘導生物合成所有催化溶劑產生同 時降低酸產生酵素活性之終端酵素(Andersch,W·,Hubert, Β·,及 Gottschalk,G. Level of enzymes involved in acetate, butyrate,acetone and butanol formation by Clostridium acetobutylicum. Eur. J. Appl. Microbiol. Biotechnol. 18:327-332,1983. Rogers, P. Genetics and biochemistry of Clostridium relevant to development of fermentation processes. Adv. Appl· Microbiol· 31:1-60,1986) o 2.6丙酮丁醇梭菌作為用於溶劑產生選擇及工程化之 模型 丙酮丁醇梭菌能夠經受習知突變方法,諸如使用如曱基 磺酸乙酯(EMS)、N-甲基Ν’-硝基N-亞硝基胍(NG)、ICR 191、亞硝酸、硝基喹啉-Ν-氧化物及三伸乙基三聚氰胺之 烷基化劑,及藉由生長於遞增濃度之丁醇上加以選擇、選 擇對烯丙醇之抗性或選擇纖維素酶、木聚糖酶或澱粉酶活 性。經由該等策略,鑑別出調節突變體,以及具有增加之 溶劑產生、對於較高溶劑濃度之更大耐性、降低之酸產生 及較高澱粉分解活性之突變體(美國專利第4,757,010號; Rogers,P.,及 Palosaari,N. Clostridium acetobutylicum mutants that produce butyraldehyde and altered quantities of solvents. Appl. Env· Microbio. 53:2761-2766,1987) ° 探索諸如丙酮丁醇梭菌之低G+C革蘭氏陽性生物體中同 源基因過度表現及異源性基因表現之研究滯後於如大腸桿 菌之較高G+C生物體之表現的研究,此係由於基於密碼子 124909.doc -21 - 200827448 使用率、胺基酸使用率及驗基含量’低G+C革蘭氏%性生 物體在遺傳學上不同。其因此要求設計新穎載體且定序且 使用適當調節序列(與具有50% GC含量之大腸桿菌相比丙 酮丁醇梭菌具有29% GC含量)。其已實現且低G+C革蘭氏 陽性生物體之研究及使用正急速進行(革蘭氏陽性/陰性穿 , 梭載體,美國專利第6,737,245號;轉座子,美國專利第 . 7,056,728號;細菌嗤菌體,Reid S. J·等人。Transformation of Clostridium acetobutylicum Protoplasts with Bacteriophage DNA. Appl Environ Microbiol. 1983 Jan;45 (1): 305-307) 0 因此,丙酮丁醇梭菌為用於本發明方法之具吸引力之宿主 生物體。 2.7丙酮丁酵梭菌中之丁酵產生 對於藉由丙酮丁醇梭菌產生丁醇,監測丁醇生產力之最 適當酵素為W/z5(CAC3298),一種醛-醇脫氫酶(步驟R,圖 1) ; CAC33 92,一種NADH依賴型丁醇脫氫酶(步驟R,圖 (》 1) ; W/z(CAP0059),一種醇脫氫酶(步驟R,圖1);及 ac//^7(CAP0162),一種醇脫氫酶/乙醛脫氫酶(步驟1〇,圖 . 1)。其屬性更充分描述於下文正信號酵素部分。 2·7·1 丁酸(丁醇產生)途徑 對於丁醇產生,葡萄糖首先藉助於糖酵解轉化為丙酮 酸。酵素甘油酸-3-填酸脫氫酶催化最後一個酵素步驟,甘 油酸-3-構酸向丙酮酸之轉化(步驟a,圖1)。接下來,丙酮 酸藉由酵素丙酮酸-鐵氧還蛋白氧化還原酶轉化成乙 醯-CoA,其中伴隨二氧化碳分子損失(步驟b,圖1)。隨後 124909.doc -22· 200827448 2個乙醯-CoA分子藉由乙醯_c〇A乙醯基轉移酶(Μ",(硫解 酶),CAP0078 ;及CAC2873)縮合成乙醯乙醯_CoA,其中 產生1個游離CoA基團(步驟I,圖1)。藉由3-羥基丁醯-CoA 脫氫酶,CAC2708)將乙醯乙醯-CoA轉化為3-輕基丁 醯<οΑ(β-羥基丁醯-CoA),一種需要將NADH氧化為NAD+ 之過程(步驟J,圖1)。3-羥基丁醯-C〇A隨後藉由巴豆酸酶 (cri,CAC2712)轉化為巴豆醯-CoA,其中伴隨水分子損失 (步驟K,圖1)。巴豆醯- CoA藉由丁醢- CoA脫氫酶, CAC2711)轉化為丁醯-CoA,其中伴隨NADH氧化為 NAD+(步驟L,圖1)。丁酿-CoA經丁酸脫氫酶(ad/ze, CAP0035 3. adhel ^ CAPO 162)及 NADH 還原為丁醛(步驟 Q,圖1)。最終,丁醛經脫氫酶,CAP0035,αd, CAP0162 ^ adh ^ CAP0059 ^ bdhA ^ CAC3299 ^ bdhB ^ CAC3298 及CAC3392)及NADPH還原為丁醇(步驟R,圖1)。 在溶劑產生開始期間,丁酸及乙酸由丙酮丁醇梭菌再吸 收且藉由ci/a/ei/Z?複合物(乙醯乙醢-CoA ··乙酸/ 丁酸:CoA 轉移酶)(步驟S,圖1)分別轉化成丁醯-CoA及乙醯-CoA。 此等中間物可隨後向下流至丁酸途徑。丁酸產生並非以溶 劑產生之起始而終止,此係由於丁醯基·磷酸鹽向丁酸之 轉化為對於丙酮丁醇梭菌合成ATP可用之少數機制之一(步 驟N,圖1)。在溶劑產生期間產生之丁酸經複合物 (乙醯乙醯-CoA ··乙酸/丁酸:CoA轉移酶)再循環恢復為丁 醯-CoA(步驟S,圖1)。 2.7.2提供丁醇產生之正反饋之信號酵素 124909.doc •23- 200827448 可藉由使用在丙酮丁醇梭菌ATCC 824之pSOLl巨型質體 上存在之〜/操縱子的轉錄調節核苷酸序列監測溶劑產生之 起始。μ/操縱子控制3種基因五,CAP0035(醛-醇脫氫 酶),ci/d,CAPO 163(A)及 ci/β,CAPO 164(B)(丁酸-乙醯乙 醯-CoA轉移酶亞單位A與B)之轉錄,隨著溶劑產生之起始 其之表現增加約 10倍。(Feustel,L·等人。Characterization and development of two reporter gene systems for Clostridium acetobutylicum. Appl· Environ. Microbiol· 70:798-803,2004)adc, CAPO 165(乙醯乙酸脫羧酶)亦存在於pSOLl巨型質體上, 隨著溶劑產生之起始,其之轉錄亦增加約10倍(Feustel,L. 等人,同上文)。 使用操縱子之轉錄調節核苷酸序列對於監測後續階 段之溶劑產生可為次最佳的,此係因為五之基因產物丁 醛/丁醇脫氫酶僅在溶劑產生起始期間具有活性。在溶劑 產生之稍後部分期間,另一存在於其自身單順反子操縱子 上之酸-醇脫氫酶接管(Petersen, D. J·等人。Molecular cloning of an alcohol (butanol) dehydrogenase gene cluster from Clostridium acetobutylicum ATCC 824. J. Bacteriol. 173:1831-1834,1991 ; Sauer,U.,及 P. Diirre· Differential induction of genes related to solvent formation during the shift from acidogenesis to solventogenesis in continuous culture of Clostridium acetobutylicum. FEMS Microbiol. Lett· 125:115-120,1995)。因此,操縱子之轉錄調節核 苷酸序列可為與報導基因偶合之更適當序列,此尤其係因 124909.doc •24- 200827448 為咸信6办方編碼之醛-醇脫氫酶負責高丁醇產生(以旧… L·’等人,同上文)。 所關注之用於監測丁醇產生之其他轉錄調節核芽酸序列 包括CAC3392(NADH-依賴型丁醇脫氫酶)及祕,CAp〇〇59(醇 脫氫酶),此係由於此等基因編碼丁醇產生之最後步驟丁 • 駿還原成丁醇中所使用之酵素。 • 另外,由於丁醛為一離開丁醇之酵素步驟且不存在丁醛 〇 再循環機制,因此可使用a^(CAP〇162,醇脫氫酶/乙醛 脫氫酶)之轉錄調節核苷酸序列。 然而,CAC3299(NADH_依賴型丁醇脫氫酶a)為用 於監測丁醇產生之不當選擇,此係由於其在指數生長期期 間表現且培養物之pH值開始下降後即達到最大值(FeusW 等人,同上文)。 2·7·3使用位於分枝點上方或下方之酵素作為信號 酵素 (J 對於丙酮丁醇梭菌中之丁醇產生,若競爭途徑下游之酵 素已缺失或下調,則緊接分枝點上方或下方之酵素可用作 • 信號酵素。例如,若丙酮產生途徑中如乙醯乙酸脫羧酶之 酵素經缺失(步驟Τ,圖1),則可使用分枝點上方緊接分枝 點之酵素乙醯-CoA乙醯基轉移酶(步驟ζ,圖1}監測丁醇產 生。類似地,位於此分枝點下方之酵素3_羥基丁醯弋〇八脫 氫酶、巴豆酸酶及丁醯-CoA脫氫酶(步驟j、K、L,圖丨)亦 可用以監測丁醇產生。 2·7·4提供丁醇產生之負反颔之信號酵素 124909.doc •25· 200827448 丁酸返徑中包含磷酸丁醯基轉移酶(p炀,CAC3〇76)及丁 酸激酶,CAC1660及〜灸,CAC3075)(步驟M&N,圖 1)之酵素活性傳遞丁醯-CoA受質轉向偏離丁酸途徑之信 號。可將此料素中之一者之轉錄調節㈣酸序列與報導 基因偶合以指示丁醇產生可能正在降低。考慮到在溶劑產 • 生期間經由丁酸途徑持續Ατρ產生之需要,使用此等轉錄 ' 調節核苷酸序列可為次最佳的。若干其他競爭途徑可使中 間物偏離丁酸途徑,且編碼各別酵素之基因可代表用於監 測丁醇產生之適用轉錄調節核苷酸序列。乳酸脫氫酶可將 使用乳酸脫氫酶之丙酮酸還原為乳酸(步驟U,圖i)。可能 無需監測丙酮酸轉移,此係由於除在鐵限制及高PH值條件 下外,丙酮丁醇梭菌中之乳酸產生最低(Bahl,H•等人。 Nutritional factor affecting the ratio of solvents produced by Clostridium acetobutylicum. Appl. Environ. Microbiol. 52:169-172,1986)。丙酮酸脫羧酶可將丙酮酸轉化成乙醛 U (步驟U,圖i)。乙醯-CoA可抽離以製造乙酸(步驟, 圖1)。乙醯-CoA亦可抽離以製造乙醇(步驟〇及p,圖丨)。 • 乙醢乙醯-CoA可藉助於乙醯乙醯_c〇A:乙酸/丁酸_c〇A轉 移酶及乙醯乙酸脫羧酶轉化為丙酮(步驟8及丁,圖丨)。 2·7·5使用多個信號酵素 在用於產生丁醇之丙酮丁醇梭菌之分批培養物中,可將 若干使用具有不同光譜發射之螢光素酶之構築體併入各種 途徑中以指示醱酵之進行。使用磷酸轉乙醯基酶, CAC1742,步驟G,圖1)、乙酸激酶(似μ,CAC1743,步 124909.doc -26- 200827448 驟Η,圖1)、磷酸丁醯基轉移酶⑺紿,CAC3076,步驟M, 圖1)或丁酸激酶(CAC1660及,CAC3075,步驟N,圖^ 之調節序列的構築體將傳遞培養物之酸生成階段之起始及 活躍之信號。此構築體之信號強度可隨後用以保持培養物 平衡以獲得所需酸濃度及細胞塊。此構築體之信號強度降 • 低結合利用丁酸途徑中酵素的轉錄調節核苷酸序列之構築 • 體之信號的出現指示向溶劑產生之過渡發生。視需要可調 ζ^ 郎培養條件以延遲此過渡或促進此過渡。培養物處於溶劑 產生階段後,利用丁酸酵素轉錄調節核苷酸序列之構築體 之信號強度可隨後用以監測與控制此階段培養物之最大溶 劑產生。 或者’在丙酮丁醇梭菌用於產生丁醇之分批培養物中, 可利用若干具有相同螢光素酶之構築體。由於螢光素酶之 光譜發射為pH依賴型,在酸性環境中存在紅移,因此此係 可能的(Feustel,L·等人,同上文)。因此,在使用如磷酸丁 〇 酿基轉移酶(户紿,CAC3076,步驟Μ,圖1)之酵素的轉錄 調節核苷酸序列之情況下,若在溶劑產生起始時其轉錄幾 • 乎完全受抑,則螢光素酶信號將會在酸產生階段開始時見 到。由於pH值降低,發射峰將自pH 6.8下之560 mm移至 pH值為約5下之617 nm。若第二構築體使用如在溶劑產生 起始後表現之之基因的轉錄調節核苷酸序列,則由於 尸A構築體所產生之螢光素酶衰減或變為非活性,信號強 度應降低且發射光譜遷移。隨後螢光素酶信號之強度增 加其中隨者溶劑產生進行在較為中性之pH值下可見持續 124909.doc -27- 200827448 遷移返回至發射峰。 在用於產生丁醇之丙酮丁醇梭菌之連續培養物中,可將 若干使用具有不同光譜發射之螢光素酶之構築體併入各種 途徑中以指示醱酵之狀態。利用磷酸轉乙醯基酶, CAC 1742,步驟G圖1)、乙酸激酶(似,CAC 1743,步驟 Η,圖1)、磷酸丁醯基轉移酶〇紿,CAC3 076,步驟Μ,圖 1)或丁 酸激酶,CAC1660 及,CAC3075,步驟Ν, 圖1)之調節序列的構築體之信號的出現將指示培養物之參 數移動偏離彼等維持溶劑產生階段之培養物所需之參數。 隨後可採取措施以調節培養條件以使培養物返回至溶劑產 生階段。由於藉助於丁酸激酶活性合成ATP之連續需要, 故使用磷酸丁醯基轉移酶紿)或丁酸激酶之轉錄調節核苷 酸序列可為次最佳的。 表1 :選擇與酸產生或溶劑產生有關之丙酮丁醇梭菌酵素 字母 基因ID 名稱 定義 G CAC1742 pta 填酸轉乙醯基酶(Phosphotransacetylase)[另一來 源稱為磷酸乙醯基轉移酶(Phosphate acetyltransferase)] Η CAC1743 askA 乙酸激酶 I CAC2873 乙醯-CoA乙醯基轉移酶 I CAP0078 thil 乙醯輔酶A乙醯基轉系酶[硫解酶] J CAC2708 hbd 亦列為Hdb β·羥基丁醯-CoA脫氫酶[亦列為3-羥基丁醯-CoA脫氫酶] Κ CAC2712 crt 巴豆酸酶[3-羥基丁醯-CoA脫水酶] L CAC2711 bed 丁醯-CoA脫氫酶 Μ CAC3076 ptb 磷酸丁醯基轉移酶 Ν CAC1660 丁酸激酶 Ν CAC3075 buk 丁酸激酶,BUK 0 CAP0162 adhel 醇脫氫酶/乙醛脫氫酶[醛脫氫酶ΓΝ乂 D+η 0 CAP0035 adhe 醛-醇脫氫酶[ADHE1] ~' Ρ CAPO 162 adhel 醇脫氫酶/乙醛脫氫酶[醛脫氫醢ΓΝΑΓ)+η 124909.doc -28 - 200827448 P CAP0036 未表徵,枯草芽孢桿菌(B. subtillis)之YgaT基 因之直系同源物 P CAC3298 bdhB NADH依賴型丁醇脫氫酶b [BDHII] P CAC3299 bdhA NADH依賴型丁醇脫氫酶a [BDHI] P CAP0059 adh 醇脫氫酶 Q CAP0162 adhel 醇脫氫酶/乙醛脫氫酶[醛脫氫酶(NAD+)] Q CAP0035 adhe 醛-醇脫氫酶[ADHE1] R CAP0059 adh 醇脫氫酶 R CAC3298 bdhB NADH依賴型丁醇脫氫酶B [BDH II] R CAC3299 bdhA NADH依賴型丁醇脫氫酶a [BDH I] R CAC3392 NADH依賴型丁醇脫氫酶 R CAP0162 adhel 醇脫氫酶/乙醛脫氫酶[酸脫氫酶(NAD+)] R CAP0035 adhe 醛-醇脫氫酶[ADHE1] S CAPO 163(A) ctfa 丁酸-乙醯乙酸CoA轉移酶亞單位A S CAP0164(B) ctfb 丁酸-乙醯乙酸CoA轉移酶亞單位B T CAPO 165 adc 乙醯乙酸脫羧酶 U CAP0025 pdc 丙酮酸脫羧酶 2.8 丁酸途徑 對於藉由丙酮丁醇梭菌產生丁酸,用於監測丁酸生產力 之最適當酵素為乙酸激酶,CAC1743,步驟Η,圖1) 及丁酸激酶CAC1660及〜灸CAC3075,步驟Ν,圖 1)。其屬性更充分描述於下文正信號酵素部分。 丁酸產生途徑如下。葡萄糖首先藉助於糖酵解轉化為丙 酮酸。酵素甘油醛-3-磷酸脫氫酶催化最後之酵素步驟,甘 油醛-3-磷酸向丙酮酸之轉化(步驟A,圖1)。接下來,丙酮 酸藉由酵素丙酮酸-鐵氧還蛋白氧化還原酶轉化成乙醯-CoA,其中伴隨二氧化碳分子損失(步驟B,圖1)。隨後2個 乙醯-CoA分子藉由乙醯-CoA乙醯基轉移酶(ί/π7,(硫解 酶),CAP0078 ;及CAC2873,步驟I,圖1)縮合成乙醯乙 醯輔酶Α,其中產生1個游離CoA基團。乙醯乙醯-CoA藉由 3-羥基丁醯-CoA脫氫酶(ZzW,CAC2708,步驟J,圖1)轉化 124909.doc -29- 200827448 為3-羥基丁醯-CoA(P-羥基丁醯-CoA),一種需要NADH氧 化成NAD+之過程。3-羥基丁醯_CoA隨後藉由巴豆酸酶 (cri,CAC2712,步驟K,圖1)轉化為巴豆醯CoA,其中伴 隨水分子損失。巴豆醯CoA藉由丁醯-CoA脫氫酶, CAC2711,步驟L,圖1)轉化為丁醯-CoA,其中伴隨 NADH氧化為NAD+。丁醯-CoA經磷酸丁醯基轉移酶〇紿, CAC3076,步驟Μ,圖1)磷酸化以產生丁醯基磷酸。最 後,丁醯基磷酸藉由丁酸激酶(CAC1660及kA:,CAC3075, 步驟Ν,圖1)轉化為丁酸,其中產生一分子ΑΤΡ。 2.8.1提供丁酸產生之正反饋的信號酵素 在酸產生期間,編碼分別負責催化乙酸及丁酸產生之最 後步驟的酵素乙酸激酶(ad,步驟Η,圖1)及丁酸激酶 ,步驟Ν,圖1)之基因之表現較高(Durre,Ρ.等人 Transcriptional regulation of solventogenesis in Clostridium acetobutylicum. J. Mol. Microbiol. Biotechnol. 4:295-300, 2002)。因此,其轉錄調節核苷酸序列代表用於建構信號 酵素構築體之理想選擇。此外,由於乙酸激酶及丁酸激酶 之受質乙醯基-磷酸酯及丁醯基-磷酸酯分別並不作為競爭 反應之受質,因此產生此等中間化合物之酵素之轉錄調節 核苷酸序列亦可用以監測酸產生狀態,尤其由於此等酵素 構酸轉乙醯基酶CpM,步驟G,圖1)及鱗酸丁醯基轉移酶 Cp紿,步驟Μ,圖1)亦在酸產生期間高度表現。 2.8.2提供丁酸產生之負反饋的信號酵素 124909.doc -30- 200827448 若干競爭途徑可使中間物偏離丁酸途徑。乳酸脫氫酶可 使用乳酸脫氫酶將丙酮酸還原為乳酸(步驟u,圖1)。丙酮 酸脫羧酶可將丙酮酸轉化成乙醛(步驟u,圖1)。由此,抽 離丙酮酸以形成乙醇。乙醯-CoA可抽離以製造乙酸(步驟G 及Η,圖1)。乙醯_c〇A亦可抽離以製造乙醇(步驟〇及p, 圖1)。乙醯乙醯-CoA可藉助於乙醯乙醯-CoA:乙酸/丁酸-CoA轉移酶及乙醯乙酸脫叛酶而轉化為丙酮(步驟s及τ, 圖丨)。丁醯-C〇A亦可分流以產生丁醇(步驟q及r,圖丨)。 另外,丁酸可藉由乙醯乙醯-CoA ··乙酸/丁酸_c〇A轉移 酶再循環成丁醯-CoA,且自其分流以合成丁醇(步驟s,圖 1) 〇 用於信號酵素構築體之轉錄調節核苷酸序列之2個最適 當來源為酵素乙醯乙醯_CoA:乙酸/ 丁酸-C〇A轉移酶及丁 醛脫氫酶之基因的轉錄調節核苷酸序列。乙醯乙醯·c〇A : CJ 乙酸/ 丁酸_C〇A轉移酶(步驟S,圖1)將再吸收之丁酸轉化 成丁醯-CoA,其可隨後分流至丁酸途徑。丁搭脫氫酶(步 驟R圖丨)為丁酸途控中之第一個酵素且將丁醯-C〇A還原 為丁醛,其為丁醇之直接前驅物。轉錄調節核苷酸序列之 一替代來源為將丁醯-CoA還原為丁醛(自丁醇中單步移除 =不能偏離至競爭用途或再循環之受質)之丁醢心八脫氮 酶(步驟Q,圖1)之轉錄調節核苷酸序列。 2.9乙醇產生途徑 124909.doc -31- 200827448 在另-實施例中’從業者使用本發明之方法來調節乙醇 之產生。對於乙醇產生,葡萄糖首先藉助於糖酵解而轉化 為丙酮酸。酵素甘油駿-3_磷酸脫氣酶催化最後一個酵素步 驟,甘油醛-3-磷酸向丙酮酸之轉化(圖i步驟A)。由此,由 於梭菌為少數具有丙酮酸脫羧基化之細菌種類中之一者, • 因此丙酮酸可流經2個獨立之乙醇產生途徑。在一途徑 , 中,丙酮酸藉由酵素丙酮酸-鐵氧還蛋白氧化還原酶轉化 f) 成乙醯-C〇A,其中伴隨二氧化碳分子損失(步驟B,圖丨)。 乙醯-CoA隨後藉由乙醛脫氫酶(步驟〇,圖1)&nadh轉化 為乙醛。最後,乙醛經脫氫酶,CAC3298 ; , CAC3299 ;及可能地,CAP0162 及 CAP0035,步驟 P,圖1)及NADH還原為乙醇。在另一途徑中,丙酮酸經丙 酮酸脫羧酶(步驟U,圖1)脫羧基化以形成乙醛,其隨後經 脫氫酶,CAC3298 ; ,CAC3299 ;及可能地 ’ CAP0162 及 CAP0035,步驟 p,圖 i)及 nadh還原 (j 為乙醇。 2.9.1提供乙醇產生之正反餚的信號酵素 . 可藉由設計具有與報導基因偶合的脫氫酶之轉錄調節核 苷酸序列之構築體來直接監測乙醇產生。即使此等酵素為 乙醇產生途徑之最終酵素且對於中間物乙醛而言不存在競 爭用途,但此方法可發出超出實際乙醇產生比例之信號, 此係由於脫氫酶亦用於丁酸途徑以將丁醛還原為丁醇。或 者’可經由使用2個各使用其各別轉錄調節核苷酸序列之 構築體藉由同時監測丙酮酸脫叛酶及乙駿活性較佳測定乙 124909.doc -32- 200827448 醇產生。 2·9·2提供乙酵產生之負反饋的信號酵素 若干競爭途徑可使中間物偏離乙醇途徑。乳酸脫氫酶可 使用乳酸脫氫酶將丙酮酸還原為乳酸(步驟U,圖1)。乙 醯-CoA可抽離以製造乙酸(步驟g及Η,圖1)。乙醯<〇八亦 可藉由乙醯-Co A乙醯基轉移酶轉化為乙醯乙醯-C〇 a(步驟 k i,圖丨)。由此乙醢-CoA可轉化為丙酮(步驟s及τ,圖i)、
() 丁酸(步驟 J、K、L、Μ&Ν,圖 1)或丁醇(步驟J、K、L、Q 及R,圖1)。“號酵素構築體可經設計使用碟酸轉乙酿基 酶(步驟G,圖1)及乙醯-CoA乙醯基轉移酶(步驟j,圖丨)之 轉錄調節核苷酸序列以監測受質乙醯_(:〇八自乙醇途徑之偏 離。可能無需監測丙酮酸轉移,除非培養條件具有鐵限制 及局pH值。 2.10丙酮途徑 在另一實施例中,從業者使用本發明之方法來調節丙鲖 1 之產生。對於丙酮產生,葡萄糖首先藉助於糖酵解轉化為 丙酮酸。酵素甘油醛_3_磷酸脫氫酶催化最後一個酵素步 驟,甘油醛-3-磷酸向丙酮酸之轉化(步驟a,圖丨)。接下 來,丙酮酸藉由酵素丙酮酸-鐵氧還蛋白氧化還原酶轉化 成乙醯-CoA,其中伴隨二氧化碳分子損失(步驟B,圖丨)。 隨後2個乙醯_CoA分子藉由乙醯_c〇A乙醯基轉移酶(^以, (硫解酶),CAP0078 ;及CAC2873)縮合成乙醯乙醯_輔酶 A,其中產生1個游離CoA基團(步驟j,圖1}。藉由乙醯乙 醯-CoA :乙酸/丁酸coA轉移酶將乙醯乙醯_c〇a轉化為乙 124909.doc -33- 200827448 醯乙酸(步驟S,圖1)。藉由乙醯乙酸脫羧酶將乙醯乙酸轉 化為丙酮,其中產生一分子二氧化碳(步驟τ,圖1)。 2.10.1提供丙酮產生之正反饋的信號酵素 在pSOLl巨型質體上具有,CAP0165(乙醯乙酸脫羧 酶’步驟T,圖1),其在與操縱子相反之方向上自其自 身啟動子轉錄。在溶劑產生階段起始時發生乙醯乙酸脫羧 酶之轉錄’且酵素活性在整個溶劑產生階段穩定存在 (Gerischer,U. ’ 及 Durre,P. mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis· J. Bact· 174:426-433,1992)。另外,乙醯 乙酸脫羧酶為丙酮途徑之最後一個酵素。因此,使用 之轉錄調節核苷酸序列對於監測丙酮產生而言係理想的。 對於丙酮之分批培養物產生,由於酵素乙醯乙醯_c〇A:乙 酸/丁酸:CoA轉移酶之亞單位將在酸產生階段期間產生之 終產物乙酸轉化為乙醯-CoA,其中其可充當乙醯-CoA乙 醯基轉移酶之受質以產生乙醯乙醯-CoA,因此酵素乙醯乙 醯-CoA :乙酸/丁酸:c〇A轉移酶之亞單位可適用。乙醯 乙醯-CoA :乙酸/ 丁酸:c〇A轉移酶可隨後將乙醯乙醯_ CoA轉化成乙醯乙酸,其為丙酮合成途徑之最後一個中間 物(步驟S、I、S、T,圖1)。類似地,在連續溶劑產生培 養物中乙醯乙醯-CoA :乙酸/丁酸:c〇A轉移酶活性可提 供關於乙醯乙酸產生速率之資訊,且因此間接提供關於丙 _產生速率之資訊。 2·1〇·2提供丙酮產生之負反饋的信號酵素 124909.doc -34- 200827448 若干競爭途徑可使中間物偏離丙酮途徑。乳酸脫氫酶可 使用乳酸脫氫酶將丙酮酸還原為乳酸(步驟U,圖1)。然而 除在鐵限制及高pH值條件下外此為最低的。乙醯_〇〇八可 抽離以製造乙酸(步驟G及H,圖1)。乙醯-CoA亦可抽離以 製造乙醇(步驟〇及P,圖1)。藉由乙醯乙酸脫羧酶轉化為 丙酮之受質乙醯乙醯-C〇A亦可藉由乙醯乙醯_c〇a··乙酸/ 丁酸:C〇A轉移酶轉化為丁醯-CoA,其為產生丁酸或丁醇 f) 之中間物(步驟1、K、L,因而分別為Μ及N或Q及R之分枝 ,、、i 圖1)。自乙酿乙酿- CoA起始之丁酸途徑中之其他酵素 的活性可適用,因為其將提供關於對 丙酬I產生不可用之中 間物及目標產生之資訊(步驟J、K、L、M、N、卩及尺,圖 1)與經過分枝點之將提供關於培養物之特定醱酵階段之 資訊的酵素(步驟Μ、Ν' Q及R,_相比,使用丁酸途徑 中分枝點前之酵素3_羥基丁醯_c〇A、巴豆酸酶及丁醯_c〇a 脫氫酶(步驟J、K及L,圖1)將提供在所有時刻(酸產生及 (j 溶劑產生)關於受質轉移之資訊。因此,較佳使用3-羥基丁 醯CoA、巴丑酸酶及丁醯-C〇A脫氫酶之轉錄調節核苷酸 • 序列。應記住丁酸可藉由乙醯乙醯-CoA :乙酸/ 丁酸: C〇A轉移酶再循環成乙醯乙酸,其可充當丙嗣產生之受 貝口此,基於羥基丁醯-CoA、巴豆酸酶及丁醯-(^〇八脫 氳酶M S文丁醯基轉移酶及丁酸激酶之轉錄調節核苷酸序 列之信號酵素可提供過高信號(步驟j、K、L、m&n,圖 1) 〇 2·11其他微生物中之溶劑產生 124909.doc -35- 200827448 如運動醱酵單胞菌及釀酒酵母之乙醇產生生物體使一分 子之葡萄糖醱酵成兩分子之乙醇及兩分子之co2。需要兩 個酵素步驟。丙酮酸脫羧酶首先將丙酮酸裂解為乙醛及二 氧化碳。隨後乙醇脫氫酶藉由將NADH之氫當量轉移至乙 駿使NAD+再生,由此產生乙醇。運動醱酵單胞菌為植物 • 體液及蜂蜜中常見之細菌,其依賴於恩納杜道夫途徑作為 • 醋酵途徑。每個葡萄糖分子此較短途徑僅得到一個ATP。 運動酵單胞菌具有兩個醇脫氫酶同工酵素,其在酸酵期 間催化乙醛還原為乙醇,伴隨有NADH氧化為NAD+。 釀酒酵母產生乙醇為眾所周知且對於每個葡萄糖分子產 生淨產生兩分子ATP。運動醱酵單胞菌與釀酒酵母均充當 用於在其他微生物中產生乙醇之異源基因之來源。
2.11.1大腸桿菌中之溶劑產生 細菌大腸桿菌並非天然具有酵素丙酮酸脫羧酶,且因此 當其厭氧性生長時,產生最小量之乙醇連同一起產生混合 Ci 酸(25 mM葡萄糖上之醱酵生長得到6.5 mM乙醇、8.2 mM 乙酸、6.5 mM乳酸、0.5 mM丁二酸及約1 mM甲酸,留下 10.4 mM殘餘葡萄糖)Brau及 Sahm (1986a) Arch. Microbiol. 144:296-301, (1986b) Arch. Microbiol. 146:105-110。當將 自運動醋酵單胞菌選殖之編碼醇脫氫酶π(α^5)及丙酮酸 脫竣酶⑺心)之基因引入且表現於大腸桿菌中時,25瓜以葡 萄糖之初始濃度完全轉化得到高達41.5 mM乙醇,而幾乎 未形成酸。其他研究人員已解釋此研究(c〇nway等人 (1987a) J. BaCteriol· 169:2591-2597; Neale 等人(1987) 124909.doc -36· 200827448
Nucleic Acids Res. 15:1752-1761 ; Ingram及 Conway [1988] Appl. Environ. Microbiol. 54:397-404 ; Ingram 等人(1987) Appl· Environ. Microbiol. 53:2420-2425) 〇 厭氧及好氧條件 下乙醇產生之程度與運動醱酵單胞菌乙醇產生基因之表現 量直接相關。因此,使用適當轉錄調節核苷酸序列,可設 計對應於乙醇產生異源構築體之信號酵素構築體,其隨後 可用於保持培養物平衡以提高乙醇產生之比率及量。 該技術不侷限於大腸桿菌,因為後續研究藉由使用兩種 其他腸細菌菊歐氏桿菌及植生克雷 伯菌(尺增加乙醇自己St、戊酶及糖混 合物之產量來證明此方法之一般適用性(Tolan &Finn. Appl. Environ. Microbiol. 53:2033-2038, 2039-2044,1987 ; Beall 等人,1993 ; Ingram及 Conway,1988 ; Wood及 Ingram, 1992)〇 2.12合成途徑 術語合成途徑包括產生亦稱為天然產物之次級代謝產物 的天然、先前已存在之途徑,該等天然產物諸如脂族、芳 族及雜芳族有機酸、生物鹼、類萜、聚酮化合物、酚、環 烯醚萜、類固醇、皂苷、肽、香精油、樹脂及香脂。另 外,合成途徑亦包括經由遺傳工程、細胞融合、接合或其 他方式完全或部分引入生物體中之途徑。例如經由使用編 碼來自運動醱酵單胞菌之乙醇脫氫酶II及丙酮酸脫羧酶之 異源蛋白質之質體將乙醇產生途徑引入大腸桿菌中。或經 由表現來源於植物黃花蒿α心wa L.)之與釀酒酵 124909.doc -37- 200827448 母之甲羥戊酸類異戊二烯途徑偶合的合成紫穗槐-4,11-二 烯(amorpha-4,l Ι-diene)合成酶基因工程化大腸桿菌之類萜 途徑(Martin,V. J. JL 等人 Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature Biotech. 21:796-802, 2003 ;美國專利申請公開案 2004/0005678 Al)。 3.報導鱧構築體 3.1製備用作信號酵素之螢光素酶表現載體之方法
Ο 除非另有所述,否則本發明之實踐將採用此項技術能力 内之習知化學、生物化學、分子生物學、免疫學及藥理學 方法。該等技術於文獻中充分說明。參見例如Remington's Pharmaceutical Sciences,第十八版(Easton,Pa.: Mack Publishing Company, 1990) ; Methods In Enzymology (S. Colowick 及 N. Kaplan,編,Academic Press, Inc·);及 Handbook of Experimental Immunology,第 I-IV卷(D. M. Weir及 C. C. Blackwell,編,1986,Blackwell Scientific Publications) ; Ausubel, F. M. 5 等人,Current Protocols in
Molecular Biology,John Wiley and Sons,Inc·,Media,Pa. (1995.) ; Sambrook,J.,等人,Molecular Cloning: A Laboratory Manual,第三版,(Cold Spring Harbor Laboratory (Cold Spring Harbor,Ν·Υ·) (2001)) 〇 根據本發明,將包含具有所關注基因之轉錄調節核苷酸 序列的表現卡匣之信號酵素構築體插入適當載體中,其隨 後用以轉化預定宿主,該核苷酸序列與報導基因及相關調 節序列及連接子操作性連接。 124909.doc -38 - 200827448 在-態樣中’本發明提供適用於監測生物體中生物化學 途:之目標產生之報導體構築體。在某些實施例中,該等 構築體用以在微生物培養期間即時提供該資訊。該等構築 體包括包含轉錄調節核苦酸序列之重組核酸分子,例如與 編碼發光報導體之基因操作性連接的啟動子,其中該轉錄 . 冑節核㈣序列亦調節其表現報導目標之產生的酵素之表 . 現。 (、 本發明尤其涵蓋該系統之兩個實施例。在第一實施例 中,將報導體構築體與宿主基因及其轉錄調節核普酸序列 分隔開。因A生物冑含有平行調節構築冑··一者控制酵素 之表現且複本控制報導體之表現。由於轉錄調節核苷酸序 列相同,因此報導體之表現量反映信號酵素在該系統中之 表現量。術語’’轉錄調節核苷酸序列,,涵蓋所有負責控制基 因表現之核苷酸序列。其包括啟動子及增強子序列,及其 中基因阻抑蛋白質及基因活化蛋白質結合之序列。其進一 U 步包括初級應答蛋白質結合以活化二級應答蛋白質之轉錄 的區域。此外術語"轉錄調節核苷酸序列,,涵蓋保持轉錄調 . 節活性的經修飾核苷酸序列。另外,術語,,轉錄調節核苷 酸序列”包括來自其他生物體之同源轉錄調節核苷酸序 列,以便若該同源序列取代天然序列,則其將以類似方式 起作用。 在第二實施例中,將報導體與天然轉錄調節核苷酸序列 偶合以便編碼信號酵素之基因及編碼報導體之基因處於同 一核酸區段控制下。 124909.doc -39- 200827448 3.2轉錄調節核苷酸序列 信號酵素之轉錄調節核苷酸序列必須與預定宿主相容。 根據本發明,最佳轉錄調節核苷酸序列為彼等來自宿主生 物體之核苷酸序列。對於監測丙酮丁醇梭菌之酸產生及溶 劑產生基因之表現,可易於獲得此等基因之大多數轉錄調 節核苷酸序列。參見表2。經由分析轉錄調節核苷酸序 列,可設計適當引子以便所關注之轉錄調節核苷酸序列可 藉由使用聚合酶鏈反應(PCR)技術自染色體組DNA選殖 出。可經由使用利用丙酮丁醇梭菌ATCC 824之序列基因組 之計算方法來鑑別表2中未列舉之基因之轉錄調節序列 (Paredes,C. J·等人。Transcriptional organization of the
Clostridium acetobutylicum genome, Nuc. Acids Res. 32:1973-1981)。或者,由於已知酸產生及溶劑產生基因之 序列且可經由諸如TIGR或國家生物技術資訊中心(National Center for Biotechnology Information)(NCBI,www.ncbi.nlm.nih.gov) 之基於網際網路之服務獲得,可使用來源於所關注之基因 序列之引子經由諸如cDN A引子延伸反應之標準分子生物 學技術偶合反轉錄來鑑別轉錄調節核苷酸序列。 表2:丙酮丁醇梭菌之選擇基因之轉錄調節核苷酸序列的來源 基因ID 方向 註釋 IR 長度 說明 參考文獻 CAC1742 + pta 264 磷酸轉乙醯基酶 Boynton. Appl. Environ. Microbiol. 1996 CAC1743 + askA 11 乙酸激酶 Boynton. Appl. Environ. Microbiol. 1996 CAC2708 - hbd 104 NAD依賴型β-經基 丁醢-CoA脫氫酶 Boynton. J. Bacteriol. 1996 CAC2711 bed 13 丁醯-CoA脫氫酶 Boynton. J. Bacteriol. 199 124909.doc -40- 200827448 CAC2712 crt 175 巴豆酸酶(3-羥基丁 醯-CoA脫水酶) Boynton. J. Bacteriol. 199 CAC2873 326 乙醯-CoA乙醯基轉 移酶 Stim-Hemdon. Gene. 1995 CAC3075 buk 27 丁酸激酶BUK Walter. Gene. 1993 CAC3076 ptb 108 磷酸丁醯基轉移酶 Walter. Gene. 1993 CAC3298 麵 bdhB 276 NADH依賴型丁醇脫 氫酶 B(BDH II) Walter. J. Bacteriol. 1992 CAC3299 - bdhA 147 NADH依賴型丁醇脫 氫酶 A(BDH I) Walter. J. Bacteriol. 1992 CAP0035 adhe 476 醛-醇脫氫酶ADHE1 Fontaine. J. Bacteriol· 2002 CAP0078 - thil 105 乙酿輔酶A乙醢基轉 移酶(硫解酶) Winzer. J. Mol. Microbiol. Biotechnol·,2000 CAP0162 + adhel 666 醛脫氫酶(NAD+) Nair. J. Bacteriol. 1994 Fischer. J. Bacteriol. 175:6959-6969,1993 CAP0163 + ctfa 63 丁酸·乙醯乙酸CoA 轉移酶亞單元A Nair. J. Bacteriol. 1994 Fischer. J. Bacteriol. 175:6959-6969,1993 CAP0164 + ctfb 4 丁酸·乙醯乙酸CoA 轉移酶亞單元A Nair. J. Bacteriol. 1994 Fischer. J. Bacteriol. 175:6959-6969,1993 CAP0165 - adc 232 乙醯乙酸脫羧酶 Gerischer. J· Bacteriol· 172, 1990 Gerischer. J. Bacteriol. 174:426-433,1992
a) 基因ID · ΤIG R之糸統基因代碼。 b) 方向:編碼鏈 c) 註解:根據TIGR之基因符號。 d) IR長度:上游基因間區域之長度。 e) 說明:基因功能之說明。 3.3產生光之分子 視應用而定適用於實施本發明之產生光之分子可採用多 種形式中之任一種。其共有以下特徵:其為發光的,亦即 其在紫外(UV)、可見及/或紅外(IR)中由於電子激發態至較 低能態、通常基態之躍遷而自原子或分子發射電磁輻射。 124909.doc -41 200827448 產生光之分子之實例包括諸如螢光分子之光致發光分子、 化學發光化合物、填光化合物及生物發光分子。 在某些實施例中,發光報導體為自足的。如本文中所 用,若發光報導體在不添加外源有機受質之情況下產生 光,則其為π自足的’’。因此,例如螢光報導體為”自足的n。 產生微生物螢光素酶之lux操縱子由於含有產生必要受質 之酵素亦產生自足報導體。相反,產生哺乳動物螢光素酶 之luc基因需要添加諸如螢光素且通常ATP之受質以生物發 光。因此其為非自足的。自足報導體在本發明之方法中提 供特定優勢,此係由於添加外源受質為昂貴的且在監測及 調節培養物狀態中引入低效率。 3.3.1生物發光蛋白質 生物發光分子不同於螢光分子之處在於其無需輸入輻射 能量來發光。相反,生物發光分子利用諸如ATP之化學能 來產生光。與螢光分子相比,生物發光分子之優勢為在信 號中幾乎不存在背景。僅偵側到之光為由外源性生物發光 分子產生之光。相反,用以激發螢光分子之光通常產生干 擾信號量測之背景螢光。 已知數種類型之生物發光分子。其包括螢光素酶家族 (de Wet, J. R,等人,Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7:725-737, 1987) 及水母發光蛋白(aequorin)家族(Prasher,等人.Cloning and expression of the cDNA coding for Aequorin, a bioluminescent calcium-binding protein. Biochem Biophys Res Commun 126: 124909.doc -42- 200827448 1259-1268,1985)。已在多種原核及真核生物中鑑別出螢光 素酶家族之成員。原核螢光素酶由稱為lux操縱子 (luxCDABE)之5基因複合物之兩個亞單元(luxAB)編碼。其 餘三個基因包含luxCDE亞單元且編碼負責生物合成用於發 光反應由螢光素酶所用的醛受質之脂肪酸還原酶。 真核螢光素酶("luc”)通常由單個基因編碼(de Wet,J· R.,等人,Proc· Natl. Acad. Sci. U.S.A. 82:7870-7873, 1985 ; de Wet,J. R,等人,Mol. Cell· Biol· 7:725-737, 1987)。含有螢光素酶系統之例示性真核生物體為北美洲 營火義(Photinus pyralis)。已廣泛地研究蠻火A榮先青酶 且其廣泛用於ATP檢定中。已選殖且表現編碼另一叩頭蟲 物種向光叩頭蟲之螢光素酶 (lucOR)的 cDNA(Wood,等人.Complementary DNA coding click beetle luciferases can elicit bioluminescence of different colors. Science 244:700-702,1989)。此甲蟲之特 殊之處在於物種之不同成員發射不同顏色之生物發光。分 離出彼此具有95-99%同源性之4類純系。其在546 nm(綠 光)、560 nm(黃綠光)、578 nm(黃光)及593 nm(橙光)下發 光。 螢光素酶以及水母發光蛋白樣分子需要能量來源,諸如 ATP、NAD(P)H、氧化受質,諸如螢光素(長鏈脂肪醛)或 水母三嗓(coelentrizine)及氧。在lux操縱子的情況下,編 碼合成醛受質之酵素之基因與螢光素酶同時表現。在經 lux信號酵素構築體轉化之細胞中,氧為生物發光之唯一 124909.doc -43- 200827448 外在要求。該等構築體在除氧外無需添加外源性化合物的 意義上為自足的。相反,以luc信號酵素轉化之細胞將需 要添加外源性受質且通常需要添加ATP以發光。 編碼獲自原先稱為發光致病桿菌(Xenorhabdus luninescens)之土壤細菌發光桿菌(Photorhabdus luminescens)之lux操縱子的質體構築體(Frackman等人, Cloning, organization, and expression of the bioluminescence genes of Xenorhabdus luninescens. J. Bacteriol. 172”5767-5773,1990)在37°C下賦予經轉化之大腸桿菌最佳生物發光 (Xi,等人.Cloning and nucleotide sequences of lux genes and characterization of Luciferase of Xenorhabdus luninescens from a human wound. J. Bacter. 173:1399-1405,1991) 〇 該 序列可獲自GenBank,其寄存編號為M90092。與來自發光 桿菌(P. luminescens)之螢光素酶相對比,自發光原核及真 核生物體分離之其他螢光素酶在較低溫度下具有最佳生物 發光(Campbell,A. K. Chemiluminescence,Principles and
Applications in Biology and Medicine. Ellis Horwood, Chichester,UK. 1988) o
為促進發光桿菌lux在丙酮丁醇梭菌中之表現,將野生 型lux操縱子(luxCDABE)之核苷酸序列再次工程化以具有 69%之AT含量。此伴隨有利用遺傳密碼子之簡幷性以便在 簡幷位置包括C或G之密碼子可經編碼相同胺基酸但在簡 幷位置具有A或T之密碼子置換。丙酮丁醇梭菌優化之lux 操縱子之個別基因的序列以及其相應胺基酸序列於SEQ ID 124909.doc -44- 200827448 NO· 1-10中給出。可類似修飾其他發光蛋白質以便其經優 化以在丙酮丁醇梭菌及其他具有6〇_8〇。/。範圍内之高Μ含 量之生物體中表現。 已鑑別出多種其他螢光素酶編碼基因,其包括(但不限 於)下列:Sherf,Β· An〇d,κ· ν·,美國專利第 5,670,356 號;Kazami,;·等人,美國專利第 5,_,123號; Zenno, S·等人,美國專利第 5,618,722 號;w〇〇d, κ· V,美 國專利第5,650,289號;W(K)d,κ· ν’美國專利第5,64l,64i 號;Kajiyama Ν·及Nakano, ε·,美國專利第 5,229,285號; Cormier,M. J·及 L〇renz,W· w•,美國專利第 5,292,658 號; Connier·,M· J·及 L〇renz,W· w·,美國專利第 5,418,155 號; de Wet,J. R·等人,Molec· Cell· Biol. 7:725-737,1987 ;
Tatsumi,Η· Ν·等人,Biochim· Bi〇phys AcU 1131:16i_ 165,1992,及 Wood,Κ· V·等人,Science 244:700-702, 1989,其全部以引入的方式併入本文中。該等螢光素酶編 碼基因可經本文中所描述之方法修飾以產生適用於(例如) 革蘭氏陽性微生物之多肽序列及/或表現卡匣。 3·3·2螢光蛋白質 螢光為物質自單個電子激發態之發光,移除輻射源後其 持續時間極短。發射螢光之波長比激發照射之波長長(斯 托克定律(Stokes1 Law)),此係因為部分激發光經螢光分子 轉化為熱。 來自激發照射源之背景螢光及漫射光可使螢光分子之使 用變得複雜。可能需要連同使用激發濾光器一起屏蔽照射 124909.doc -45- 200827448 源以阻斷大多數具有與螢光部分所發射光子類似波長之光 子。類似地,可連同"ί貞測器使用二次遽光器(barrier filter) 以篩選出大多數具有螢光分子所發射之彼等其他波長的光 子。或者,可使用產生接近適當激發波長而不接近螢光發 射波長之高強度光之雷射以激發螢光部分。 ♦ 螢光分子包括小分子,諸如螢光素以及螢光蛋白質,諸 . 如綠色螢光蛋白(GFP)(Chalfie等人,Morin等人)、2,4-二 氧四氫蝶啶(lumazine)及黃色螢光蛋白(YFP)(〇’Kane,等人, Daubner,等人)。在性質上,通常發現螢光蛋白質與螢光 素酶結合且藉由接受來自酵素結合之激發態氧化螢光素的 能量在此等生物體中充當最終生物發光發射體(Ward等人 (1979) J· Biol. Chem. 254:781-788 ; Ward 等人(1978) Photochem. Photobiol. 27:389-396 ; Ward 等人(1982) Biochemistry 21:4535-4540)。其可用於本發明之系統中以 增加偵測器對生物發光產生系統之靈敏度且亦以使發射光 Q 之波長遷移至更適當波長以用於偵測目的。 最佳表徵之GFP為彼拿自水母物種多管水母 (Aequorea)、尤其維多利亞水母(Aequorea victoria)及福氏 多管水母(Aequorea forskalea)及海腎(Renilla reniformis)分 離者(Ward 等人 Biochemistry 21:4535-4540; 1982 ;
Prendergast 等人 Biochemistry 17:3448-3453,1978)。在維 多利亞水母中,添加鈣後GFP吸收水母發光蛋白產生之光 且發射綠色螢光,其發射波長為約510 nm(Ward等人 Photochem. Photobiol. Rev 4:1 -57,1979) 〇 124909.doc -46· 200827448 多管水母GFP編碼其蛋白質序列内部固有之發色團,免 除外部受質或辅因子之需要且使得能夠遺傳編碼強螢光 (Ormo,Μ·等人 Crystal structure of the Aequorea victoria green fluorescent protein· Science 273:1392-1395,1996)。 發色團定位於桶狀結構中心且完全屏蔽避免曝露於大量溶 劑。突變誘發研究已產生具有新穎顏色、改良之螢光及其 他生物化學特性之GFP變異體。 已分離出編碼維多利亞水母GFP同型之DNA,且已測定 其核苷酸序列(Prasher (1992) Gene 111:229-233)。重組表 現之維多利亞水母GFP在包括細菌(例如參見Chalfie等人 (1994) Science 263:802-805 ; Miller 等人(1997) Gene 191:149-153)、酵母及真菌(Fey 等人(1995) Gene 165:127-130 ; Straight 等人(1996) Curr· Biol· 6:1599-1608 ; Cormack 等人(1997) Microbiology 143:303-3 1 1)之多種生物 體中保持其活體内發螢光能力。 關於維多利亞水母GFP及其突變體之專利包括下列專 利:Chalfie,M.,及 Prasher,D·美國專利第 5,491,084 號; Tsien,R.及 Heim,R.美國專利第 5,625,048 號;Tsien,R·, 及 Heim,R·美國專利第 5,777,079 號;Zolotukhin,S.等人。 美國專利第 5,874,304號;Anderson,M.,及 Herzenberg,L· Α·專利第5,968,738號;Cormack,Β· Ρ·等人。美國專利第 5,804,387 號;Tsien,R.及 Heim,R·美國專利第 6,066,476 號;Chalfle,M.,及 Prasher,D·美國專利第 6,146,826號; 及Tsien,R.等人美國專利第7,005,511號。與該等螢光編碼 124909.doc -47- 200827448 基因有關之專利可經本文中所描述之方法修改以產生適用 於(例如)革蘭氏陽性微生物之多肽序列及/或表現卡匣。 3.4比色或螢光反應 作為光產生分子之替代物,催化比色或螢光反應或合成 比色或螢光受質之酵素亦適用於實施本發明,且視應用而 定可採用多種形式中之任一種。當用以分析諸如醱酵肉湯 之複雜樣品時,由於酵素對其受質具有極佳特異性,因此 使用編碼催化比色或螢光反應之酵素的報導體構築體可為 有利的。另外,當將較多受質轉化成比色或螢光產物時, 比色或螢光反應之信號強度隨時間增加。 一種預期適用作信號酵素之比色酵素為由細菌基因/acZ 產生之β-半乳糖苷酶。此酵素將無色受質X-gal(5-溴-4-氣-3-吲哚-b_D-吡喃半乳糖苷)裂解成半乳糠及藍色不溶性產 物。細菌/(2CZ基因可在丙酮丁醇梭菌中使用,此係因為經 證明丙酮丁醇梭菌並不具有β·半乳糖普酶(Yu,P.-L.等人 Differential induction of β-galactosidase and phospho-β-galactosidase activities in the fermentation of whey permeate by Clostridium acetobutylicum. Appl. Microbiol. Biotechnol. 26:254-257. 1987)。經證明來自一種具有與梭 菌物種類似之密碼子使用率的低G+C含量生物體高溫產硫 嗜熱厭氧芽孢桿菌(Thermoanaerobacterium thermosulfurigenes) 之McZ基因在丙酮丁醇梭菌中良好充當報導體(Burchhardt, G.? and H. Bahl. Cloning and analysis of the β-galactosidase-encoding gene from Clostridium thermosulfurogenes EMI. Gene 106: 13- 124909.doc • 48- 200827448 1991)。可使用之其他酵素包括編碼β-葡糖醛酸酶之基 因(Girbal, L.等人 Development of a sensitive gene expression reporter system and an inducible promoter-repressor system for Clostridium acetobutylicum. Appl. Environ. Microbiol· 69:4985-4988,2003)及可能地來自醣丁 酸梭菌之編碼β-1,4-内切葡聚糖酶之基因(Quixley,Κ· 等人 Construction of a reporter gene vector for Clostridium beijerinckii using a Clostridium endoglucanase gene. J. Mol. Microbiol. Biotechnol· 2:53-57,2000) o 3.5 表現卡匣 將欲監測之酵素之所需轉錄調節核苷酸序列與編碼報導 體酵素之基因以及適當轉譯調節元件(例如革蘭氏陽性薛-道氏(Shine-Dalgarno)序列)、短的隨機核苷酸序列及可選 擇標記操作性連接以形成所謂的表現卡匣。根據說明書之 教示,用於製備表現卡匣之個別組分及用於裝配該等組分 之方法為分子生物學技術中所熟知(參見例如Ausubel, F. Μ.,等人或Sambrook,等人)。適用於本發明之表現卡匣 之實例包括報導卡匣(Girbal,L.等人,同上文)及/acZ 報導卡匣(Tummala,S· Β·等人。Development and characterization of a gene expression reporter system for Clostridium acetobutylicum ATCC 824, Appl. Envir. Mircobiol. 65:3793-3799,1999)〇本發明之一較佳實施例使用在CDABE之野生 型排列中具有發光桿菌lux之表現卡匣,其已經優化以在 丙酮丁醇梭菌中表現且在各lux基因之5’具有革蘭氏陽性細 124909.doc •49- 200827448 菌薛-道氏序列。SEQIDNO: u。另一較佳實施例使用具 有發光桿菌lux基因之表現卡匣,其已經優化以在丙酮丁 醇梭菌中表現且各lux基因之5,具有革蘭氏陽性細菌薛·道 氏序列,但排列在諸如1uxABCDE之非野生型序列中(美國 專利第6,737,245號)。 . 不同於真核iuc操縱子,由於細菌lux操縱子含有内源性 • 產生駿叉質之基因,因此該操縱子為自足的。因此,螢光 素酶及内源性醛受質之同時共產生允許即時量測生物發光 而無需在監測生物發光信號強度前添加外源醛。然而,可 使用luxAB構築體且視使用iuc操縱子之信號酵素構築體所 需在量測生物發光之前添加醛受質。本發明之一較佳實施 例使用螢光素酶表現卡匣,其中發光桿菌之lux操縱子以 與美國專利第6,737,245號類似的方式,與丙酮丁醇梭菌之 醱酵途徑中酵素之適當轉錄調節核苷酸序列操作性連接。 本發明之另一較佳實施例使用具有編碼螢光蛋白與丙酮丁 U 醇梭菌之醱酵途徑中酵素之適當轉錄調節核苷酸序列操作 性連接之基因的表現卡匣。 3·6穿梭載體 隨後將表現卡匣插入”穿梭載體”中,其為可在兩種或兩 種以上宿主中複製之質體。欲用於革蘭氏陰性與革蘭氏陽 性生物體之穿梭載體要求穿梭載體含有各類別之複製起 點。穿梭載體之實例包括pAUL_A載體(Chakraborty,等人 (1992) J. Bacteriol. 174:568 574)、ρΜΚ4及 pSUM 系列(美 國專利第 6,737,245 號)及 pIMPl(Mermelstein,L· D·等人。 124909.doc -50- 200827448
Bio/Technology 1〇:190_195, 1992)。其他載體為熟習此項 技術者所熟知且易於由目錄號獲得。 、 3·7染色體整合 代替以質體轉化生物體,信號酵素可整合入宿主之染色 體中。與基於質體之結構相比,使用報導體構築體之染色 * 冑整合提供若干優勢,其包括較高穩定性及消除為保持質 . 體而對生物體維持選擇壓力之抗生素之使用。一種實現染 色體整合之方法使用含有諸如氯黴素基因之抗生素抗性2 因上游之所需基因的DNA片段及目標生物體之同源1)]^八的 片段。此DNA片段可連接以形成無複製子之環且用於轉 化。例如,在大腸桿菌之情況下可靶向基因,且在克雷 伯菌(尺/eh紿//幻中可連接短的隨機心片段以促進同源 重組。以此方式,乙醇產生基因已經染色體整合入大腸桿 菌中(Ohta等人 Appl. Environ. Microbiol· 57: 893-900, 1991)。 所整合報導體之複本數可由用於選擇方法中之抗生素的 〇 濃度來控制。例如,當將低濃度抗生素用於選擇時,發現 /、有單複本整合之純系,儘管頻率極低。儘管此對於許 #基因而言可為不利的,但考慮到光量測中所使用侦測器 之同莖敏度,螢光素酶之低複本數可為理想的。在單一步 驟中藉由對§有更鬲濃度抗生素之板加以選擇可實現較高 表現量。 染色體整合之另一方法使用諸如轉座子之可轉座元件, 其和:供工程化卡g之引入。 3·8與所監測酵素之調節控制平行的信號酵素 124909.doc -51 - 200827448 由於存在兩個獨立轉錄調節核苦酸序列,因此經轉化宿 主内之穿梭載體上之信號酵素的表現將與欲監測原生酵素 之表現天然平行。染色體整合亦將產生平行調節控制,除 非-者能夠引入與原生基因串聯之信號酵素序列。 3.9具有與所監測酵素串聯之調節控制之信號酵素 —種將信號酵素置於與原生酵素之調節控制相同的調節 制下之方法為選擇使用位於内源性f體上之操縱子,如 位於PS0L1巨型質體上的扣/。此處,可將質體加以分離, 切除操縱子且經報導基因與待監測之原生基因串聯插入之 含有新穎操縱子之表現卡E置換。於適當宿主中轉化及擴 土曰後,可隨後分離質體且隨後用以轉化丙酮丁醇梭菌之 PSOL1質體缺陷型菌株。 3.10丙酮丁酵梭菌之轉化 多種用於將信號酵素構築體引入細胞或細胞原生質體之 方法對於熟習此項技術者而言為已知的且包括(但不限於) 〇 下列方法:脂質介導之轉移(例如使用脂質體,包括中性 及陽離子型脂質)、直接注入(例如顯微注射)、細胞融合、 • 微粒轟擊(例如基因槍法,諸如DNA粒子轟擊)、共沈澱(例 如與磷酸鈣或乙酸鋰共沈澱)、DEAE·葡聚糖或聚乙二醇 介導之轉移' 病毒載體介導之轉移、電穿孔及接合。 電穿孔為轉化丙酮丁醇梭菌之較佳方法。理想地,使用 自中期對數生長期製備之電穿孔感受態丙酮丁醇梭菌。電 穿孔後’在37°C於如2χΥΤ肉湯之適當肉湯中同時在氮氣 氛下培育細胞。恢復期後,將細胞轉移至厭氧手套箱中, 124909.doc •52- 200827448 隨後將連續稀釋液塗鋪於補充有必要抗生素濃度之如 2xYT瓊脂平板的營養板上。 3·11偵測含有信號酵素構築體之具有螢光素酶之純系 Γ
可藉由在暗室中手動視覺檢測或藉由使用諸如併入電耦 合設備(CCD)攝影機者之影像偵測系統來鑑別含有來源於 完全luxCDABE操縱子之信號酵素構築體的微生物菌落。 由於需要氧用於生物發光反應,因此可能需要平板曝露於 低濃度之氧中以便偵測陽性菌落。獲自luc及luxAB之表現 卡匣要求添加外源受質以便產生光。在本發明之一較佳實 加例中,$質為酸。當投與細胞時,可將駿以蒸氣形式應 用於培養基周圍之氣氛中或直接應用於培養基。 4·細胞及培養物 —便用信號酵素適用於監測所有類型之輯或合成途徑。 >可為❺生型,其中其天然地產生所需目寺票,或其已 經受突^誘發及陽性選擇以過度產生所需目標。或者了宿 :了:則經工程化以表現所需醱酵或合成途徑所需之酵 素、、可呈,過度表現酸酵或合成途徑所需 需之異源酵素叫 中。m2 合成途徑酵素同時引人宿主細胞 在门時引入之情況下,信號酵素可盥 合成途徑酵素處於㈣操酵之撥酵或 縱子上。此外,宿主亦可經遺傳 所關注之潑酵或合成途徑下降=;由此迫使受質自 峄在丙鋼丁醇梭菌情況下, 124909.doc -53- 200827448 預期用於本發明之野生型菌株包括來自美國組織培養物收 藏中心(American Tissue Culture Collection)(ATCC)之 ATCC 824及ATCC 43084,及來自德國微生物採集和培養 有限責任公司(Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Germany)之 DSM 792 及 DSM 1731。預期用於本發明之丙酮丁醇梭菌之高丁醇產生突變 體包括諸如來自ATCC之ATCC 39058及ATCC 55025(美國 5,192,673)的菌株。另一預期用於本發明之高產菌株為 B643(Contag,Ρ· R.等人,Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and expression in Escherichia coli. Appl. Environ. Microbiol. 56:3760-3765,1990)。 另一預期用於本發明之高產突變體為B 1 8,其係來源於上 文之B643。預期在用於產生丁醇之丙酮丁醇梭菌中過度表 現之酵素包括丁醛脫氫酶及丁醇脫氫酶。預期在丙酮丁醇 梭菌中下調或缺失之競爭醱酵途徑之酵素包括丙酮酸脫羧 酶、乳酸脫氫酶及乙酸激酶。 預期用於本發明之拜氏梭菌之菌株包括菌株ATCC 25752、ATCC 51743 及 BA101、ATCC PTA 1550(美國 6,3 5 8,717及美國申請案第10/945,551號)。預期用於本發明 之梭菌之其他物種包括醣丁酸梭菌菌株ATCC BAA-117 ; 及腓尼基梭菌菌株ATCC 43978。 本發明之細胞培養物之特徵在於其以商業上有價值之量 產生合成或醱酵途徑目標且其亦產生傳遞目標產生之狀態 信號的發光報導體。 124909.doc -54- 200827448 在某些實施例中,商業上有價值之量之目標包括彼等在 100 1酸酵罐中產生之目標。在其他實施例中,在容量為 100至500 1之醱酵罐中產生商業上有價值之量之目標。在 其他實施例中,在500 is !,〇〇〇!容量之醱酵罐中產生商業 上有彳貝值之量之目標。在其他實施例中,在L000 1至2〇〇〇 • i容量之酸酵罐中產生商業上有價值之量之目標。在某些 . 其他實施例中,在2,000 1至5,000 1容量之醱酵罐中產生商 () 業上有彳貝值之量之目標。在其他實施例中,在5000 1至 10,000 1容量之醱酵罐中產生商業上有價值之量之目標。 在其他實施例中,在10,000 1至50,000 i容量之醱酵罐中產 生商業上有價值之量之目標。在某些其他實施例中,在 5〇,〇00 1至200,〇〇〇 1容量之醱酵罐中產生商業上有價值之 篁之目標。在其他實施例中,在2〇〇,〇〇〇 1至4〇〇 〇〇〇 !容量 之酸酵罐中產生商業上有價值之量之目標。在某些實施例 中,在400,000 1至8〇〇,〇〇〇 1容量之酸酵罐中產生商業上有 〇 彳貝值之量之目標。在其他實施例中,在80〇,〇〇〇 1至 2,000,000 1容量之醱酵罐中產生商業上有價值之量之目 , 標。在某些實施例中,在2,000,000 1至4,〇〇0,00〇 !容量之 醱酵罐中產生商業上有價值之量之目標。在其他實施例 中,在4,000,〇〇〇 1至8〇〇〇,〇〇〇丨容量之醱酵罐中產生商業 上有價值之量之目標。 4.1受質 本發明之受質為可經酵素促轉化成中間化合物之碳基化 合物。如本文中所用,術語"碳受質”係指含有至少一個碳 124909.doc -55- 200827448 原子之物曾,甘^r _ g ^ ^ φ 八u經酵素促轉化為用於後續轉化成所需碳 不的中間物。例示性 粉、糊精及糖。 厌又質包括(但不限於)生質、殿 ::文中所用’"生質"係指含纖維素及,或澱粉的原料, 草包厂但不限於)木片、玉米結桿、稻穀、草、草料、皮爾 ΓΡΓΓ叫、馬鈐薯、塊莖、根、全粉碎玉米、葡萄 r =、穀物、小麥、大麥、裸麥、高梁、麵皮、禾 ::含有糖之原料(例如糖蜜、果實物質、甘薦或、 生質之特定物質。本發明不受限於任何用作 在本發明之較佳實施财,該原、料為含 澱粉原料(例如穗軸、全粉碎王乎主半 或禾穀及其混合物)。在特广/物、高粱及/ 寺較佳實細例中,該術語係指 麻。-丨何植物來源之任何含澱粉物質,包括食品加工 :物,諸如杏仁及其他堅果殼,果園及葡萄園之修枝及剪 枝及如葡萄之收穫水果。 >如本文中所用,”澱粉,,係指任何含澱粉物質。詳言之, 該術語係指各種植物基物質’包括(但不限於)小:、二 麥、馬鈴薯、甘薯、木薯澱粉、玉米 梁、裸麥及麵皮。實際上,希望本發明不受限二: 類型及/或來源之澱粉。-般而言,該術語係指任何包2 植物之複合多醣碳水化合物、包含直鏈澱粉及支鏈殺於3 具有下式之物質:(c6Hl0〇5)x,其中,,χ"可為任何數字:” ,如本文中所用,"纖維素"係指任何含有纖維素之物· 詳言之,該術語係指具有下式之葡萄糖(或”纖維二糖:)之 124909.doc -56 - 200827448
苷鍵。纖維素與木質素組合形成”木質纖維素,,。 併如=中所用,·,半纖維素"係指任何含有半纖維素之物 質。詳言之,該術語係指具有木糖基、葡糖 基、阿拉伯糖基或甘露糖基殘基之雜聚物。土 礼糖 適當受質包括(但不限於)含有可轉化為糖之组分的經斤 理材料(例如纖維素生質、糖原、澱粉及其各種形式,= 如玉米澱粉、小麥殿粉、玉㈣體及小麥固體卜在本發 明之開發期間,以玉米、高粱及小麥澱粉得到良好結果: 儘官亦發現包括取自其他榖物及塊莖(例如甘薯、馬鈴
薯、稻縠及木薯澱粉)的澱粉之其他來源亦適用於本發 明。可購得各種澱粉。 X 可自包括木質纖維材料之多種來源獲得可醱酵糖。木質 纖維材料可獲自木質纖維廢物(例如植物殘體及廢紙)。適 當植物殘體之實例包括(但不限於)任何植物材料,諸如 莖、葉片、果殼、外皮、穗轴及其類似物,以及玉米秸 桿榨/查、木材、木片、木聚及鑛屬。廢紙之實例包括 (但不限於)任何類型之拋棄紙張(例如影印紙、電腦列印 、、、氏筆忑本紙、6己事本紙、打字機紙及其類似物)以及報 紙、雜誌、硬紙板及紙基包裝材料。 替代酸酵文質為乳清,其為移除絡蛋白後含有約4至$ % 乳糖(可由梭菌直接鏺酵之二醣)之溶液。此受質廣泛可得 124909.doc -57- 200827448 且乳清用於溶劑產生之醱酵用途將解決當前乳清廢棄問 題。 近來,經證明自居民垃圾中收集之擠壓有機廢物含有28 至39%總糖,其可由丙酮丁醇梭菌醱酵用於溶劑產生 (Lopez-Contreras A. M·, Utilisation of saccharides in • extruded domestic organic waste by Clostridium acetobutylicum ATCC824 for production of acetone, butanol, and ethanol. Appl. Microbiol. Biotechnol. 54:162- c 167,2000)。居民有機廢物自處置場所之轉移將有助於減 輕現有處置場所之壓力且提供堆肥之較高價值替代方法。 將糖轉化成乙醇之條件為在此項技術中所已知。溫度通 常在約25°C與35°C之間(例如25°C與35°C之間,且尤其在 3 0°C )。轉化介質之適用pH值範圍經提供在4·〇與6.0之間、 4.5與6.0之間及pH 5.5與5.8之間。然而,希望本發明不限 於任何特定溫度及/或pH條件,此係由於此等條件取決於 〇 所涉及之受質、酵素、中間物及/或目標。 4.2介質及碳受質 • 本發明之轉化介質必須含有適當碳受質。適當碳受質包 括(但不限於)生質、單醣(例如葡萄糖及果糖)、二醣(例如 乳糖及蔗糖)、寡醣(例如澱粉及纖維素)以及其混合物,及 諸如乾酪乳清滲透物、玉米漿、甜菜糖蜜及大麥芽之可再 生原料之未經純化之混合物。在其他實施例中,碳受質包 含諸如一氧化碳或甲醇之一碳受質,已證明其經代謝轉化 為關鍵生物化學中間物。 124909.doc -58· 200827448 已報導在甲基營養型酵母(Yamada等人Agric. Biol.
Chem·,53:541-542,1989)及細菌(Hunter 等人 Biochem·, 24:4148-415 5,1985)中自單碳源產生甘油。此等生物體可 吸收範圍在由甲烷至甲酸之氧化態中之單碳化合物且產生 甘油。在一些實施例中,碳吸收途徑係經由核酮糖-單磷 酸、經由絲胺酸或經由木酮糖-單磷酸(G〇ttschalk,
Bacterial Metabolism,第二版,Springer·Vedag,New
York,1986)。核酮糖單磷酸途徑涉及甲酸與核酮糖5_碌酸 之縮合以形成變成果糖且最終變成3-碳產物甘油醛·3_磷酸 之6碳糖。同樣地,絲胺酸途徑經由亞甲基四氫葉酸吸收 一碳化合物至糖酵解途徑中。 除使用一碳及二碳受質外,已知甲基營養型生物體利用 諸如甲胺、葡糖胺之多種其他含碳化合物及多種胺基酸用 於代謝活動。舉例而言,已知曱基營養型酵母利用來自甲 胺之碳以形成海藻糖或甘油(Belli〇n等人於Murrell等人 (編)7th Microb· Growth Cl Compd· Int. Symp·,415-432, Interxept,Andover,UK,1993中)。類似地,各種假絲酵母 物種代謝丙胺酸或油酸(Sulter等人Arch. Micr〇bi〇1., 153:485-489, 1990)。因此,本發明利用之碳源涵蓋多種含 碳受質’且其僅受宿主生物體之需求限制。 儘官預期所有以上提及之碳受質及其混合物將用於本發 月之方法中,但較佳碳受質包括單醣、二醣、寡醣、多醣 反义質在更尤其較佳之實施例中,碳受質係選自由 葡萄糖、果糖、蔗糖及諸如甲醇及一氧化碳之單碳受質組 124909.doc -59- 200827448 成之群。在-最尤其較佳之實施例中,受質為葡萄糖。 如在此項技術中已知’除適當碳源之外,醱酵介質亦必 須含有適當氮源、無機鹽、辅因子、緩衝劑及其他適於培 養物生長且促進產生所需目標(例如甘油)所必需的酵素途 徑之組分。在一些實施例中,鹽及/或維生素Bi2或其前驅 ^ 物適用於本發明。 5·監測及調節方法 (、 在鉍生物生長及培養期間,各種生物化學途徑之動力學 變化,使各種目標之產生率變化。例如在丙酮丁醇梭菌之 分批培養物中,諸如乙酸及丁酸之酸的初始產生使培養物 之pH值降低,然而當未解離丁酸之濃度達到9 mM後移動 發生’其中丙_丁醇梭菌再吸收所分泌之酸且轉入諸如丁 醇及丙酮之溶劑之產生中。丁醇對細胞具有毒性效應且其 積累最終抑制產生其之酵素的表現。藉由將報導體置於各 種生物化學途控之策略點,可監測該等途徑之狀態且若需 y 要,可使培養條件"保持平衡,,以誘導且維持產生最大量之 產物的狀態。在觀察到丁醇對培養物抑制效應之情況下, 可開始自醱酵肉湯移除丁醇或可將水或培養基添加至醱酵 罐中以將所累積之丁醇稀釋至低於抑制臨限。 由報導體所產生之信號的強度來傳遞生物化學途徑之狀 態的信號。此轉而反映信號酵素構築體之轉錄活性。發光 報導體尤其具有吸引力,此係因為其即時產生與提供關於 醱酵或合成途徑之狀態的直接資訊之基因表現程度有關之 信號。在丙酮丁醇梭菌培養物中使用信號酵素允許立即調 124909.doc -60- 200827448 節培養調節以維持或誘導高生產力。 5·1偵測培養物中之光 本發明涵蓋若干量測微生物培養物中之光的方法。習知 醱酵罐可具有一或多個位於槽側面上之埠孔以便該等埠孔 在醱酵肉湯之初始位準之下。隨後可將諸如光電倍增管 • (PMT)或(:(:〇攝影機之偵測光之構件安裝在配備有透明窗 • 口之埠孔外,但其經定位以偵測經由該埠孔窗口發射之 ( 光或者,可在將醱酵罐滅菌之前,經由埠孔或其他開口 將外邛女裝之PMT或CCD攝影機與光纖電親或其他類型之 放置在醱酵罐内部的光導連接。光纖電纜可與經設計於醱 酵授摔器之葉輪内的流動單元連接。 亦可藉由將偵測器放置於醱酵罐内實現光量測。偵測器 可安裝在固定位置或拴住,且依賴於電線應答資料獲取及 分析電子設備之信號。或者’偵測器可使用無線通訊系 統,其除使偵侧器安裝在固定位置或拴住之選擇外,還將 Ο 允許偵測器自由浮動或在其自身動力下操作以在醱酵肉湯 内移動。债測器可進一步經設計為與CCD成像器及冷卻元 件之積體微流控晶片。 另外,可將培養基流連續抽離醱酵罐且導入光偵侧裝 置。此處樣品流可間歇或持續流經位於光偵侧裝置内部之 流動單元。此處,若需要增強介質之發光,則混合室可經 放置以便ATP或氧可添加至樣品流中。或者,若需要可將 稀釋劑添加至混合室中之樣品中以降低信號強度。 此外,可經由手動或自動採樣埠將樣品定期抽離醱酵 124909.doc -61- 200827448 罐,且隨後分析發光。 5.2處理光信號 本發明之一重要態樣係使用高靈敏度構件來使得能夠快 速量測醱酵肉湯之生物發光,以便所獲得之信號可用於即 時監測及控制培養物。該設備需能夠偵測且計數個別光子 且隨時間如以閃爍計數器形式累積總計數。最靈敏計數設 備採用光電倍增管(PMT),其中進入PMT之光激發光電陰 極中之電子,使得光電子發射,當其向偵測器加速時光電 子釋放遞增之偵侧到之電子級聯。自諸如Hamamatsu之供 應商可獲得多種PMT。藉由採用濾光器、光栅及其他光譜 設備結合PMT可獲得光譜資訊。 不太敏感之設備包括電耦合設備(CCD)攝影機。其可經 冷卻以降低背景雜訊或其可含有以與PMT類似之方式起作 用的微通道增強器以放大入射光子所產生之信號。例示性 基於微通道增強器之單光子偵側設備為自Hamamatsu獲得 之C2400系列。其他潛在計數技術包括積體CCD、電子倍 增CCD、雪崩光電二極體(avalanche photodiode)及互補金 屬氧化物半導體(CMOS)影像感應器。 為方便起見PMT與CCD均可用於含有全部必要電源及電 子電路之模組中。舉例而言,PMT模組通常含有整合於單 個封裝中之高壓電源、分壓電路、信號轉換電路、光子計 數電路、CPU介面及冷卻裝置。可易於獲得允許光子計數 信號與電腦整合之軟體,由此允許信號用於監測與控制醱 酵過程之演算法中。 124909.doc -62- 200827448 5·3測定生物化學途徑之狀態:電腦軟體 測定生物化學途徑之狀態依賴於報導體報導之信號酵素 之性質。視信號酵素是否催化朝向目標或朝向產生另一終 產物或反向循環至該途徑的中間物之分枝之轉化而定,該 #號可與目標之產生正相關或負相關。在該等兩種替代形 式之間,信號之絕對水準提供有關所需產物產生之資訊, 且強度隨時間改變之信號之動力學亦提供有關產物產生是 Ο 否增加或降低之資訊。另外,亦可計算動力學之變化速率 且用以監測與控制醱酵。 儘管此資訊可經人工處理且根據其採取措施,但在某些 貝施例中該 > 訊係由電腦處理。因此,本發明之軟體將包 括以關於各報導體之信號水準之輸入資料形式接收的代 碼,執行確定培養物狀態之作為(至少)該水準或水準之函 數的廣开法之代碼,及確定該培養條件應如何改變以將彼 。養物保持在所需狀態之代碼,及指示該系統對培養物進 行適當改變以實現該條件之代碼,其為調節溫度、添加營 養物、自培養物移除產物、降低培養物密度或將使培養物 改變為所需狀態之任何其他改變。 5·4調節培養物中之途徑活性 1藉由使用㈣酵素即時監測酵素表現且因此監測醋酵途 任中之活性的能力向操作者或醱酵過程控制者提供調節條 :以,物"保持,,在所需終產物的最高生產力之特定階 :::力。一種利用信號酵素之即時信號傳遞能力以控制 ^之方法係採用用以控制常見高細胞密度大腸桿菌醱 124909.doc -63- 200827448 酵之即時信號方法。此處細胞通常以分批模式生長至中間 細胞密度,其後起始進料策略。進料策略可歸類為兩種主 要類別:開環(非反饋)及閉環(反饋)(美國專利第Μ55』92 號)。開環進料策略通常為預先確定之碳/營養物添加進料 概況。通常使用之進料時程包括怪定或遞增之進料率味 定、逐步或指數)以便跟上遞增之細胞密度。儘管該等簡 單預先破定之進料概況已成功應用於某些情況下,但主要 缺點為缺乏基於培養物之代謝反饋的進料率調節。因此, 開環進料策略可因在其偏離其,,預期"增長模式時,向培養 物過度進料或未充分進料而失敗。 另一方面,閉環進料策略通常依賴於指示培養物之代謝 狀態的量測。大腸桿菌之兩種最通常量測之在線變數為溶 解氧(DO)濃度及pH值。在監測!)〇情況下,上升之表示 氧消耗減少’該氧消耗減少又係基於營養物限制或耗盡。 當DO升高至臨限值以上或變化率超過臨限值時,過程控 制者將增加營養物進料率。相反,當D〇下降低至所需= 定點或變化率超過臨限值時,過程控制將減少營養物進= 率以反映代謝需求。類似地’可單獨或與d〇量測組合使 用培養物PH值之變化或培養物阳值之變化率以調整營養 物進料添加至酸酵罐中之速率。 氧化還原電勢為可量取用以監測與控制細菌酸酵之另 :變數。視需要’可經由添加還原劑提高酸酵肉湯之氧化 遇原電勢。可以氧化還原探針摘測厭氧培養物中低於即 探針之<1 ppm靈敏度的微量氧濃度。 124909.doc -64- 200827448 孤則σ養物代謝活性之其他方法包括分析醱酵肉湯及廢 氣。儘管兩種方法可在線進行,但其不能現場執行且將不 提供關於與醱酵途徑有關之酵素的基因表現之資訊。相 反,該分析僅將提供關於培養物之一般代謝狀態的資訊。 因為υ酵素提供培養物代謝活性之即時狀態,因此可 採用配口白知冋翁度細胞培養系統之及pH值控制使用 . @相同過&控制决异法以與信號酵素系統―^使用。其將 尤其有利於監測盆中Γ)Π 、日,丨《τ» _ f、 ,、TD〇監測不可能之厭氧培養物。就丙 ㈣丁醇梭®中之丁醇產生舉例而言,培養物穩定地處於溶 劑產生階段中後,丁醇產生之大多數中間物將來自如葡萄 糖之原料的持續新陳代謝。針對丁酸途徑之末端使用諸如 讓(將丁搭還原4 丁醇之搭_醇脫氫酶)之信號酵素提供關 於丁醇產生之狀恕且因此提供培養物代謝率。信號強度及 信號強度之變化率可隨後用以以與大腸㈣培養物_ DO 監測大致相同的方式控制培養物之進料率。此可在培養物 〇 %始產生★劑時’藉由監測信號酵素之初始表現在丙_ 丁 醇梭菌刀批培養物中進行。當來自酸產生階段之有機酸經 #吸收時,強度將存在初始增力口。當㈣酸之濃度降 ㈣’酵素活性將下降,同時向過程控制者傳遞信號以起 始培養物進料或增加現有進料率。此後,遞增之信號強度 才曰示丁醇產生;^加且因此培養物代謝率增加。過程控制隨 後將逐漸增加進料率,同時持續監測酵素之信號強度。若 信號強度持續增加’則過程控制者可持續增加進料率,只 要信號酵素之信㈣度的變化率在增b。若注意到信號酵 124909.doc -65- 200827448 素之信號強度的變化率下降’則過程控制者將減少進料率 以不向培養物中過度進料且引起受質抑制及丁醇產生率降 低。猎由持續監測信號酵素信號且調節進料率以反映信號 酵素所提供之資訊,則培養物將處於最高丁醇生產力狀 態。
Ο 分批進料方法之替代方法為連續分財法,纟中通常自 酸酵罐中同時移除酿酵肉湯,且添加新鮮營養物或水以維 持醱酵罐體積及所需細胞密度。由於連續醱酵方法代表穩 匕其亦可經由使用—或多種信號酵素來監測及控制。信 號強度之任何降低或增加表示偏離已預先存在之穩態,且 視所需醱酵參數而定,該等信號可指示操縱者或過程控制 者其:為調節醱酵條件之時間。在維持穩態中連續移除醱 酵肉湯之要求提供簡便方式以採用在線量測信號酵素監 測。 信號酵素亦可用於監測醱酵或合成途徑中之分解代謝物 阻抑。一些酵素對所存在之代謝產物之濃度敏感,其中代 謝產物能夠與酵素之操縱子結合且阻斷基因之轉錄。當代 謝產物濃度增加時,酵素之基因轉錄之速率降低。藉助於 利用相同轉錄調節核苷酸序列之信號酵素構築體,信號酵 素之4唬強度將按比例下降。當醱酵過程控制者偵測到信 唬酵素之信號強度降低時,過程控制可採取措施以對抗抑 制性代謝產物之積累。例如,若代謝產物為分泌於介質中 之目標’則過程控制者可起始目標自培養基中之移除。若 代謝產物為中間產物,則可經由添加水或新鮮培養基增加 124909.doc -66 - 200827448 培養物之總體積來降低阻抑物之細胞内濃度。 對於具有不同誘導型醱酵或合成途徑之生物體,信號酵 素之使用可指示該途徑是否具有活性,且亦指示活性之強 度,由此提供調整培養條件之時機。例如,以丙酮丁醇梭 菌,在分批培養物之酸產生階段中,若溶劑產生途徑中之 4吕號酵素開始指示彼途徑中之活性,則必要時操縱者或過 程控制者可向培養基中添加作為受質之丙酮酸。此誘導酸 產生酵素之表現,由此延長酸產生階段(11111611以α· Μ·等 人。Effect of pyruvate on giucose metab〇lism in cl〇stridium acetobutylicum. Biochimie. 69:1183-1190, 1987)。此舉可進行以 提供更多隨後用於再吸收及以增加之產量轉化溶劑的有機 酸? 類似地,若發現溫度或pH值影響特定醱酵或合成途徑之 生產力,則信號酵素之使用可用於使生產力最大化。例 如,若發現丙酮丁醇梭菌之特定菌株在一種溫度下產生較 多有機酸’但在另一溫度下相對於其他溶劑產生較高濃度 之丁醇,則當溶劑產生移動發生時,信號酵素之使用可指 示以便可以最大丁醇生產力之及時方式調節培養物之溫 度。 6·系統及設備 6.1培養容器 適用於丙酮丁醇梭菌之分批醱酵的醱酵罐為此項技術中 所熟知(Beesch,S. C. Acetone-butanol fermentation of sugars. Eng. Proc· Dev· 44:1677-1682,1952 ; Beesch,S· C· 124909.doc -67- 200827448
Acetone-butanol fermentation of starches. Appl. Microbiol. 1:85-96, 1953 ; Killeffer, D. H. Butanol and acetone from corn. A description of the fermentation process. Ind. Eng. Chem. 19:46-50,1927 ; MuCutchan W. N.,及 Hickey,R. J.
The butanol-acetone fermentations. Ind. Ferment. 1:347-388,1954)。欲使用之醱酵罐通常具有50,000至200,000加 侖之容量且無機械攪拌系統。藉由噴射亦經由維持醱酵罐 内之正壓力用來防止培養物污染之無菌二氧化碳來促進醱 酵罐内含物之混合。 分批進料醱酵方法亦可用於丙酮丁醇梭菌醱酵。在某些 實施例中,在50,000 1至200,000 1容量之醱酵罐中產生商 業上有價值之量之目標產物。在其他實施例中,在 200.000 1至400,000 1容量之醱酵罐中產生商業上有價值之 量之目標產物。在某些其他實施例中,在400,000 1至 800.000 1容量之醱酵罐中產生商業上有價值之量之目標產 物。在其他實施例中,在800,000 1至2,000,000 1容量之醱 酵罐中產生商業上有價值之量之目標。在某些實施例中, 在2,000,000 1至4,000,000 1容量之醱酵罐中產生商業上有 價值之量之目標。在其他實施例中,在4,000,000 1至 8,000,000 1容量之醱酵罐中產生商業上有價值之量之目 標。 用於丙酮丁醇梭菌之連續醱酵的醱酵罐亦為此項技術中 已知(美國專利第4,424,275號及美國專利第4,568,643號)。 因為可經由連續培養,伴隨移除含溶劑之醱酵肉湯維持高 124909.doc •68- 200827448 密度、穩態培養物,因此可使用較小容積之醱酵罐。在某 些實施例中,在50,000 1至200,000 1容量之醱酵罐中產生 商業上有價值之量之目標產物。在其他實施例中,在 200,000 1至40〇,〇〇〇 1容量之醱酵罐中產生商業上有價值之 量之目標產物。在某些其他實施例中,在4〇〇,〇〇〇 1至 • 800,000 1容量之醱酵罐中產生商業上有價值之量之目標產 物。在其他實施例中,在800,000 1至2,〇〇〇,000 1容量之醱 ’罐中產生商業上有價值之量之目標。在某些實施例中^ 在2,000,000 1至4,〇〇〇,〇〇〇 1容量之醱酵罐中產生商業上有 價值之量之目標。在其他實施例中,在4,〇〇〇,〇〇〇 !至 8,000,000 1容量之醱酵罐中產生商業上有價值之量之目 標。 上文之醱酵方法亦可使用如WO 81/01012中所揭示之固 定化細胞。固定產生簡化產物回收之無細胞醱酵肉湯,且 可增加細胞密度,由此增加溶劑之產生率。 〇 6·2量測光之電子設備 PMT及CCD偵測模組為市售的且可如上所述在無廣泛改 . 冑之情況下無需定製使用。其可結合MU或其他光譜設 備使用以分析特定波長。另外,光學纖維組件亦可購^ = 將光子轉化為電子信號。 6.3資訊學/軟體 因此,本發明之系統可包括—或多個電腦,其包含在單 夺U在夕個時間點或在一定時間内連續存取代表報 導體信號強度的資料之代碼,及執行將數據轉換成有關培 124909.doc -69- 200827448 養物中一或多個±物化學途徑狀態之資訊的演算法之代 碼。操縱♦或過程㈣者可使用此資訊來調節培養條件以 增加、維持或減緩產物產生之水準。 6.4回應電腦信號改變培養條件之裝置 調節或改變培養條件之裝置為此項技術中所熟知且包括 致動螺線管或其他置於加壓管線上之閥門,及使用泵用於 未加屢流體。通常將諸如彼等含有葡萄糖或氨者之進料管 線保持在壓力T,如諸如空氣、氮氣或二氧化碳之氣體管 線般。另夕卜將諸如冷卻或熱水或蒸汽之公用事業管線加 自過程控制者發出信號後,螺線管經致動且閥位置改 變以打開或關閉管線。以此方式,可另外將#養液添加至 酸酵罐中或可將熱水導向„罐護套中以增加培養物之溫 未力壓g線通彳包含特定、非A量進料組分且依賴於系 來轉移液體。通常與無_橡膠或其㈣㈣組合使用螺 動栗。對於少量之液體’蠕動驅動可用以計量來自注射写 之液體。過程控制者通常將給予電路電壓錢其斷開,由 此打開或關閉電動系。過程控制者可以類似方式指引系自 醱酵罐移除醱酵肉湯。 :由過程控制者控制之其他醱酵參數包括醱酵罐之通風 逮率、攪拌速率及内部大氣壓。 6·5收集產物之構件 ::構件可用於自醱酵肉湯中分離溶劑,其包括以溶劑 、’"卒取(美國專利第4,424,275號及美國專利第4,568,643 124909.doc 200827448 號)、使用碳氟化合物(美國專利第4,777,135號)、使用吸收 材料(美國專利第4,520,104號)、使用全蒸發膜(美國專利第 5,755,967號)及使用汽提氣(美國專利申請案第1〇/945,551 號)。 本發明之一實施例使用蒸汽壓縮蒸餾系統(美國專利第 4,671,856、4,769,113、4,869,067、4,902,197、4,919,592、 4’978’429、5,597,453及5,968,321號)。對於丙酮丁醇梭菌之分 批醱酵,收集已用醱酵介質中所含之溶劑首先要求離心肉 湯以移除細胞及顆粒物質。隨後將經澄清之肉湯送至蒸餾 系統中,其令該經澄清之肉湯進入熱交換器且藉由自流出 之蒸餾產物及廢液轉移的熱預熱。將經預熱之肉湯脫氣且 饋入/、有父替形成於藉由密封塾隔開的堆疊金屬板之間的 逆流蒸發及冷凝室之板式蒸發器/冷凝器。介質進入蒸發 至在此處其沸騰。離開蒸發室之經加熱之蒸汽穿過移除 務狀物之網且隨後藉由低壓壓縮機加壓。將加壓蒸汽傳遞 至冷凝至,在此處其濃縮為蒸餾產物,向沸騰室中之肉湯 ”、、且後自系統中排出。同樣地收集含有溶解固體 之未蒸發肉湯且自系統中排出。 、子於丙酮丁醇梭菌之連續培養物,可離心自醱酵罐抽離 之醱酵肉湯以濃縮細胞及顆粒物質。若需要可將所濃縮之 、、田胞及物質再次添加返回至醱酵罐中以增加細胞密度或進 步,酵部分醱酵之受f。或者,若經澄清之醱酵肉湯含 、口合生可曝酵文質’則可將經澄清之醱酵肉湯再次添加 返回至醱酵罐中。t需要自介質中收集溶劑時,可利用兩 124909.doc -71- 200827448 種朿略。一 & 考為儲存經澄清之醱酵肉 起始蒸餾操作。H 直至存在合理量以 汽蒸餘系統中。 3之_肉濟連續饋入蒸 可使由丁醇構成含有溶劑混合 歷自發相分離。浮面於干…田 肉•基於比重經 之溶劑層與剩餘水性部分分離。❹輔助使含有丁醇 6·6生物燃料設施 :二月之一實施例包含生物燃料設施。在此實施例中, i料傳、/生f上為糖、糊精”1粉或生f之原料。隨後將 1性質=生=料碼頭接收站’其中將原料隔離且根據 、、=子。右需要,則可抽取部分原料不用於儲存,以 ㈣加工成培養物受f介質。如培養條件所要求,隨後可 將受質介質饋入酸酵生物之分批或連續培養物中。達到溶 劑之目標濃度後,隨後將潑酵肉湯饋入一或多個蒸汽壓縮 =顧系統中中將溶劑與肉湯分離。將溶劑就地饋入罐 場:以暫時儲存。若可醱酵受質剩餘,則可將已用肉湯再 循環返回至醱酵罐。將累積之微生物及未醱酵受質加工成 動物飼料或處理微生物以獲得工業酵素。在裝卸碼頭,到 達之槽罐式卡車裝載溶劑、動物飼料或酵素。 6·7培養物之目標產物 本發明亦提供培養物之目標產物。藉由本發明之方法產 生之組合物不同於藉由其他方法產生之組合物之處在於在 某些產生階段,其具有微量其來源,亦即來自培養物受質 之化合物及產生其之微生物生物體。以相同方式,其缺乏 124909.doc •72- 200827448 通常存在於使用不同培養受 不同微生物物種產生之組人 發明之組合物亦將具有微量 合物的化合物。 質或不同丙酮丁醇梭菌菌種或 物中的微量化合物。另外,本 不同於非醱酵方法所產生之組 在實施例中#由本發明之工程化丙嗣丁醇梭菌產生 之丁醇將3有不同於藉由其他丙_ 了醇梭㈣株及酸酵方 法所產生之τ醇的微量化合物,此係因為本發明之丙嗣丁
醇梭菌醱酵方法將利用獲自莧菜及/或甜高粱之生質,而 非依賴於己或玉米漿。 6·7·1·食品級芳香及香料萃取劑及化妝品及醫藥加 工助劑 在實施例中本發明之目標適用作芳香及香料萃取劑 或適用於化妝品及醫藥加工。一種該芳香及香才斗萃取劑及 加工助劑為丁醇。本發明之丁醇組合物不同於獲自石油來 源之丁醇組合物。|發明之丁醇組合物包含來自❻酮丁醇 梭菌醱酵肉湯之餾份,且因此可含有醱酵來源之諸如低分 子量醇、醛或酯之共餾份。石油衍生之丁醇係主要藉助於 經由使合成氣(一氧化碳及氫)與丙烯反應之羰基合成法製 造之丁醛之氫化來製造。此方法產生在8:1至1〇:1比率範圍 内的正丁醇及異丁醇。因此,預期石油衍生之丁醇的主要 微量污染物為異丁醇。基於可經由使用氣相層析(gc)或與 質譜儀(MS)聯合之GC區分的微量組合物,本發明之丁醇 可與石油衍生之丁醇加以區分。 7·商業方法 124909.doc -73· 200827448 在本發明之一實施例中,在生物技術公司與煉油公司之 間形成合資企業。生物技術公司具有能夠將生質醱酵成溶 劑之專有生物工程化菌株。該等溶劑具有用作燃料或燃料 添加劑之用途。煉油公司具有石油化工專門技術且亦參與 適用作燃料之成品石油化學品之製造。生物技術許可煉油 Α司使用專有生物工程化菌株。希望煉油公司構建生質酸 酵設備。生物技術公司向合資企業提供醱酵及方法開發專 門技術,而煉油公司提供工程專門技術。煉油公司進一步 支援合資企業之擴大及方法開發實驗。煉油公司購買合資 企業生質醱酵設備所產生的溶劑,且因此等收益生物技術 公司接收專利權稅。 “本文中已展不J^描述本發明之較佳實施例,但對於 …、白此項技術者而吕該等實施例顯然僅以實例方式提供。 在不悖離本發明之情況下,熟習此項技術者現將進行多種 變更、變化及取代。應瞭解本文中所描述之本發明實施例 Ο 之各種替代可用於實施本發明 ^布望下列申請專利範圍定 義本發明之範疇且由此涵蓋哕笙 盍该4睛未項範疇内之方法及結 構及其等效物。 124909.doc -74- 200827448 序列表 SEQ ID NO: 1·經優化之luxA核苷酸序列
ATGAAATTTGGATTATTTTTTCTTAATTTTATAAATAGTACAACTATTCAAGAACAGTCAATAGCAAGAATGCA
GGAGATTACAGAGTATGTTGATAAGCTAAATTTTGAGCAGATTCTTGTATGTGAAAATCATTTTTCAGATAATG
GTGTTGTAGGTGCTCCTTTAACTGTTAGTGGTTTTTTATTAGGACTTACAGAAAAAATTAAGATAGGTTCATTA
AATCATGTAATTACTACACATCATCCAGTTAGAATAGCAGAAGAGGCTTGCCTTTTAGATCAACTITCTGAAGG
AAGATTTATATTAGGTTTTAGTGATTGTGAAAGAAAAGATGAGATGCACTTTTTTAATAGACCTGAACAATATC
AACAACAACTTTTTGAAGAGTGCTATGATATTATAAATGACGCATTAACTACAGGATATTGTAATCCAAATGG
AGATTTTTATAATTTTCCTAAAATTTCAGTAAATCCACATGCTTATACTCAGAATGGTCCTAGAAAGTATGTTAC
AGCAACTTCTTGTCATGTAGTTGAATGGGCAGCTAAGAAGGGTATACCATTAATTTTTAAATGGGATGATAGTA
ATGAAGTAAAACATGAGTATGCTAAGAGATATCAAGCAATAGCTGGTGAATATGGAGTTGATCTTGCAGAAAT
TGATCATCAATTAATGATATTAGTTAATTATTCAGAGGATTCTGAAAAAGCTAAGGAAGAGACAAGAGCATTT
ATAAGTGATTATATTTTAGCTATGCACCCTAATGAAAATTTTGAAAAAAAACTTGAGGAAATAATAACTGAAA
ATTCAGTTGGTGATTATATGGAGTGCACAACTGCTGCAAAACTTGCAATGGAAAAATGTGGAGCTAAAGGTAT
TCTTTTATCTTTTGAAAGTATGTCAGATTTTACACATCAGATTAATGCAATAGATATAGTAAATGATAATATTA
AGAAATATCATATGTAA SEQ ID NO: 2-經優化之LuxA胺基酸序列
MKFGNFLLTYQPPQFSQTEVMKRLVKLGRISEECGFDTVWLLEHHFTEFGLLGNPYVAAAYLLGATKKLNVGTAAI
VLPTAHPVRQLEEVNLLDQMSKGRFRFGICRGLYNKDFRVFGTDMNNSRALMECWYKLIRNGMTEGYMEADNEH
KFHKVKVLPTAYSQGGAPIYWAESASTTEWAAQHGLPMILSWIINTNEKKAQIELYNEVAQEYGHDIHNIDHCLS
mSVDHDSMKAKEICRNFLGHWYDSYVNATTIFDDSDKTKGYDFNKGQWRDFVLKGHKNTNRRVDYSYEINPVG
TPQECIDIIQTDIDATGISNICCGFEANGTVDEIISSMKLFQSDVMPFLKEKQRSLLY SEQ ID NCh 3-經優化之luxB核苷酸序列
ATGAAATTTGGATTATTTTTTCTTAATTTTATAAATAGTACAACTATTCAAGAACAGTCAATAGCAAGAATGCA
GGAGATTACAGAGTATGTTGATAAGCTAAATTTTGAGCAGATTCTTGTATGTGAAAATCATTTTTCAGATAATG
GTGTTGTAGGTGCTCCTTTAACTGTTAGTGGTTTTTTATTAGGACTTACAGAAAAAATTAAGATAGGTTCATTA
AATCATGTAATTACTACACATCATCCAGTTAGAATAGCAGAAGAGGCTTGCCTTTTAGATCAACTTTCTGAAGG
AAGATTTATATTAGGTTTTAGTGATTGTGAAAGAAAAGATGAGATGCACTTTTTTAATAGACCTGAACAATATC
AACAACAACTTTTTGAAGAGTGCTATGATATTATAAATGACGCATTAACTACAGGATATTGTAATCCAAATGG
AGATTTTTATAATTTTCCTAAAATTTCAGTAAATCCACATGCTTATACTCAGAATGGTCCTAGAAAGTATGTTAC
AGCAACTTCTTGTCATGTAGTTGAATGGGCAGCTAAGAAGGGTATACCATTAATTTTTAAATGGGATGATAGTA
ATGAAGTAAAACATGAGTATGCTAAGAGATATCAAGCAATAGCTGGTGAATATGGAGTTGATCTTGCAGAAAT
TGATCATCAATTAATGATATTAGTTAATTATTCAGAGGATTCTGAAAAAGCTAAGGAAGAGACAAGAGCATTT
ATAAGTGATTATATTTTAGCTATGCACCCTAATGAAAATTTTGAAAAAAAACTTGAGGAAATAATAACTGAAA
ATTCAGTTGGTGATTATATGGAGTGCACAACTGCTGCAAAACTTGCAATGGAAAAATGTGGAGCTAAAGGTAT
TCTTTTATCTTTTGAAAGTATGTCAGATTTTACACATCAGATTAATGCAATAGATATAGTAAATGATAATATTA
AGAAATATCATATGTAA -75- 124909.doc 200827448 SEQ ID NO: 4·經優化之LuxB胺基酸序列
ΜΚίΌΙ^Ρ1^ΝΕΙΝ3ΤΤΚ3Ε(38ΙΑΚΜ(3ΕΙΤΕΥνϋίαΝΡΈ(2ΙΕναΕΝΗΡ30Ν〇νν〇ΑΡΙ^Τν80ΡΙΧΟί 丁 EKIKIGSLNHVIT 丁 HHPVRIAEEACLLDQLSEGRFILGFSDCERKDEMHFFNRPEQYQQQLFEECYDIINDALTTGYCNPNGDFYNFPKISV NPHAYTQNGPRKYVTATSCHVVEWAAKKGIPLIFKWDDSNEVKHEYAKRYQAIAGEYGVDLAEIDHQLMLVNYS EDSEKAKEETRAFISDYILAMHPNENFEKKLEEIITENSVGDYMECTTAAKLAMEKCGAKGILLSFESMSDFTHQINA SEQ ID NO: 5-經優化之luxe核苷酸序列
ATGAATAAAAAGATATCATTTATTATAAATGGAAGAGTTGAAATATTTCCTGAGTCAGATGATTTAGTACAATC
TATAAATTTTGGTGATAATTCTGTTCATCTTCCAGTACTTAATGATTCACAGGTTAAGAATATTATAGATTATAA
TGAGAATAATGAGCTTCAGCTTCATAATATTATAAATTTTCTTTATACAGTAGGACAGAGATGGAAGAATGAG
u
GAGTATAGCAGAAGAAGAACTTATATAAGAGATCTTAAGAGATATATGGGTTATAGTGAGGAAATGGCAAAA
TTAGAAGCTAATTGGATTTCAATGATATTATGTTCTAAGGGAGGTTTATATGATTTAGTTAAAAATGAATTAGG
AAGTAGACATATTATGGATGAATGGTTACCTCAAGATGAATCATATATAAGAGCATTTCCAAAAGGTAAAAGT
GTACATCTTTTAACAGGAAATGTTCCTTTAAGTGGAGTACTTTCAATTTTAAGAGCTATACTTACTAAAAATCA
GTGCATTATAAAGACATCTAGTACTGATCCATTTACAGCAAATGCTTTAGCACTTAGTTTTATAGATGTTGATC
CTCATCATCCAGTAACTAGATCTTTAAGTGTTGTATATTGGCAACATCAAGGTGATATTTCACTTGCTAAAGAA
ATAATGCAACATGCAGATGTTGTAGTTGCTTGGGGAGGTGAAGATGCAATTAATTGGGCTGTAAAGCACGCAC
CTCCAGATATAGATGTTATGAAATTTGGACCTAAAAAGTCTTTTTGTATTATAGATAATCCAGTAGATTTAGTT
AGTGCTGCAACAGGTGCTGCACATGATGTATGCTTTTATGATCAGCAGGCTTGTTTTTCAACTCAAAATATATA
TTATATGGGATCACATTATGAAGAATTTAAACTTGCATTAATTGAAAAACTTAATTTATATGCTCATATACTTCC
AAATACAAAGAAAGATTTTGATGAAAAGGCAGCTTATAGTTTAGTTCAGAAAGAATGTTTATTTGCAGGACTT
AAAGTAGAAGTTGATGTACATCAAAGATGGATGGTTATTGAATCAAATGCTGGTGTAGAATTAAATCAGCCAC
TTGGAAGATGCGTTTATTTACATCATGTAGATAATATAGAGCAAATTTTACCTTATGTTAGAAAGAATAAAACT
CAAACAATATCTGTATTTCCATGGGAAGCAGCTTTAAAGTATAGAGATCTTTTAGCACTTAAAGGTGCTGAAAG
AATTGTTGAGGCAGGAATGAATAATATATTTAGAGTAGGTGGTGCTCATGATGGAATGAGGCCTTTACAGAGA
CTTGTTACTTATATAAGTCATGAAAGACCAAGTCATTATACAGCAAAAGATGTAGCTGTAGAGATTGAGCAAA
CTAGATTTTTAGAAGAAGATAAGTTTTTAGTATTTGTTCCTTAA SEQ ID NO: 6-經優化之LuxC胺基酸序列
MNKKISFIINGRVEIFPESDDLVQSINFGDNSVHLPVLNDSQVKNIIDYNENNELQLHNIINFLYTVGQRWKNEEYSRR
RTYIRDLKRYMGYSEEMAKLEANWISMILCSKGGLYDLVKNELGSRHIMDEWLPQDESYIRAFPKGKSVHLLTGNV
PLSGVLSILRAILTKNQCIIKTSSTDPFTANALALSFIDVDPHHPVTRSLSWYWQHQGDISLAKEIMQHADWVAWG
GEDAJNWAVKHAPPDIDVMKFGPKKSFCnDNPVDLVSAATGAAHDVCFYDQQACFSTQNIYYMGSHYEEFKLALI
EKLNLYAHILPNTKKDFDEKAAYSLVQKECLFAGLKVEVDVHQRWMVIESNAGVELNQPLGRCVYLHHVDNIEQI
LPYVRKNKTQTISVFPWEAALKYRDLLALKGAERIVEAGMNNIFRVGGAHDGMRPLQRLVTYISHERPSHYTAKDV
AVEIEQTRFLEEDKFLVFVP 76- 124909.doc 200827448 SEQ ID NO: 7-經優化之luxD核苷酸序列
ATGGAAAATAAAAGTAGATATAAGACAATAGATCATGTTATTTGTGTAGAGGAGAATAGAAAGATACATGTTT
GGGAAACTTTACCTAAAGAAAATTCACCAAAAAGAAAAAATACACTTATTATAGCATCTGGATTTGCTAGAAG
AATGGATCATTTTGCTGGTTTAGCTGAATATTTATCTCAAAATGGATTTCATGTAATTAGATATGATTCATTACA
TCATGTTGGTTTAAGTTCAGGAACTATAGATGAATTTACAATGTCAATTGGTAAGCAGAGTTTACTTGCAGTAG
TTGATTGGTTAAATACTAGAAAAATAAATAATCTTGGAATGTTAGCTAGTTCATTATCTGCAAGAATAGCTTAT
GCAAGTCTTTCAGAGATTAATGTATCTTTTCTTATAACAGCTGTTGGTGTAGTTAATTTAAGATATACTTTAGAA
AGAGCACTTGGATTTGATTATCTTAGCCTTCCTATTGATGAATTACCAGATAATCTTGATTTTGAGGGACATAA
GTTAGGTGCTGAAGTATTTGCAAGAGATTGCTTTGATTCAGGATGGGAAGATCTTACATCTACTATAAATAGTA
TGATGCACTTAGATATTCCTTTTATAGCTTTTACAGCAAATAATGATGATTGGGTTAAACAAGATGAGGTAATT
ACTCTTCTTTCTAGTATAAGAAGTCATCAGTGTAAAATATATTCACTTTTAGGTTCTAGTCATGATCTTGGAGAA
AATTTAGTTGTATTAAGAAATTTTTATCAATCAGTTACAAAGGCTGCAATTGCTATGGATAATGGTTGCCTTGA
TATAGATGTAGATATTATAGAACCATCTTTTGAGCATTTAACTATTGCAGCTGTTAATGAAAGAAGAATGAAAA
TAGAAATAGAGAATCAAGTAATTAGTTTAAGTTAA SEQ ID NO: 8-經優化之LuxD胺基酸序列
MENKSRYKTIDHVICVEENRKIHVWETLPKENSPKRKNTLnASGFARRMDHFAGLAEYLSQNGFHVIRYDSLHHVG
LSSGTIDEFTMSIGKQSLLAWDWLNTRKINNLGMLASSLSARIAYASLSEINVSFLITAVGVVNLRYTLERALGFDY
LSLPIDELPDNLDFEGHKLGAEVFARDCFDSGWEDLTSTINSMMHLDIPFIAFTANNDDWVKQDEVITLLSSIRSHQC
KIYSLLGSSHDLGENLWLRNFYQSVTKAAIAMDNGCLDIDVDIIEPSFEHLTIAAVNERRMKIEIENQVISLS SEQ ID NO: 9-經優化之luxE核苷酸序列
ATGACATCTTATGTTGATAAACAAGAAATAACTGCAAGTTCAGAGATTGATGATTTAATATTTAGTTCAGATCC
TCTTGTATGGTCTTATGATGAACAGGAAAAGATTAGAAAAAAGTTAGTTCTTGATGCTTTTAGACATCATTATA
AACATTGTCAAGAGTATAGACATTATTGCCAGGCACATAAAGTAGATGATAATATAACAGAAATTGATGATAT
ACCAGTTTTTCCTACTTCAGTATTTAAGTTTACAAGATTACTTACTTCAAATGAAAATGAGATTGAATCATGGTT
TACAAGTTCAGGAACTAATGGTTTAAAATCTCAAGTTCCAAGAGATAGACTTAGTATAGAAAGACTTTTAGGA
TCAGTATCTTATGGTATGAAGTATATAGGAAGTTGGTTTGATCATCAAATGGAGTTAGTTAATCTTGGTCCTGA
TAGATTTAATGCTCATAATATTTGGTTTAAATATGTAATGTCACTTGTAGAACTTTTATATCCAACAAGTTTTAC
TGTAACAGAAGAGCATATAGATTTTGTTCAGACTTTAAATAGTCTTGAAAGAATTAAACATCAAGGAAAGGAT
ATATGTTTAATTGGTTCACCTTATTTTATATATCTTTTATGCAGATATATGAAAGATAAGAATATTTCTTTTAGT
GGAGATAAATCACTTTATATAATAACTGGAGGTGGATGGAAATCTTATGAAAAGGAGAGTTTAAAAAGAAATG
ATTTTAATCATCTTTTATTTGATACTTTTAATCTTTCAAATATTAATCAAATAAGAGATATTTTTAATCAGGTAG
AATTAAATACATGTTTTTTTGAGGATGAAATGCAAAGAAAACATGTTCCACCTTGGGTATATGCAAGGGCTCTT
GATCCAGAAACTTTAAAGCCTGTTCCAGATGGTATGCCTGGACTTATGTCTTATATGGATGCTTCAAGTACTAG
TTATCCAGCTTTTATAGTAACTGATGATATTGGTATAATAAGTAGAGAATATGGACAATATCCTGGAGTTTTAG
TTGAGATTTTAAGAAGAGTTAATACAAGAAAACAGAAGGGTTGTGCACTTTCATTAACTGAGGCTTTTGGATCT
TGA -77- 124909.doc 200827448 SEQ ID NO: 10-經優化之LuxE胺基酸序列
MTSYVDKQEITASSEIDDLIFSSDPLVWSYDEQEKIRKKLVLDAFRHHYKHCQEYRHYCQAHKVDDNITEIDDIPVFP
TSVFKFTRLLTSNENEIESWFTSSGTNGLKSQVPRDRLSIERLLGSVSYGMKYIGSWFDHQMELVNLGPDRFNAHNI
WFKYVMSLVELLYPTSFTVTEEHIDFVQTLNSLERIKHQGKDICLIGSPYFIYLLCRYMKDKNISFSGDKSLYIITGGG
WKSYEKESLKRNDFNHLLFDTFNLSNtNQIRDIFNQVELNTCFFEDEMQRKHVPPWVYARALDPETLKPVPDGMPG
LMSYMDASSTSYPAFIVTDDIGIISREYGQYPGVLVEILRRVNTRKQKGCALSLTEAFGS SEQ ID NO: 11·由革蘭氏陽性(gram-positive)核糖體結合位 點分開之經優化之LuxCDABE基因
U aattcgaattctcagactcaaatagaacaggattctaaagacttaagagcagctgtagatcgtgattttagtacgatagagccaacattgegaaattatggggcaacggaagcacaactt gaagacgccagagccaaaatacacaagcttaaccaagaacagaggttatacaaatgacagttaatacagaggcactaataaacagcctaggcaagtcctaccaagaaatttttgatga agggctaattccttataggaataagccaagtggttctcctggggtgcctaatatttgtattgacatggtgaaagaggggatttttttgtcgtttgaacggaatagtaaaatattaaacgaaatt actttaagattgcttagagacgataaagctttgtttatatttccaaatgaattgccatcaccgttgaagcattctatggataggggatgggttagagaaaatttaggtgatctgattaaatceat accaccgagacaaattttaaaaaggcagtttggttggaaagatctatatcgttttacggatgaaatcagtatgcagatttcttatgatttacgtgaacaggttaattcagtgactttcttgcttac atcagacgtgagttggtaatttaatatatatacccttcatccttcaagttgctgctttgttggctgctttctctcaccccagtcacatagttatctatgctcctggggattcgttcacttgccgcxjg cgctgcaacttgaaatctattgggtatatgctattggtaattatggaaaattgcctgatttatatataacttaacttgtaaaccegataataatttacatgaatattatcacgtataaaaaaattgc gattcttttaatttgaaatagttcaatttaattgaaactttttattaacaaatcttgttgatgtgaaaattttcgtttgctattttaacagatattgttaaacggagaaggcagcatgttgatgattcact eagccagactgacagttttaagcggaaaattgcagagtatgatcgcattctgataaaggttacaggtcactcgcaaccagaatttcatctttgtatattttgttttgttatttacgttgcagcaa gacaaaaatagaagaaacaaatatttatacaacccgtttgcaagagggttaaacagcaatttaagttgaaattgccctattaaatggagcatgcggatcctcgactttttaacaaaatatatt gataaaaataataggatccgggcccctcgagaggaggatggcaaatatgaataaaaagatatcatttattataaatggaagagttgaaatatttcctgagtcagatgatttagtacaatcta taaattttggtgataattctgttcatcttccagtacttaatgattcacaggttaagaatattatagattataatgagaataatgagcttcagcttcataatattataaattttctttatacagtaggaca gagatggaagaatgaggagtatagcagaagaagaacttatataagagatcttaagagatatatgggttatagtgaggaaatggcaaaattagaagctaattggatttcaatgatattatgtt ctaagggaggtttatatgatttagttaaaaatgaattaggaagtagacatattatggatgaatggttacctcaagatgaatcatatataagagcatttccaaaaggtaaaagtgtacatctttta acaggaaatgttcctttaagtggagtactttcaattttaagagctatacttactaaaaatcagtgcattataaagacatctagtactgatccatttacagcaaatgctttagcacttagttttatag gatgcaattaattgggctgtaaagcacgcacctccagatatagatgttatgaaatttggacctaaaaagtctttttgtattatagataatccagtagatttagttagtgctgcaacaggtgctgc acatgatgtatgcttttatgatcagcaggcttgtttttcaactc ⑽ aatatetattatatgggatcacattatgaagaa伽aacttg^^ aatacaaagaaagattttgatgaaaaggcagcttatagtttagttcagaaagaatgtttatttgcaggacttaaagtagaagttgatgtacatcaaagatggatggttattgaatcaaatgctg gtgtagaattaaatcagccacttggaagatgc 抑 atttecatcatgtagat ⑽tatagagcaaat伽ccttatgttaga 姐 gaataaaacte^ gctttaaagtatagagatcttttagcacttaaaggtgctgaaagaattgttgaggcaggaatgaataatatatttagagtaggtggtgctcatgatggaatgaggcctttacagagacttgtt acttatataagtcatgaaagaccaagtcattatacagcaaaagatgtagctgtagagattgagcaaactagatttttagaagaagataagtttttagtatttgttccttaataggaggtaaaag aatatggaaaataaaagtagatataagacaatagatcatgttatttgtgtagaggagaatagaaagatacatgtttgggaaactttacctaaagaaaattcaccaaaaagaaaaaatacact tattatagcatctggatttgctagaagaatggatcattttgctggtttagctgaatatttatctcaaaatggatttcatgtaattagatatgattcattacatcatgttggtttaagttcaggaactat 78- 124909.doc 200827448 ttgagggacataagttaggtgctgaagtatttgcaagagattgctttgattcaggatgggaagatcttacatctactataaatagtatgatgcacttagatattccttttatagcttttacagcaa ataatgatgattgggttaaacaagatgaggtaattactcttctttctagtataagaagtcatcagtgtaaaatatattcacttttaggttctagtcatgatcttggagaaaatttagttgtattaaga aatttttatcaatcagttacaaaggctgcaattgctatggataatggttgccttgatatagatgtagatattatagaaccatcttttgagcatttaactattgcagctgttaatgaaagaagaatg aaaatagaaatagagaatcaagtaattagtttaagttaaaacctataccaatagatttcgagttgcagcgcggcggcaagtgaacgcattcccaggagcatagataactctgtgactggg gtgcgtgaaagcagccaacaaagcagcaacttgaaggatgaagggtatattgggatagatagttaactctatcactcaaatagaaatatactgcaggcggccgcaggaggactctcta tgaaatttggaaattttttacttacatatcaacctccacagtttagtcaaactgaagttatgaagagattagtaaaacttggtagaatatcagaggaatgtggatttgatacagtttggttacttg aacatcattttactgagtttggtcttttaggaaatccttatgtagcagctgcatatttacttggtgctacaaagaaattaaatgtaggtacagcagctattgttttacctacagcacatcctgttag acagttagaagaagtaaatcttttagatcaaatgtctaaaggtagatttagatttggaatatgcagaggattatataataaggattttagagtttttggtactgatatgaataatagtagggctct tatggagtgttggtataaattaattagaaatggaatgacagaaggttatatggaagcagataatgagcatataaagtttcataaagtaaaagtacttccaactgcttattcacagggaggtg cacctatttatgtagttgctgaatctgcaagtacaactgaatgggctgcacagcatggattaccaatgatactttcatggattataaatacaaatgagaagaaagctcaaatagaattatata atgaagtagcacaagagtatggacatgatattcataatatagatcattgcctttcttatattactagtgttgatcatgattcaatgaaagctaaagaaatatgtagaaattttttaggtcattggta tgattcttatgtaaatgcaacaactatttttgatgatagtgataaaacaaagggatatgattttaataaaggtcagtggagagattttgttcttaaaggacataagaatactaatagaagagtag attattcatatgaaataaatcctgttggaactccacaagagtgtattgatataatacaaactgatattgatgctacaggaatatctaatatttgctgtggatttgaagcaaatggtactgtagatg aaataattagtagtatgaagttatttcagtctgatgttatgccttttcttaaggagaaacaaagaagtttactttattagctaaggaggaaaatgaaatgaaatttggattattttttcttaattttata aatagtacaactattcaagaacagtcaatagcaagaatgcaggagattacagagtatgttgataagctaaattttgagcagattcttgtatgtgaaaatcatttttcagataatggtgttgtag
gtgctcctttaactgttagtggttttttattaggacttacagaaaaaattaagataggttcattaaatcatgtaattactacacatcatccagttagaatagcagaagaggcttgccttttagatca cgcattaactacaggatattgtaatccaaatggagatttttataattttcctaaaatttcagtaaatccacatgcttatactcagaatggtcctagaaagtatgttacagcaacttcttgtcatgta gttgaatgggcagctaagaagggtataccattaatttttaaatgggatgatagtaatgaagtaaaacatgagtatgctaagagatatoaagcaatagctggtgaatatggagttgatcttgc agaaattgatcatcaattaatgatattagttaattattcagaggattctgaaaaagctaaggaagagacaagagcatttataagtgattatattttagctatgcaccctaatgaaaattttgaaa aaaaacttgaggaaataataactgaaaattcagttggtgattatatggagtgcacaactgctgcaaaacttgcaatggaaaaatgtggagctaaaggtattcttttatcttttgaaagtatgtc agattttacacatcagattaatgcaatagatatagtaaatgataatattaagaaatatcatatgtaatataccctatggatttcaaggtgcatcgcgacggcaagggagcgaatccccggga gcatatacccaatagatttcaagttgcagtgcggcggcaagtgaacgcatccccaggagcatagataactatgtgactggggtaagtgaacgcagccaacaaagcagcagcttgaaa gatgaagggtatagataacgatgtgaccggggtgcgtgaacgcagccaacaaagaggcaacttgaaagataacgggtataaaagggtatagcagtcactctgccatatcctttaatatt agctgccggctagcaggaggtaaaacaggtatgacatcttatgttgata ⑽caagaaataactgcaagUcagag^ aacaggaaaagattagaaaaaagttagttcttgatgcttttagacatcattataaacattgtcaagagtatagacattattgccaggcacataaagtagatgataatataacagaaattgatg atataccagtttttcctacttcagtatttaagtttacaagattacttacttcaaatgaaaatgagattgaatcatggtttacaagttcaggaactaatggtttaaaatctcaagttccaagagatag acttagtatagaaagacttttaggatcagtatcttatggtatgaagtatataggaagttggtttgatcatcaaatggagttagttaatcttggtcctgatagatttaatgctcataatatttggttta aatatgtaatgtcacttgtagaacttttatatccaacaagttttactgtaacagaagagcatatagattttgttcagactttaaatagtcttgaaagaattaaacatcaaggaaaggatatatgttt aattggttcaccttattttatatatcttttatgcagatatatgaaagataagaatatttcttttagtggagataaatcactttatataataactggaggtggatggaaatcttatgaaaaggagagtt taaaaagaaatgattttaatcatct伽mgatecttttaatc伽臟tattaatc 臟teagagate腿 aatcaggtagaa伽 atecatgtttttttgagga^^ tgttccaccttgggtatatgcaagggctcttgatc^agaaactttaaagcctgttccagatggtatgcctgga gatgatattggtataataagtagagaatatggacaatatcctggagttttagttgagattttaagaagagttaatacaagaaaacagaagggttgtgcactttcattaactgaggcttttggat cttgaatgcatgtcgactctagagcatgctagtttctttggaaagaggagcagtcaaaggctcatttgttcaatgcttttgcgaaacgttttgtcgaactctaggcgaaggttctcgactttcc cxjgcatcaggggtatatacaagtaaaaaagctcagggggtaaacctgagcttgggatgttgatttttaagtatgagatacatgggcggatttaaataacggagtcagtttggaaatatcaa cggtcttttctgctttatcgaggctataagtttcttgcagttttaaccacaaccgcggagagctgccaagtacttgtgacagttttattgccatctctggcgtgactgctgctttacacgatacta aacgttgaaccgtagagggagcaacattcaatgcccgcgctaagttcacgaattc 【圖式簡單說明】 圖1描述多種在產酸或產溶劑階段期間具有活性之丙_ 丁醇梭菌中之生化途徑。由以 下字母識別催化特定反應之酵素:(A)甘油醛3-磷酸脫氫 酶;(B)丙酮酸-鐵氧還蛋白氧化還原酶;(C)NADH-鐵氧還 -79- 124909.doc 200827448 蛋白氧化還原酶;(D)NADPH-鐵氧還蛋白氧化還原酶; (E)NADH-紅素氧還蛋白氧化還原酶;(F)氫化酶;(G)磷酸 轉乙醯基酶(磷酸乙醯基轉移酶,CAC1742) ; (H)乙酸 激酶,CAC1743) ; (I)乙醯_CoA乙醯基轉移酶(硫解 酶)(ί/π·/,CAP0078 及 CAC2873); (J)3-羥基丁醯-CoA 脫氫 酶;(K)巴豆酸酶(3-羥基丁醯-CoA脫水酶、β-羥基丁醯-CoA脫氫酶,CAC2708) ; (L) 丁醯-CoA脫氫酶, CAC2711) ; (Μ)磷酸 丁醯基轉移酶 〇7炀,CAC3076) ; (N) 丁 酸激酶CAC3075及CAC1660); (Ο)乙醛脫氫酶(可能 為 adhel,CAP0162^ adhe ^ CAP0035) ; (P)乙醇脫氫酶 (adhel , CAP0162 ; bdhB , CAC3298 ;及 bdhA , CAC3299) ; (Q)T ^ m Si ^ (adhel , CAP0162 A adhe ^ CAP0035) ; (R) T S| 1¾ lL SI {adhel , CAP0162 ; adhe, CAP0035 ; adh , CAP0059 ; bdhB , CAC3298 ; bdhA , CAC3299 ;及CAC3392) ; (S)丁酸乙醯乙酸CoA轉移酶(乙 醯乙醯-CoA :乙酸 /丁酸·· CoA轉移酶,CAP0163(A) 反ctfb,CAP0164(B) ; (T)乙醯乙酸脫羧酶〇☆, CAP0165) ; (U)丙酮酸脫羧酶〇?☆,CAP0025)。所選酵素 進一步詳述於表1中。其他可見於易於可用之參考材料, 諸如基因組研究院(The Institute for Genomic Research)之 網站(www.tigr.org) 〇 124909.doc -80 · 200827448 序列表 <110>美商科寶科技公司 <12〇>微生物酵素途徑之即時監測 \ <130> 32897-701.601 <140〉 096134723 <141> 2007-09-17 <150> 60/882,834 ' <151> 2006-12-29 <160> 11 <170> Patentln Ver. 3.3
<210〉 1 <211> 975 <212> DNA 丙酮丁醇梭桿菌 ( <400〉 1 atgaaatttg gattattttt tcttaatttt ataaatagta caactattca agaacagtca 60 atagcaagaa tgcaggagat tacagagtat gttgataagc taaattttga gcagattctt 120 gtatgtgaaa atcatttttc agataatggt gttgtaggtg ctcctttaac tgttagtggt 180 tttttattag gacttacaga aaaaattaag ataggttcat taaatcatgt aattactaca 240 catcatccag ttagaatagc agaagaggct tgccttttag atcaactttc tgaaggaaga 300 tttatattag gttttagtga ttgtgaaaga aaagatgaga tgcacttttt taatagacct 360 gaacaatatc aacaacaact ttttgaagag tgctatgata ttataaatga cgcattaact 420 acaggatatt gtaatccaaa tggagatttt tataattttc ctaaaatttc agtaaatcca 480 catgcttata ctcagaatgg tcctagaaag tatgttacag caacttcttg tcatgtagtt 540 gaatgggcag ctaagaaggg tataccatta atttttaaat gggatgatag taatgaagta 600 aaacatgagt atgctaagag atatcaagca atagctggtg aatatggagt tgatcttgca 660 gaaattgatc atcaattaat gatattagtt aattattcag aggattctga aaaagctaag 720 gaagagacaa gagcatttat aagtgattat attttagcta tgcaccctaa tgaaaatttt 780 gaaaaaaaac ttgaggaaat aataactgaa aattcagttg gtgattatat ggagtgcaca 840 actgctgcaa aacttgcaat ggaaaaatgt ggagctaaag gtattctttt atcttttgaa 900 agtatgtcag attttacaca tcagattaat gcaatagata tagtaaatga taatattaag 960 aaatatcata tgtaa 975
<210〉 2 <211> 360 <212〉 PRT <213>丙酮丁醇梭桿菌 <400〉 2
Met Lys Phe Gly Asn Phe Leu Leu Thr Tyr Gin Pro Pro Gin Phe Ser 15 10 15
Gin Thr Glu Val Met Lys Arg Leu Val Lys Leu Gly Arg lie Ser Glu 20 25 30
Glu Cys Gly Phe Asp Thr Val Trp Leu Leu Glu His His Phe Thr Glu 35 40 45
Phe Gly Leu Leu Gly Asn Pro Tyr Val Ala Ala Ala Tyr Leu Leu Gly 50 55 60
Ala Thr Lys Lys Leu Asn Val Gly Thr Ala Ala lie Val Leu Pro Thr 65 70 75 80
Ala His Pro Val Arg Gin Leu Glu Glu Val Asn Leu Leu Asp Gin Met 85 90 95
Ser Lys Gly Arg Phe Arg Phe Gly lie Cys Arg Gly Leu Tyr Asn Lys 100 105 110
Asp Phe Arg Val Phe Gly Thr Asp Met Asn Asn Ser Arg Ala Leu Met 115 120 125
Glu Cys Trp Tyr Lys Leu lie Arg Asn Gly Met Thr Glu Gly Tyr Met 130 135 140 124909-序列表.doc 200827448
Glu Ala Asp Asn Glu His lie Lys Phe His Lys Val Lys Val Leu Pro 145 150 155 160
Thr Ala Tyr Ser Gin Gly Gly Ala Pro lie Tyr Val Val Ala Glu Ser 165 170 175
Ala Ser Thr Thr Glu Trp Ala Ala Gin His Gly Leu Pro Met lie Leu 180 185 190
Ser Trp lie lie Asn Thr Asn Glu Lys Lys Ala Gin lie Glu Leu Tyr 195 200 205
Asn Glu Val Ala Gin Glu Tyr Gly His Asp lie His Asn lie Asp His 210 215 220
Cys Leu Ser Tyr He Thr Ser Val Asp His Asp Ser Met Lys Ala Lys 225 230 235 240
Glu lie Cys Arg Asn Phe Leu Gly His Trp Tyr Asp Ser Tyr Val Asn 245 250 255
Ala Thr Thr lie Phe Asp Asp Ser Asp Lys Thr Lys Gly Tyr Asp Phe 260 265 270
o
Asn Lys Gly Gin Trp Arg Asp Phe Val Leu Lys Gly His Lys Asn Thr 275 280 285
Asn Arg Arg Val Asp Tyr Ser Tyr Glu lie Asn Pro Val Gly Thr Pro 290 295 300
Gin Glu Cys lie Asp lie lie Gin Thr Asp lie Asp Ala Thr Gly lie 305 310 315 320
Ser Asn lie Cys Cys Gly Phe Glu Ala Asn Gly Thr Val Asp Glu lie 325 330 335 lie Ser Ser Met Lys Leu Phe Gin Ser Asp Val Met Pro Phe Leu Lys 340 345 350
Glu Lys Gin Arg Ser Leu Leu Tyr 355 360
<210〉 3 <211> 975 <212> DNA <213>丙酮丁醇梭桿菌 ; <400〉 3 atgaaatttg gattattttt tcttaatttt ataaatagta caactattca agaacagtca 60 atagcaagaa tgcaggagat tacagagtat gttgataagc taaattttga gcagattctt 120 gtatgtgaaa atcatttttc agataatggt gttgtaggtg ctcctttaac tgttagtggt 180 tttttattag gacttacaga aaaaattaag ataggttcat taaatcatgt aattactaca 240 catcatccag ttagaatagc agaagaggct tgccttttag atcaactttc tgaaggaaga 300 tttatattag gttttagtga ttgtgaaaga aaagatgaga tgcacttttt taatagacct 360 gaacaatatc aacaacaact ttttgaagag tgctatgata ttataaatga cgcattaact 420 acaggatatt gtaatccaaa tggagatttt tataattttc ctaaaatttc agtaaatcca 480 catgcttata ctcagaatgg tcctagaaag tatgttacag caacttcttg tcatgtagtt 540 gaatgggcag ctaagaaggg tataccatta atttttaaat gggatgatag taatgaagta 600 aaacatgagt atgctaagag atatcaagca atagctggtg aatatggagt tgatcttgca 660 gaaattgatc atcaattaat gatattagtt aattattcag aggattctga aaaagctaag 720 gaagagacaa gagcatttat aagtgattat attttagcta tgcaccctaa tgaaaatttt 780 gaaaaaaaac ttgaggaaat aataactgaa aattcagttg gtgattatat ggagtgcaca 840 actgctgcaa aacttgcaat ggaaaaatgt ggagctaaag gtattctttt atcttttgaa 900 agtatgtcag attttacaca tcagattaat gcaatagata tagtaaatga taatattaag 960 aaatatcata tgtaa 975
<210> 4 <211> 324 <212> PRT <213>丙酮丁醇梭桿菌 <400> 4 124909-序列表.doc 200827448
Met Lys Phe Gly Leu Phe Phe Leu Asn Phe lie Asn Ser Thr Thr lie 15 10 15
Gin Glu Gin Ser lie Ala Arg Met Gin Glu lie Thr Glu Tyr Val Asp 20 25 30
Lys Leu Asn Phe Glu Gin lie Leu Val Cys Glu Asn His Phe Ser Asp 35 40 45
Asn Gly Val Val Gly Ala Pro Leu Thr Val Ser Gly Phe Leu Leu Gly 50 55 60
Leu Thr Glu Lys lie Lys lie Gly Ser Leu Asn His Val lie Thr Thr 65 70 75 80
His His Pro Val Arg lie Ala Glu Glu Ala Cys Leu Leu Asp Gin Leu 85 90 95
Ser Glu Gly Arg Phe lie Leu Gly Phe Ser Asp Cys Glu Arg Lys Asp 100 105 110
Glu Met His Phe Phe Asn Arg Pro Glu Gin Tyr Gin Gin Gin Leu Phe 115 120 125
Glu Glu Cys Tyr Asp lie lie Asn Asp Ala Leu Thr Thr Gly Tyr Cys 130 135 140
Asn Pro Asn Gly Asp Phe Tyr Asn Phe Pro Lys lie Ser Val Asn Pro 145 150 155 160
His Ala Tyr Thr Gin Asn Gly Pro Arg Lys Tyr Val Thr Ala Thr Ser 165 170 175
Cys His Val Val Glu Trp Ala Ala Lys Lys Gly lie Pro Leu lie Phe 180 185 190
Lys Trp Asp Asp Ser Asn Glu Val Lys His Glu Tyr Ala Lys Arg Tyr 195 200 205
Gin Ala lie Ala Gly Glu Tyr Gly Val Asp Leu Ala Glu lie Asp His 210 215 220
Gin Leu Met lie Leu Val Asn Tyr Ser Glu Asp Ser Glu Lys Ala Lys 225 230 235 240
Glu Glu Thr Arg Ala Phe lie Ser Asp Tyr lie Leu Ala Met His Pro 245 250 255
Asn Glu Asn Phe Glu Lys Lys Leu Glu Glu lie lie Thr Glu Asn Ser 260 265 270
Val Gly Asp Tyr Met Glu Cys Thr Thr Ala Ala Lys Leu Ala Met Glu 275 280 285
Lys Cys Gly Ala Lys Gly lie Leu Leu Ser Phe Glu Ser Met Ser Asp 290 295 300
Phe Thr His Gin lie Asn Ala He Asp lie Val Asn Asp Asn lie Lys 305 310 315 320
Lys Tyr His Met
<210〉 5 <211> 1443 <212> DNA •丙酮丁醇梭桿菌 <400> 5 atgaataaaa agatatcatt tattataaat ggaagagttg aaatatttcc tgagtcagat 60 gatttagtac aatctataaa ttttggtgat aattctgttc atcttccagt acttaatgat 120 tcacaggtta agaatattat agattataat gagaataatg agcttcagct tcataatatt 180 ataaattttc tttatacagt aggacagaga tggaagaatg aggagtatag cagaagaaga 240 acttatataa gagatcttaa gagatatatg ggttatagtg aggaaatggc aaaattagaa 300 124909·序列表.doc 200827448 gctaattgga tttcaatgat attatgttct aagggaggtt tatatgattt agttaaaaat 360 gaattaggaa gtagacatat tatggatgaa tggttacctc aagatgaatc atatataaga 420 gcatttccaa aaggtaaaag tgtacatctt ttaacaggaa atgttccttt aagtggagta 480 ctttcaattt taagagctat acttactaaa aatcagtgca ttataaagac atctagtact 540 gatccattta cagcaaatgc tttagcactt agttttatag atgttgatcc tcatcatcca 600 gtaactagat ctttaagtgt tgtatattgg caacatcaag gtgatatttc acttgctaaa 660 gaaataatgc aacatgcaga tgttgtagtt gcttggggag gtgaagatgc aattaattgg 720 gctgtaaagc acgcacctcc agatatagat gttatgaaat ttggacctaa aaagtctttt 780 tgtattatag ataatccagt agatttagtt agtgctgcaa caggtgctgc acatgatgta 840 tgcttttatg atcagcaggc ttgtttttca actcaaaata tatattatat gggatcacat 900 tatgaagaat ttaaacttgc attaattgaa aaacttaatt tatatgctca tatacttcca 960 aatacaaaga aagattttga tgaaaaggca gcttatagtt tagttcagaa agaatgttta 1020 tttgcaggac ttaaagtaga agttgatgta catcaaagat ggatggttat tgaatcaaat 1080 gctggtgtag aattaaatca gccacttgga agatgcgttt atttacatca tgtagataat 1140 atagagcaaa ttttacctta tgttagaaag aataaaactc aaacaatatc tgtatttcca 1200 tgggaagcag ctttaaagta tagagatctt ttagcactta aaggtgctga aagaattgtt 1260 gaggcaggaa tgaataatat atttagagta ggtggtgctc atgatggaat gaggccttta 1320 cagagacttg ttacttatat aagtcatgaa agaccaagtc attatacagc aaaagatgta 1380 gctgtagaga ttgagcaaac tagattttta gaagaagata agtttttagt atttgttcct 1440 taa 1443
ij
<210〉6 <211> 480 <212> PRT <213>丙酮丁醇梭桿菌 <400〉 6
Met Asn Lys Lys lie Ser Phe lie lie Asn Gly Arg Val Glu lie Phe 15 10 15
Pro Glu Ser Asp Asp Leu Val Gin Ser lie Asn Phe Gly Asp Asn Ser 20 25 30
Val His Leu Pro Val Leu Asn Asp Ser Gin Val Lys Asn lie lie Asp 35 40 45
Tyr Asn Glu Asn Asn Glu Leu Gin Leu His Asn lie lie Asn Phe Leu 50 55 60
Tyr Thr Val Gly Gin Arg Trp Lys Asn Glu Glu Tyr Ser Arg Arg Arg 65 70 75 80
Thr Tyr lie Arg Asp Leu Lys Arg Tyr Met Gly Tyr Ser Glu Glu Met 85 90 95
Ala Lys Leu Glu Ala Asn Trp lie Ser Met lie Leu Cys Ser Lys Gly 100 105 110
Gly Leu Tyr Asp Leu Val Lys Asn Glu Leu Gly Ser Arg His lie Met 115 120 125
Asp Glu Trp Leu Pro Gin Asp Glu Ser Tyr lie Arg Ala Phe Pro Lys 130 135 140
Gly Lys Ser Val His Leu Leu Thr Gly Asn Val Pro Leu Ser Gly Val 145 150 155 160
Leu Ser lie Leu Arg Ala lie Leu Thr Lys Asn Gin Cys lie lie Lys 165 170 175
Thr Ser Ser Thr Asp Pro Phe Thr Ala Asn Ala Leu Ala Leu Ser Phe 180 185 190
He Asp Val Asp Pro His His Pro Val Thr Arg Ser Leu Ser Val Val 195 200 205
Tyr Trp Gin His Gin Gly Asp lie Ser Leu Ala Lys Glu lie Met Gin 210 215 220
His Ala Asp Val Val Val Ala Trp Gly Gly Glu Asp Ala lie Asn Trp 225 230 235 240
Ala Val Lys His Ala Pro Pro Asp lie Asp Val Met Lys Phe Gly Pro 245 250 255 •4 124909-序列表.d〇c 200827448
Lys Lys Ser Phe Cys lie lie Asp Asn Pro Val Asp Leu Val Ser Ala 260 265 270
Ala Thr Gly Ala Ala His Asp Val Cys Phe Tyr Asp Gin Gin Ala Cys 275 280 285
Phe Ser Thr Gin Asn lie Tyr Tyr Met Gly Ser His Tyr Glu Glu Phe 290 295 300
Lys Leu Ala Leu lie Glu Lys Leu Asn Leu Tyr Ala His lie Leu Pro 305 310 315 320
Asn Thr Lys Lys Asp Phe Asp Glu Lys Ala Ala Tyr Ser Leu Val Gin 325 330 335
Lys Glu Cys Leu Phe Ala Gly Leu Lys Val Glu Val Asp Val His Gin 340 345 350
Arg Trp Met Val lie Glu Ser Asn Ala Gly Val Glu Leu Asn Gin Pro 355 360 365
Leu Gly Arg Cys Val Tyr Leu His His Val Asp Asn lie Glu Gin lie 370 375 380
Leu Pro Tyr Val Arg Lys Asn Lys Thr Gin Thr lie Ser Val Phe Pro 385 390 395 400
Trp Glu Ala Ala Leu Lys Tyr Arg Asp Leu Leu Ala Leu Lys Gly Ala 405 410 415
Glu Arg lie Val Glu Ala Gly Met Asn Asn lie Phe Arg Val Gly Gly 420 425 430
Ala His Asp Gly Met Arg Pro Leu Gin Arg Leu Val Thr Tyr lie Ser 435 440 445
His Glu Arg Pro Ser His Tyr Thr Ala Lys Asp Val Ala Val Glu lie 450 455 460
Glu Gin Thr Arg Phe Leu Glu Glu Asp Lys Phe Leu Val Phe Val Pro 465 470 475 480
<210〉 7 <211> 924 <212> DNA <213>丙酮丁醇梭桿菌 <400> 7 atggaaaata aaagtagata taagacaata gatcatgtta tttgtgtaga ggagaataga 60 aagatacatg tttgggaaac tttacctaaa gaaaattcac caaaaagaaa aaatacactt 120 attatagcat ctggatttgc tagaagaatg gatcattttg ctggtttagc tgaatattta 180 tctcaaaatg gatttcatgt aattagatat gattcattac atcatgttgg tttaagttca 240 ggaactatag atgaatttac aatgtcaatt ggtaagcaga gtttacttgc agtagttgat 300 tggttaaata ctagaaaaat aaataatctt ggaatgttag ctagttcatt atctgcaaga 360 atagcttatg caagtctttc agagattaat gtatcttttc ttataacagc tgttggtgta 420 gttaatttaa gatatacttt agaaagagca cttggatttg attatcttag ccttcctatt 480 gatgaattac cagataatct tgattttgag ggacataagt taggtgctga agtatttgca 540 agagattgct ttgattcagg atgggaagat cttacatcta ctataaatag tatgatgcac 600 ttagatattc cttttatagc ttttacagca aataatgatg attgggttaa acaagatgag 660 gtaattactc ttctttctag tataagaagt catcagtgta aaatatattc acttttaggt 720 tctagtcatg atcttggaga aaatttagtt gtattaagaa atttttatca atcagttaca 780 aaggctgcaa ttgctatgga taatggttgc cttgatatag atgtagatat tatagaacca 840 tcttttgagc atttaactat tgcagctgtt aatgaaagaa gaatgaaaat agaaatagag 900 aatcaagtaa ttagtttaag ttaa 924
<210> 8 <211〉 307 <212> PRT <213〉丙酮丁醇梭桿菌 124909-序列表.doc 200827448 <400〉 8
Met Glu Asn Lys Ser Arg Tyr Lys Thr lie Asp His Val lie Cys Val 15 10 15
Glu Glu Asn Arg Lys lie His Val Trp Glu Thr Leu Pro Lys Glu Asn 20 25 30
Ser Pro Lys Arg Lys Asn Thr Leu lie lie Ala Ser Gly Phe Ala Arg 35 40 45
Arg Met Asp His Phe Ala Gly Leu Ala Glu Tyr Leu Ser Gin Asn Gly 50 55 60
Phe His Val lie Arg Tyr Asp Ser Leu His His Val Gly Leu Ser Ser 65 70 75 80
Gly Thr lie Asp Glu Phe Thr Met Ser lie Gly Lys Gin Ser Leu Leu 85 90 95
Ala Val Val Asp Trp Leu Asn Thr Arg Lys lie Asn Asn Leu Gly Met 100 105 110 Ο
Leu Ala Ser Ser Leu Ser Ala Arg lie Ala Tyr Ala Ser Leu Ser Glu 115 120 125 lie Asn Val Ser Phe Leu lie Thr Ala Val Gly Val Val Asn Leu Arg 130 135 140
Tyr Thr Leu Glu Arg Ala Leu Gly Phe Asp Tyr Leu Ser Leu Pro lie 145 150 155 160
Asp Glu Leu Pro Asp Asn Leu Asp Phe Glu Gly His Lys Leu Gly Ala 165 170 175
Glu Val Phe Ala Arg Asp Cys Phe Asp Ser Gly Trp Glu Asp Leu Thr 180 185 190
Ser Thr lie Asn Ser Met Met His Leu Asp lie Pro Phe lie Ala Phe 195 200 205
Thr Ala Asn Asn Asp Asp Trp Val Lys Gin Asp Glu Val lie Thr Leu 210 215 220
Leu Ser Ser lie Arg Ser His Gin Cys Lys lie Tyr Ser Leu Leu Gly 225 230 235 240
Ser Ser His Asp Leu Gly Glu Asn Leu Val Val Leu Arg Asn Phe Tyr 245 250 255
Gin Ser Val Thr Lys Ala Ala lie Ala Met Asp Asn Gly Cys Leu Asp 260 265 270
He Asp Val Asp lie lie Glu Pro Ser Phe Glu His Leu Thr lie Ala 275 280 285
Ala Val Asn Glu Arg Arg Met Lys lie Glu lie Glu Asn Gin Val lie 290 295 300
Ser Leu Ser 305 <210> 9 <211〉 1113 <212> DNA <213>丙酮丁醇梭桿菌 <400〉 9 atgacatctt atgttgataa acaagaaata actgcaagtt cagagattga tgatttaata 60 tttagttcag atcctcttgt atggtcttat gatgaacagg aaaagattag aaaaaagtta 120 gttcttgatg cttttagaca tcattataaa cattgtcaag agtatagaca ttattgccag 180 gcacataaag tagatgataa tataacagaa attgatgata taccagtttt tcctacttca 240 gtatttaagt ttacaagatt acttacttca aatgaaaatg agattgaatc atggtttaca 300 agttcaggaa ctaatggttt aaaatctcaa gttccaagag atagacttag tatagaaaga 360 cttttaggat cagtatctta tggtatgaag tatataggaa gttggtttga tcatcaaatg 420 -6- 124909-序列表.doc 200827448 gagttagtta atcttggtcc tgatagattt aatgctcata atatttggtt taaatatgta 480 atgtcacttg tagaactttt atatccaaca agttttactg taacagaaga gcatatagat 540 tttgttcaga ctttaaatag tcttgaaaga attaaacatc aaggaaagga tatatgttta 600 attggttcac cttattttat atatctttta tgcagatata tgaaagataa gaatatttct 660 tttagtggag ataaatcact ttatataata actggaggtg gatggaaatc ttatgaaaag 720 gagagtttaa aaagaaatga ttttaatcat cttttatttg atacttttaa tctttcaaat 780 attaatcaaa taagagatat ttttaatcag gtagaattaa atacatgttt ttttgaggat 840 gaaatgcaaa gaaaacatgt tccaccttgg gtatatgcaa gggctcttga tccagaaact 900 ttaaagcctg ttccagatgg tatgcctgga cttatgtctt atatggatgc ttcaagtact 960 agttatccag cttttatagt aactgatgat attggtataa taagtagaga atatggacaa 1020 tatcctggag ttttagttga gattttaaga agagttaata caagaaaaca gaagggttgt 1080 gcactttcat taactgaggc ttttggatct tga 1113
<210〉 10 <211> 370 <212〉 PRT <213>丙酮丁醇梭桿菌 <400> 10
Met Thr Ser Tyr Val Asp Lys Gin Glu lie Thr Ala Ser Ser Glu lie 1 5 10 15
Asp Asp Leu lie Phe Ser Ser Asp Pro Leu Val Trp Ser Tyr Asp Glu 20 25 30
Gin Glu Lys lie Arg Lys Lys Leu Val Leu Asp Ala Phe Arg His His 35 40 45
Tyr Lys His Cys Gin Glu Tyr Arg His Tyr Cys Gin Ala His Lys Val 50 55 60
Asp Asp Asn lie Thr Glu lie Asp Asp lie Pro Val Phe Pro Thr Ser 65 70 75 80
Val Phe Lys Phe Thr Arg Leu Leu Thr Ser Asn Glu Asn Glu lie Glu 85 90 95
Ser Trp Phe Thr Ser Ser Gly Thr Asn Gly Leu Lys Ser Gin Val Pro 100 105 110
Arg Asp Arg Leu Ser lie Glu Arg Leu Leu Gly Ser Val Ser Tyr Gly 115 120 125
Met Lys Tyr lie Gly Ser Trp Phe Asp His Gin Met Glu Leu Val Asn 130 135 140
Leu Gly Pro Asp Arg Phe Asn Ala His Asn lie Trp Phe Lys Tyr Val 145 150 155 160
Met Ser Leu Val Glu Leu Leu Tyr Pro Thr Ser Phe Thr Val Thr Glu 165 170 175
Glu His lie Asp Phe Val Gin Thr Leu Asn Ser Leu Glu Arg lie Lys 180 185 190
His Gin Gly Lys Asp lie Cys Leu lie Gly Ser Pro Tyr Phe lie Tyr 195 200 205
Leu Leu Cys Arg Tyr Met Lys Asp Lys Asn lie Ser Phe Ser Gly Asp 210 215 220
Lys Ser Leu Tyr lie lie Thr Gly Gly Gly Trp Lys Ser Tyr Glu Lys 225 230 235 240
Glu Ser Leu Lys Arg Asn Asp Phe Asn His Leu Leu Phe Asp Thr Phe 245 250 255
Asn Leu Ser Asn He Asn Gin lie Arg Asp lie Phe Asn Gin Val Glu 260 265 270
Leu Asn Thr Cys Phe Phe Glu Asp Glu Met Gin Arg Lys His Val Pro 275 280 285
Pro Trp Val Tyr Ala Arg Ala Leu Asp Pro Glu Thr Leu Lys Pro Val 124909-序列表.doc 200827448 290 295 300
Pro Asp Gly Met Pro Gly Leu Met Ser Tyr Met Asp Ala Ser Ser Thf 305 310 315 320
Ser Tyr Pro Ala Phe lie Val Thr Asp Asp lie Gly lie lie Ser Arg 325 330 335
Glu Tyr Gly Gin Tyr Pro Gly Val Leu Val Glu lie Leu Arg Arg Val 340 345 350
Asn Thr Arg Lys Gin Lys Gly Cys Ala Leu Ser Leu Thr Glu Ala Phe 355 360 365
Gly Ser 370
<210〉 11 <211> 7803 <212> DNA <213>人爲序列
<220> 合成之構築體 <223>人爲序列之敘述 <400> 11 aattcgaatt ctcagactca aatagaacag gattctaaag acttaagagc agctgtagat 60 cgtgatttta gtacgataga gccaacattg agaaattatg gggcaacgga agcacaactt 120 gaagacgcca gagccaaaat acacaagctt aaccaagaac agaggttata caaatgacag 180 ttaatacaga ggcactaata aacagcctag gcaagtccta ccaagaaatt tttgatgaag 240 ggctaattcc ttataggaat aagccaagtg gttctcctgg ggtgcctaat atttgtattg 300 acatggtgaa agaggggatt tttttgtcgt ttgaacggaa tagtaaaata ttaaacgaaa 360 ttactttaag attgcttaga gacgataaag ctttgtttat atttccaaat gaattgccat 420 caccgttgaa gcattctatg gataggggat gggttagaga aaatttaggt gatctgatta 480 aatcaatacc accgagacaa attttaaaaa ggcagtttgg ttggaaagat ctatatcgtt 540 ttacggatga aatcagtatg cagatttctt atgatttacg tgaacaggtt aattcagtga 600 ctttcttgct tacatcagac gtgagttggt aatttaatat atataccctt catccttcaa 660 gttgctgctt tgttggctgc tttctctcac cccagtcaca tagttatcta tgctcctggg 720 gattcgttca cttgccgccg cgctgcaact tgaaatctat tgggtatatg ctattggtaa 780 ttatggaaaa ttgcctgatt tatatataac ttaacttgta aaccagataa taatttacat 840 gaatattatc acgtataaaa aaattgcgat tcttttaatt tgaaatagtt caatttaatt 900 gaaacttttt attaacaaat cttgttgatg tgaaaatttt cgtttgctat tttaacagat 960 attgttaaac ggagaaggca gcatgttgat gattcactca gccagactga cagttttaag 1020 cggaaaattg cagagtatga tcgcattctg ataaaggtta caggtcactc gcaaccagaa 1080 tttcatcttt gtatattttg ttttgttatt tacgttgcag caagacaaaa atagaagaaa 1140 caaatattta tacaacccgt ttgcaagagg gttaaacagc aatttaagtt gaaattgccc 1200 tattaaatgg agcatgcgga tcctcgactt tttaacaaaa tatattgata aaaataatag 1260 gatccgggcc cctcgagagg aggatggcaa atatgaataa aaagatatca tttattataa 1320 atggaagagt tgaaatattt cctgagtcag atgatttagt acaatctata aattttggtg 1380 ataattctgt tcatcttcca gtacttaatg attcacaggt taagaatatt atagattata 1440 atgagaataa tgagcttcag cttcataata ttataaattt tctttataca gtaggacaga 1500 gatggaagaa tgaggagtat agcagaagaa gaacttatat aagagatctt aagagatata 1560 tgggttatag tgaggaaatg gcaaaattag aagctaattg gatttcaatg atattatgtt 1620 ctaagggagg tttatatgat ttagttaaaa atgaattagg aagtagacat attatggatg 1680 aatggttacc tcaagatgaa tcatatataa gagcatttcc aaaaggtaaa agtgtacatc 1740 ttttaacagg aaatgttcct ttaagtggag tactttcaat tttaagagct atacttacta 1800 aaaatcagtg cattataaag acatctagta ctgatccatt tacagcaaat gctttagcac 1860 ttagttttat agatgttgat cctcatcatc cagtaactag atctttaagt gttgtatatt 1920 ggcaacatca aggtgatatt tcacttgcta aagaaataat gcaacatgca gatgttgtag 1980 ttgcttgggg aggtgaagat gcaattaatt gggctgtaaa gcacgcacct ccagatatag 2040 atgttatgaa atttggacct aaaaagtctt tttgtattat agataatcca gtagatttag 2100 ttagtgctgc aacaggtgct gcacatgatg tatgctttta tgatcagcag gcttgttttt 2160 caactcaaaa tatatattat atgggatcac attatgaaga atttaaactt gcattaattg 2220 aaaaacttaa tttatatgct catatacttc caaatacaaa gaaagatttt gatgaaaagg 2280 cagcttatag tttagttcag aaagaatgtt tatttgcagg acttaaagta gaagttgatg 2340 tacatcaaag atggatggtt attgaatcaa atgctggtgt agaattaaat cagccacttg 2400 gaagatgcgt ttatttacat catgtagata atatagagca aattttacct tatgttagaa 2460 agaataaaac tcaaacaata tctgtatttc catgggaagc agctttaaag tatagagatc 2520 ttttagcact taaaggtgct gaaagaattg ttgaggcagg aatgaataat atatttagag 2580 taggtggtgc -tcatgatgga atgaggcctt tacagagact tgttacttat ataagtcatg 2640 aaagaccaag tcattataca gcaaaagatg tagctgtaga gattgagcaa actagatttt 2700 tagaagaaga taagttttta gtatttgttc cttaatagga ggtaaaagaa tatggaaaat 2760 aaaagtagat ataagacaat agatcatgtt atttgtgtag aggagaatag aaagatacat 2820 124909-序列表.doc 200827448
U gtttgggaaa ctttacctaa agaaaattca ccaaaaagaa aaaatacact tattatagca 2880 tctggatttg ctagaagaat ggatcatttt gctggtttag ctgaatattt atctcaaaat 2940 ggatttcatg taattagata tgattcatta catcatgttg gtttaagttc aggaactau 3000 gatgaattta caatgtcaat tggtaagcag agtttacttg cagtagttga ttggttaaat 3060 actagaaaaa taaataatct tggaatgtta gctagttcat tatctgcaag aatagcttat 3120 gcaagtcttt cagagattaa tgtatctttt cttataacag ctgttggtgt agttaattta 3180 agatatactt tagaaagagc acttggattt gattatctta gccttcctat tgatgaatta 3240 ccagataatc ttgattttga gggacataag ttaggtgctg aagtatttgc aagagattgc 3300 tttgattcag gatgggaaga tcttacatct actataaata gtatgatgca cttagatatt 3360 ccttttatag cttttacagc aaataatgat gattgggtta aacaagatga ggtaattact 3420 cttctttcta gtataagaag tcatcagtgt aaaatatatt cacttttagg ttctagtcat 3480 gatcttggag aaaatttagt tgtattaaga aatttttatc aatcagttac aaaggctgca 3540 attgctatgg ataatggttg ccttgatata gatgtagata ttatagaacc atcttttgag 3600 catttaacta ttgcagctgt taatgaaaga agaatgaaaa tagaaataga gaatcaagta 3660 attagtttaa gttaaaacct ataccaatag atttcgagtt gcagcgcggc ggcaagtgaa 3720 cgcattccca ggagcataga taactctgtg actggggtgc gtgaaagcag ccaacaaagc 3780 agcaacttga aggatgaagg gtatattggg atagatagtt aactctatca ctcaaataga 3840 aatatactgc aggcggccgc aggaggactc tctatgaaat ttggaaattt tttacttaca 3900 tatcaacctc cacagtttag tcaaactgaa gttatgaaga gattagtaaa .acttggtaga 3960 atatcagagg aatgtggatt tgatacagtt tggttacttg aacatcattt tactgagttt 4020 ggtcttttag gaaatcctta tgtagcagct gcatatttac ttggtgctac aaagaaatta 4080 aatgtaggta cagcagctat tgttttacct acagcacatc ctgttagaca gttagaagaa 4140 gtaaatcttt tagatcaaat gtctaaaggt agatttagat ttggaatatg cagaggatta 4200 tataataagg attttagagt ttttggtact gatatgaata atagtagggc tcttatggag 4260 tgttggtata aattaattag aaatggaatg acagaaggtt atatggaagc agataatgag 4320 catataaagt ttcataaagt aaaagtactt ccaactgctt attcacaggg aggtgcacct 4380 atttatgtag ttgctgaatc tgcaagtaca actgaatggg ctgcacagca tggattacca 4440 atgatacttt catggattat aaatacaaat gagaagaaag ctcaaataga attatataat 4500 gaagtagcac aagagtatgg acatgatatt cataatatag atcattgcct ttcttatatt 4560 actagtgttg atcatgattc aatgaaagct aaagaaatat gtagaaattt tttaggtcat 4620 tggtatgatt cttatgtaaa tgcaacaact atttttgatg atagtgataa aacaaaggga 4680 tatgatttta ataaaggtca gtggagagat tttgttctta aaggacataa gaatactaat 4740 agaagagtag attattcata tgaaataaat cctgttggaa ctccacaaga gtgtattgat 4800 ataatacaaa ctgatattga tgctacagga atatctaata tttgctgtgg atttgaagca 4860 aatggtactg tagatgaaat aattagtagt atgaagttat ttcagtctga tgttatgcct 4920 tttcttaagg agaaacaaag aagtttactt tattagctaa ggaggaaaat gaaatgaaat 4980 ttggattatt ttttcttaat tttataaata gtacaactat tcaagaacag tcaatagcaa 5040 gaatgcagga gattacagag tatgttgata agctaaattt tgagcagatt cttgtatgtg 5100 aaaatcattt ttcagataat ggtgttgtag gtgctccttt aactgttagt ggttttttat 5160 taggacttac agaaaaaatt aagataggtt cattaaatca tgtaattact acacatcatc 5220 cagttagaat agcagaagag gcttgccttt tagatcaact ttctgaagga agatttatat 5280 taggttttag tgattgtgaa agaaaagatg agatgcactt ttttaataga cctgaacaat 5340 atcaacaaca actttttgaa gagtgctatg atattataaa tgacgcatta actacaggat 5400 attgtaatcc aaatggagat ttttataatt ttcctaaaat ttcagtaaat ccacatgctt 5460 atactcagaa tggtcctaga aagtatgtta cagcaacttc ttgtcatgta gttgaatggg 5520 cagctaagaa gggtatacca ttaattttta aatgggatga tagtaatgaa gtaaaacatg 5580 agtatgctaa gagatatcaa gcaatagctg gtgaatatgg agttgatctt gcagaaattg 5640 atcatcaatt aatgatatta gttaattatt cagaggattc tgaaaaagct aaggaagaga 5700 caagagcatt tataagtgat tatattttag ctatgcaccc taatgaaaat tttgaaaaaa 5760 aacttgagga aataataact gaaaattcag ttggtgatta tatggagtgc acaactgctg 5820 caaaacttgc aatggaaaaa tgtggagcta aaggtattct tttatctttt gaaagtatgt 5880 cagattttac acatcagatt aatgcaatag atatagtaaa tgataatatt aagaaatatc 5940 atatgtaata taccctatgg atttcaaggt gcatcgcgac ggcaagggag cgaatccccg 6000 ggagcatata cccaatagat ttcaagttgc agtgcggcgg caagtgaacg catccccagg 6060 agcatagata actatgtgac tggggtaagt gaacgcagcc aacaaagcag cagcttgaaa 15120 gatgaagggt atagataacg atgtgaccgg ggtgcgtgaa cgcagccaac aaagaggcaa 6180 cttgaaagat aacgggtata aaagggtata gcagtcactc tgccatatcc tttaatatta 6240 gctgccggct agcaggaggt aaaacaggta tgacatctta tgttgataaa caagaaataa 6300 ctgcaagttc agagattgat gatttaatat ttagttcaga tcctcttgta tggtcttatg 6360 atgaacagga aaagattaga aaaaagttag ttcttgatgc ttttagacat cattataaac 6420 attgtcaaga gtatagacat tattgccagg cacataaagt agatgataat ataacagaaa 6480 ttgatgatat accagttttt cctacttcag tatttaagtt tacaagatta cttacttcaa 6540 atgaaaatga gattgaatca tggtttacaa gttcaggaac taatggttta aaatctcaag 6600 ttccaagaga tagacttagt atagaaagac ttttaggatc agtatcttat ggtatgaagt 6660 atataggaag ttggtttgat catcaaatgg agttagttaa tcttggtcct gatagattta 6720 atgctcataa tatttggttt aaatatgtaa tgtcacttgt agaactttta tatccaacaa 6780 gttttactgt aacagaagag catatagatt ttgttcagac tttaaatagt cttgaaagaa 6840 ttaaacatca aggaaaggat atatgtttaa ttggttcacc ttattttata tatcttttat 6900 gcagatatat gaaagataag aatatttctt ttagtggaga taaatcactt tatataataa 6960 ctggaggtgg atggaaatct tatgaaaagg agagtttaaa aagaaatgat tttaatcatc 7020 ttttatttga tacttttaat ctttcaaata ttaatcaaat aagagatatt tttaatcagg 7080 tagaattaaa tacatgtttt tttgaggatg aaatgcaaag aaaacatgtt ccaccttggg 7140 tatatgcaag ggctcttgat ccagaaactt taaagcctgt tccagatggt atgcctggac 7200 ttatgtctta tatggatgct tcaagtacta gttatccagc ttttatagta actgatgata 7260 ttggtataat aagtagagaa tatggacaat atcctggagt tttagttgag attttaagaa 7320 124909-序列表.doc 200827448 gagttaatac gaatgcatgt atttgttcaa cccgcatcag tgatttttaa aacggtcttt agagctgcca cgatactaaa ttc aagaaaacag aagggttgtg cactttcatt aactgaggct tttggatctt 7380 cgactctaga gcatgctagt ttctttggaa agaggagcag tcaaaggctc 7440 tgcttttgcg aaacgttttg tcgaactcta ggcgaaggtt ctcgactttc 7500 gggtatatac aagtaaaaaa gctcaggggg taaacctgag cttgggatgt 7560 gtatgagata catgggcgga tttaaataac ggagtcagtt tggaaatatc 7620 tctgctttat cgaggctata agtttcttgc agttttaacc acaaccgcgg 7680 agtacttgtg acagttttat tgccatctct ggcgtgactg ctgctttaca 7740 cgttgaaccg tagagggagc aacattcaat gcccgcgcta agttcacgaa 7800 7803 o 124909-序列表.doc 10·
Claims (1)
- 200827448 十、申請專利範圍: i•-種重組核酸分子’其包含與編碼自^發光報導體 普酸序列操作性連接之轉錄調節料酸序列,复中,轉 錄調節核普酸序列調節傳遞細胞中酸酵或合成途經:目 標產物產生信號的基因之表現。 2.如請求们之分子,其中該轉錄調節核普酸序列為細菌 ^ 轉錄調節核苷酸序列。 (3.如請求項!之分子,其中該轉錄調節核普酸序列調節編 碼該途徑中酵素之基因之表現,且該報導體表現之改變 與該目標產物產生之改變呈正相關。 上、、員1之刀子,其中該轉錄調節核苷酸序列調節編 碼该途之分枝中酵素之基因之表現,且該報導體表現 之改變與該目標產物產生之改變呈負相關。 5·如W求項1 2之分子,其中該報導體之表現隨目標產物產 生之增加而增加或減少。 124909.doc 1 1 /項1之刀子,其中該報導體之表現隨目標產物產 生之減少而增加或減少。 7 ·如請求項1夕八 - 、<刀子,其中該途徑為醱酵途徑。 8 ·如請求項1夕八 刀子,其中該目標產物為終產物。 9 ·如請求jg R + v 、之刀子,其中該終產物為丙酮、乙醇或丁 醇。 如明求項1之分子,其中該目標產物為酸中間物。 2 ·如請求箱, γ 、之刀子,其中該酸中間物為乙酸、丁酸或乳 酸0 200827448 12.如請求们之分子,其中該途徑為選自葡糖新生、糖酵 解、恩納杜道夫途徑(Entner_D〇ud〇r〇ff pathway)或非氧 化戊醣磷酸途徑之受質利用途徑。 13·如明求項1之分子,其中該基因編碼自乙醯變化為 丁醇之途徑或彼途徑之分枝中之酵素。 14·如請求項丨之分子,其中該途徑為厭氧性途徑。 • I5·如請求項1之分子,其中該細菌將己醣、戊醣或胺基酸 % 轉化為酸或醇。 16 ·如叫求項1之分子,其中該轉錄調節核苷酸序列係來自 梭菌(Clostridium)、大腸桿菌(Ε. c〇li)、運動醱酵單胞菌 (Z· mobilis)或釀酒酵母(s· cerevisiae)。 17·如請求項丨之分子,其中該基因編碼丁醇脫氫酶、丁醛 脫氫酶、乙醇脫氫酶、酸醛脫氫酶、乙醯乙酸脫竣酶、 丁酸激酶、磷酸丁醯基轉移酶、磷酸轉乙醯基酶、乙酸 激酶、醯基CoA轉移酶、乳酸脫氫酶、丁基c〇A轉移 G 酶。 18·如請求項丨之分子,其中該自足發光報導體為發光報導 . 體。 β 19·如請求項18之分子,其中該發光報導體包含螢光素酶。 20·如睛求項19之分子,其中該螢光素酶係來自鞘翅目 、發光桿菌、弧菌(Κ/Ζ?γζ·ο)、 焉氏氣(Gaussia)、雙翅 Q (Diptera)、海賢(Renilla)。 21·如請求項18之分子,其中該自足發光報導體包含螢光報 導體。 124909.doc 200827448 22.如請求項21之分子,其中該螢光報導體包含綠色螢光蛋 白(”gfp,,)。 23·如請求項18之分子,其中該自足發光報導體包含磷光報 導體。 24· —種細胞,其包含在已誘導或抑制合成或醱酵途徑而致 影響該途徑目標產物之濃度時可指示的自足報導體構築 體。 25· —種細胞,其包含包括與編碼自足發光報導體之核苷酸 序列操作性連接之轉錄调節核苦酸序列的重組核酸分 子,其中該轉錄調節核苷酸序列調節傳遞該細胞中醱酵 或合成途徑目標產物產生之信號的基因之表現。 26. 如請求項25之細胞,其為細菌細胞。 27. 如清求項25之細胞’其為梭菌、大腸桿菌、運動醋酵單 胞菌或釀酒酵母。 28. 如請求項25之細胞,其中該細胞中該途徑之該目標產物 為終產物。 2 9 ·如請求項2 8之細胞’其中該細胞中該途徑之該終產物為 丁醇。 30·如請求項25之細胞,其中該基因編碼丁醇脫氫酶、丁駿 脫氫酶、乙醇脫氫酶、酸酸脫氫酶、乙醯乙酸脫竣酶、 丁酸激酶、磷酸丁醯基轉移酶、填酸轉乙醯基酶、乙酸 激酶、醯基CoA轉移酶、乳酸脫氫酶或丁基c〇a轉移 酶0 31·如請求項30之細胞,其進一步包含與編碼自足發光報導 124909.doc 200827448 體^核苷酸序列操作性連接之轉錄調節核苷酸序列,其 :該轉錄調節核苷酸序列調節丁醛脫氫酶之表現。 32· 一種培養物,其包含可產生商業上有價值之量的合成或 醱酵途徑目標產物及發光報導體的細胞。 33· —種方法,其包含·· (a) k養包含包括與編碼發光報導體之核苷酸序列操 .作性連接之轉錄調節核苷酸序列的重組核酸分子之細 ( ' 胞其中5亥轉錄調節核苷酸序列調節傳遞該細胞中醱酵 或口成途徑目標產物產生信號的基因之表現,由此報導 體之光發射傳遞該目標產物產生之信號; (b) 量測自該培養物中該報導體發出之光;及 (C)基於由該發射光傳遞信號之產生而改變培養條件 以調節該目標產物之產生。 34.如請求項33之方法,其中該發光報導體為自足報導體。 35·如請求項33之方法,其中該目標產物為終產物。 ϋ 36·如請求項33之方法,其中該目標產物為酸中間物。 37·如請求項33之方法,其包含即時量測發射光。 ^ 38·如請求項33之方法,其中該發射光隨目標產物產生之增 加而增加或減少。 39·如請求項33之方法,其中該發射光隨目標產物產生之減 少而增加或減少。 40·如請求項33之方法,其中係在包含窗口之培養容器中培 養該等細胞且經由該窗口量測該光。 41.如請求項33之方法,其中係在於該培養物中包含至少一 124909.doc 200827448 個可感測該發射光且直接或遠端向偵測器傳遞信號之、> 感應器之培養容器中培養該等細胞。 42. 如請求項33之方法,其中係在包含使培養液持續流經感 測該液流中該發射光之光感應器的設備之培養容器中典 養該等細胞。 ° 43. 如請求項33之方法,其中若目標產物產生減少,則改變 - 培養條件包含移除目標產物、添加營養物、稀釋該培養 物、移除細胞(合成途徑為分解代謝途徑,醱酵為代謝或 合成代謝途徑)。 44· 一種方法,其包含·· (a) 在產生目標產物之培養條件下培養重組細胞,其 中該細胞包含可產生光基信號之報導體構築體,該光基 信號之強度指示該目標產物之產生水準; (b) 在複數個不同時刻隨時間連續監測該培養物中該 信號之強度以指示在彼等時刻該目標產物之產生水準;及 〇 (c)回應目標產物產生之變化,改變該等培養條件以 將目標產物之產生設定在所需水準。 , 45· —種軟體,其包含: 、 接文關於細胞或細胞培養物狀態之資訊的代碼; 確定培養條件是否應該且應如何改變以優化目標產生 之代喝;及 傳輪關於改變該等培養條件之指示之代碼。 46.如凊求項45之軟體,其中該代碼可確定該細胞或細胞培 養物之狀態。 124909.doc 200827448 4 7. —種系統,其包含: a) 用於培養細胞之容器; b) 用於偵測該容器中細胞培養物之光的光子偵測器;及 c) 回應該偵測器所偵測到之光而改變培養條件之電腦 控制裝置。 • 48·如請求項47之系統,其進一步包含將光子轉換成電子及 , 將電子轉換成光子之設備。 ( 49.如請求項47之系統,其進一步包含包括至少一個窗口或 至少一個於該培養物中可直接或遠端向偵測器傳遞信號 之光感應器的醱酵室,或其包含對該培養物取樣之連續 流偵測器,由此該培養液流經可量測光之偵測器/感應 器。 50·如請求項47之系統,其進一步包含回應來自電腦之指示 目標產物產生之量的信號而自該容器移除該目標產物之 電腦控制裝置。 U 51· 一種組合物,其大體上由丁醇構成且含有微量來自莧菜 (amaranth)或甜高粱(sweet sorghum)或兩者之組分且大體 . 上不含石油副產品。 52. —種商業方法,其包含: a) 在至少一家生產經生物工程化之製造生物燃料之 細胞的第一公司與一家從事於煉油之第二公司之間建立 合資企業; b) 運作該合資企業,其中·· 0該第一公司提供對生產生物燃料之生物工程化 124909.doc 200827448 菌株的所有權之許可; ii) 該第二公司在該合資企業贊助針對生物燃料生 產之研究與開發;及 iii) 該第二公司購買由該合資企業生產之生物燃 料0124909.doc
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US20070218541A1 (en) * | 2004-05-18 | 2007-09-20 | Biomass Processing Technology, A Corporation | Fermenter And Fermentation Method |
US20060195935A1 (en) * | 2004-11-08 | 2006-08-31 | Chromagenics B.V. | Selection of host cells expressing protein at high levels |
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US20080078205A1 (en) * | 2006-09-28 | 2008-04-03 | Ortloff Engineers, Ltd. | Hydrocarbon Gas Processing |
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US20090081715A1 (en) * | 2007-09-07 | 2009-03-26 | Cobalt Technologies, Inc., A Delaware Corporation | Engineered Light-Emitting Reporter Genes |
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-
2007
- 2007-09-11 US US11/853,681 patent/US20080293086A1/en not_active Abandoned
- 2007-09-12 EP EP07842375A patent/EP2074213A4/en not_active Withdrawn
- 2007-09-12 WO PCT/US2007/078321 patent/WO2008082726A2/en active Application Filing
- 2007-09-17 TW TW096134723A patent/TW200827448A/zh unknown
- 2007-09-17 UY UY30598A patent/UY30598A1/es not_active Application Discontinuation
- 2007-09-17 AR ARP070104106A patent/AR062869A1/es unknown
- 2007-09-17 GB GB0718077A patent/GB2442116A/en not_active Withdrawn
-
2012
- 2012-01-18 US US13/353,233 patent/US20120264107A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
US20120264107A1 (en) | 2012-10-18 |
GB2442116A (en) | 2008-03-26 |
US20080293086A1 (en) | 2008-11-27 |
WO2008082726A8 (en) | 2008-11-20 |
EP2074213A4 (en) | 2010-11-10 |
WO2008082726A3 (en) | 2009-09-11 |
GB0718077D0 (en) | 2007-10-24 |
EP2074213A2 (en) | 2009-07-01 |
WO2008082726A2 (en) | 2008-07-10 |
AR062869A1 (es) | 2008-12-10 |
UY30598A1 (es) | 2008-05-02 |
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