基因修飾苯丙酮酸脫羧酶、其製備方法和用途與流程

2023-12-03 18:13:56

本專利申請要求2014年12月10日提交的名稱為「基因修飾苯丙酮酸脫羧酶、其製備方法和其用途」的美國專利申請第62/089,912號的權益，所述專利申請以全文引用的方式併入本文中。本發明涉及在微生物中使用生物酶產生c6-c10化合物，如醇、羧酸和烷烴的領域。更確切地說，其涉及使用一或多種工程改造硫胺素依賴性脫羧酶轉化給定2-酮酸底物的領域。表達如下文所描述的表示m461v變異體的本發明的基因修飾脫羧酶的一個特定實施例的微生物樣品已於2015年12月9日寄存於美國組織類型收集(atcc)專利庫,10801universityblvd.,manassas,va20110。地理政治和環境問題已激勵全世界的研究人員追求使用可再生途徑，包括(但不限於)使用微生物的發酵產生基於石化品的產品。但是，由於微生物通常未能在經濟上可行的速率或產率下產生許多基於石化品的產品，代謝工程改造已廣泛用於建立路徑和/或朝向所關注的路徑引導代謝物。當前，乙醇為使用微生物製得的最常見生物化學物質。但是，在生物燃料和化學行業中積極地追求產生較長鏈醇和羧酸的經濟上可行的方法。通過微生物發酵產生天然胺基酸中的成功尤其在使用胺基酸生物合成路徑產生所關注的化學品，包括較長鏈醇和羧酸中產生重大興趣。參見例如becker,j.；wittmann,c.「用於胺基酸生產的系統和合成代謝工程改造-工業菌株發展的脈搏(systemsandsyntheticmetabolicengineeringforaminoacidproduction-theheartbeatofindustrialstraindevelopment),」《生物技術當前觀點(curr.opinbiotechnol)》.,2012,23:718-726；和becker,j.；wittmann,c.「基於生物的化學品、材料和燃料的生產-穀氨酸棒狀桿菌作為通用細胞工廠(bio-basedproductionofchemicals,materialsandfuels-corynebacteriumglutamicumasversatilecellfactory),」《現代生物技術觀點(curr.opin.biotechnol.)》,2012,23:631-640。在胺基酸生物合成期間為關鍵中間物的2-酮酸能經受可用於細胞內部的化學物質的生物合成的不同類型的修飾。參見例如gronenberg,l.s.；marcheschi,r.j.；liao,j.c.「原核生物中的下一代生物燃料工程改造(nextgenerationbiofuelengineeringinprokaryotes),」《化學生物學新見(curr.opin.chem.biol.)》,2013,17:462-471。在一個實例中，美國專利8,232,089描述表達產生異丁醇的代謝路徑的重組酵母菌，所述路徑包括在與異丁醇產生基因共表達時將2-酮基異戊酸酯轉化為異丁醛的巴西固氮螺菌脫羧酶。在另一實例中，美國專利8,298,798描述通過2-酮酸的脫羧，接著通過乳酸乳桿菌(l.lactis)酮基異戊酸酯脫羧酶和酵母菌醇脫氫酶adh6的表達來還原產生的醛而在大腸桿菌(e.coli)細胞中產生直鏈和分支鏈醇。也參見atsumi,s.；hanai,t.；liao,j.c.「用於合成分支鏈高級醇作為生物燃料的非發酵路徑(non-fermentativepathwaysforsynthesisofbranched-chainhigheralcoholsasbiofuels)」《自然(nature)》,2008,451:86-89；marcheschi,r.j.；li,h.；zhang,k.；noey,e.l.；kim,s.；chaubey,a.；houk,k.n.；liao,j.c.「用於碳鏈延長的合成遞歸「+1」路徑(syntheticrecursive「+1」pathwayforcarbonchainelongation),」《acs化學生物學(acschem.biol)》.,2012,7:689-697；和zhang,k.；sawaya,m.r.；eisenberg,d.s.；liao,j.c.「用於生物合成非天然醇的擴張代謝(expandingmetabolismforbiosynthesisofnonnaturalalcohols),」《美國國家科學院院刊(proc.natl.acad.sci.u.s.a.)》,2008,105:20653-20658。已經由脫羧酶和醛去氫酶的表達展示2-酮酸中間物在細胞內部轉化為羧酸。參見例如xiong,m.；deng,j.；woodruff,a.p.；zhu,m.；zhou,j.；park,s.w.；li,h.；yao,f.「脂族酮的生物催化方法(abio-catalyticapproachtoaliphaticketones),」2012,《科學報導(sci.rep.)》2:311；和zhang,k.；woodruff,a.p.；xiong,m.；zhou,j.；dhande,.,k.「用於生產平臺化學異丁酸的合成代謝路徑(asyntheticmetabolicpathwayforproductionoftheplatformchemicalisobutyricacid),」《化學與可持續性、能源與材料(chemsuschem)》,2011,4:1068-1070。經由大腸桿菌的leua基因產物的工程改造延長細胞內部的2-酮酸的長度的可行性也已擴展可產生自2-酮酸的生物化學物質的範圍。參見例如atsumi,s.,同上.,和zhang,k.,同上。在大腸桿菌中，leuabcd基因延長2-酮酸的長度一個碳單元，如在亮氨酸生物合成期間所觀測，其中其共同起作用以將2-酮基異戊酸酯(5碳酸)轉化為2-酮基異己酸酯(6碳酸)。marcheschi等人.《acs化學生物學》.,2012,7:689-697描述leua的活性位點的擴展和c4酮酸、2-酮基丁酸酸[2-酮基丁酸酯]延長為c9酮酸、2-酮基壬酸[2-酮基壬酸酯]。儘管有可能使用代謝工程改造在微生物中產生變化長度的醇和羧酸，但到目前為止尚未展示特定c6-c8醇或酸的產生，優選地以按總醇產物計的大於20重量％(wt％)、更優選地大於30wt％的量。若干因素似乎決定產生自細胞內部的2-酮酸的醇/酸的特異性。脫羧酶接受變化長度的2-酮酸的雜亂性導致變化長度的醛，其接著通過對應的共表達醛或醇脫氫酶氧化或還原。因此，較高水準的雜亂性，即較低水準的特異性導致較高數量的產物。這轉而可意味著尤其所需的特定產物的較低產率。經由2-酮酸特定產生醇或羧酸也可受就leuabcd來說的脫羧酶的表達量不利地影響。較高水準的具有廣泛底物特異性的脫羧酶傾向於與leua基因產物競爭2-酮酸中間物且進而限制路徑延長2-酮酸的能力。這可導致形成比可能需要短的醇或羧酸，再次導致非所需產物和/或產物混合物。一般來說，改良工業微生物的方法從傳統菌株改良(csi)的隨機方法到代謝工程改造的高度合理方法變動。csi一般對於緩解產物抑制或提高產率有效，但在產生能夠產生完全新穎產物的菌株中效果小得多。此外，csi為時間和資源密集型的。為了獲得對於抑制性發酵產物具有高耐受性的菌株，有必要通過在增加的抑制劑濃度存在下在培養基中連續培養菌株而連續篩選和選擇突變體。這通常與使用化學誘變劑和/或紫外線(uv)輻射的誘導性突變誘髮結合進行。但是，常規的培養物篩選過程是冗長、耗時並且通常徒勞的。代謝修飾一般在創造產生新產物的菌株中更有效。這是因為基因，且在一些情況下甚至整個路徑可在生物體之間轉移(重組方法)和/或酶可經修飾(工程改造方法)。這些方法避免csi的一些缺點。包含重組和工程改造方法兩者的術語代謝工程改造為廣泛用於設計菌株以通過代謝通量分布的改變而實現代謝物過度產生中的較高效率的定向且通常較快的方法。到目前為止此操作的大部分是關於使用具有充分研究的遺傳學和生理學的生物體(例如大腸桿菌、酵母菌和融合瘤細胞)來產生次級代謝物(如抗生素)、胺基酸(例如賴氨酸)和異源蛋白質。代謝通量分布的化學計量分析提供適當代謝修飾、最優培養基調配和饋入策略和生物工藝優化的指導。但是，此方法仍需要發酵細胞中的代謝和調節網絡的深入知識。儘管這些合理方法已在涉及單個基因或單一基因簇內的若干基因的情況下取得成功，但其通常在涉及更複雜或在很大程度上未知的代謝途徑的情況下無效。這是因為這通常每次靶向一個基因，且因此未能預測給定路徑中的多個基因之間的複雜相互作用。通過修飾基因密碼的所述部分，即生物體dna進行酶修飾，其對應於酶的表達。酶修飾可產生完全新穎的官能團且可用於提高所需中間物或產物的特異性或效率。另外，已知某些酶為雜亂的且可發現進行超出其已知自然角色的任務。此類酶也可經修飾以進行新轉化，但到目前為止，此方法的成功頻繁地限於在商業上不可行的產率。參見例如zhang,k.,同上。在路徑中修飾多個酶可理論上用作使特異性和/或催化效率最大化的技術。已知在某些條件下產生辛醇的生物體的一個實例為梭菌屬(clostridium)。梭菌屬的各種物種(例如丙酮丁醇梭菌(c.acetobutylicum)、艱難梭菌(c.difficile)和克氏梭菌(c.kluyveri))用於wo2012135731中。公開案描述通過工程改造的梭菌屬物種產生少量正辛醇，連同其它產物，且將對於正辛醇的不佳特異性歸因於生物體表達或過度表達β-酮硫解酶(例如bktb)、乙醯基coa乙醯基轉移酶(例如atob)、3-羥基丁醯基-coa去氫酶(例如來自梭菌屬的hbd，或paahi)巴豆酸酶(例如crt)和反烯醯基-coa還原酶(例如ter)的能力。一般來說，工程改造修飾是針對生物體的coa路徑以產生高級醇，且此路徑避免發現於許多梭菌屬物種中的涉及氧敏感性酶和中間物的丁醇產生路徑。顯示為已經由本發明產生的正辛醇的量太小而在商業上不可行。也參見例如lee,j.y.；jang,y.s.；lee,j.；papoutsakis,e.t.；lee,s.y.「丙酮丁醇梭桿菌m5的代謝工程改造以用於高度選擇性丁醇生產(metabolicengineeringofclostridiumacetobutylicumm5forhighlyselectivebutanolproduction),」《生物技術學刊(biotechnol.)》2009,4:1432-1440；和wang,y.；blaschek,h.p.「通過用拜氏梭菌發酵而優化從熱帶玉米稈汁的丁醇生產(optimizationofbutanolproductionfromtropicalmaizestalkjuicebyfermentationwithclostridiumbeijerinckii),」《生物資源技術(bioresour.technol.)》,2011,102:9985-9990。當前研究的基因工程改造的一種應用是在能源領域。關於未來獲得和使用化石燃料的稀缺、成本和環境影響的擔憂已刺激在採用廉價、可再生生物質作為由其製得的燃料和化學品兩者的替代來源中的興趣。由於原油價格變得更易變，基於生物的化學品和工業產品已變為其石油衍生性對應物的有吸引力的替代方案。使用厭氧微生物的發酵方法提供將生物質和農業廢棄物轉化為有用產物的有前景的途徑，且同時整改可在處置低價值農業商品和食品加工副產物/廢棄物中遇到的問題。可由低成本生物質進料製備的有用產物中的一些為有機酸和醇，包括辛醇。c6-c10醇尤其用作製備作為許多行業中的高度所需進料化學品的烷烴、烯烴和醛的低成本起始物質。這些行業包括用作溶液聚合的共單體，和洗滌劑行業，其使用烷基化酚的這些前驅物來生產洗滌劑前驅物。這些醇也可用作表面活性劑；潤膚劑；化妝品和食品行業種的增稠劑；殺蟲劑；和多種其它應用。在一個實施例中，本發明提供一種基因修飾微生物的方法，其包含(a)選擇產生c7-c112-酮酸的微生物；和(b)插入編碼對應於seqid4、8、14、16、18、28、30、32、34、36、38、40、42、46、52、54、56、62、64、66、68或76的胺基酸序列，或與其至少90％同源的胺基酸序列的非天然核酸序列；使得非天然苯丙酮酸脫羧酶表達於微生物中。在另一實施例中，本發明提供基因修飾微生物。在另一實施例中，本發明提供一種製備c6-c10醛、c6-c10醇、c6-c10羧酸或c6-c10烷烴的方法，其包含以下步驟：(a)使2-酮基丁酸酯或2-酮基異戊酸酯、異丙基蘋果酸合成酶、異丙基蘋果酸異構酶和異丙基蘋果酸去氫酶在使得2-酮基丁酸酯或2-酮基異戊酸酯轉化為c7-c112-酮酸的條件下接觸；(b)使c7-c112-酮酸與苯丙酮酸脫羧酶在使得c7-c112-酮酸轉化為碳原子比轉化的c7-c112-酮酸少一個的c6-c10醛的條件下接觸，所述苯丙酮酸脫羧酶由編碼對應於seqid4、8、14、16、18、28、30、32、34、36、38、40、42、46、52、54、56、62、64、66、68或76的胺基酸序列，或與其至少90％同源的胺基酸序列的非天然核酸序列表示；和(c)任選地使c6-c10醛與(1)醇脫氫酶在形成c6-c10醇的條件下接觸；或(2)醛去氫酶在形成c6-c10羧酸的條件下接觸；或(3)脂肪醛脫羧酶在形成c6-c10烷烴的條件下接觸；進行所述方法以使得在微生物內部或外部和好氧或厭氧條件下獨立地進行每一步驟和子步驟。在另一實施例中，本發明提供一種多肽，其包含對應於seqid8、14、16、18、28、30、32、34、36、38、40、42、46、52、54、56、62、64、66、68或76，或與其至少90％同源的胺基酸序列，主要由所述胺基酸序列組成，或由所述胺基酸序列組成。在另一實施例中，本發明提供一種基因修飾微生物，其包含(a)2-酮酸源；(b)將2-酮酸轉化為c7-c11醛的野生型代謝路徑；和(c)由編碼與基因庫寄存編號l26240對應的胺基酸序列，或與其至少80％同源的胺基酸序列的核酸序列表示的非天然苯丙酮酸脫羧酶；此類序列任選地經以下各者修飾：(1)經纈氨酸取代met-380；或(2)經纈氨酸、亮氨酸或異亮氨酸取代phe-385；或(3)經纈氨酸、亮氨酸、丙氨酸或半胱氨酸取代met-461；或(4)經纈氨酸或亮氨酸取代phe-465；或(5)經甘氨酸、丙氨酸、纈氨酸或亮氨酸取代phe-532；或(6)經纈氨酸、亮氨酸、異亮氨酸、丙氨酸或甘氨酸取代gln-536；或(7)如(1)-(6)中所述的兩個或三個取代的任何組合對於下文描述的序列，每一奇數編號的序列標識號(seqid)顯示核苷酸或核酸序列，且每一偶數編號的seqid顯示對應編碼的胺基酸序列。核酸序列編碼胺基酸序列，其中術語「胺基酸序列」等效於「多肽」或「蛋白質」。所有提到的蛋白質(偶數編號的)seqid承認有可能使用替代密碼子產生給定胺基酸序列的事實。seqid1和2表示附接於胺基酸序列的起點的13個胺基酸的組氨酸標籤。seqid3和4表示對應於基因庫寄存編號l26240的野生型巴西固氮螺菌(azospirillumbrasilense)苯丙酮酸脫羧酶基因。seqid5和6表示seqid3和4的基因序列，但abppdc中的met-380在位置380處經纈氨酸置換(基於無his-tag的胺基酸序列；在13個胺基酸的his-tag的情況下，這將為位置393)。修飾帶有名稱m380v，因此遵守行業標準，其中胺基酸修飾定義為原始單字母胺基酸密碼，接著為胺基酸位置，接著為新胺基酸單字母密碼。seqid7和8表示f385l。seqid9和10表示f385v。seqid11和12表示f385i。seqid13和14表示m461c。seqid15和16表示m461v。seqid17和18表示m461l。seqid19和20表示m461a。seqid21和22表示f465l。seqid23和24表示f532a。seqid25和26表示f532g。seqid27和28表示f532v。seqid29和30表示f532l。seqid31和32表示q536g。seqid33和34表示q536a。seqid35和36表示q536l。seqid37和38表示q536i。seqid39和40表示q536v。seqid41和42表示f532v/q536v。seqid43和44表示m380l/m461v。seqid45和46表示m380v/m461v。seqid47和48表示f385v/m461v。seqid49和50表示f385l/m461v。seqid51和52表示f532a/q536v。seqid53和54表示f532v/q536a。seqid55和56表示f385l/q536v。seqid57和58表示f385v/q536v。seqid59和60表示m461v/q536v。seqid61和62表示m461l/q536v。seqid63和64表示m461a/q536v。seqid65和66表示m461v/f532v。seqid67和68表示f465l/q536v。seqid69和70表示f465v/q536v。seqid71和72表示f465l/f532v。seqid73和74表示f532a/q536a。seqid75和76表示m461v/f532v/q536v。seqid77和78表示m380v/m461v/q536v。seqid79和80表示f385l/m461l/q536v。seqid81和82表示m380v/f385v/m461v。圖1說明通過遞歸(迭代)活性，接著脫羧為比迭代路徑中在其之前的2-酮酸短一個碳原子的醛的鏈延長。圖2說明使用異丙基蘋果酸合成酶、異丙基蘋果酸異構酶、異丙基蘋果酸去氫酶和醇脫氫酶(adh6)以及abppdc的f385l變異體(seqid8)的組合從2-酮基丁酸酯體外產生直鏈c5-c8醇。圖3說明使用異丙基蘋果酸合成酶、異丙基蘋果酸異構酶、異丙基蘋果酸去氫酶和醇脫氫酶(adh6)以及abppdc的f385l變異體(seqid8)的組合從2-酮基異戊酸酯體外產生分支鏈c5-c8醇。圖4說明使用異丙基蘋果酸合成酶、異丙基蘋果酸異構酶、異丙基蘋果酸去氫酶和醇脫氫酶(adh6)以及abppdc的f385l變異體(seqid8)的組合從3-甲基-2-酮基戊酸酯體外產生分支鏈c5-c8醇。圖5說明含有「+1路徑」酶以及abppdc野生型(wt)、abppdc變異體、kivdwt(雷特氏乳球菌酮基異戊酸酯脫羧酶)且不含脫羧酶的大腸桿菌的血清瓶發酵的平均醇分布。adh6也包含於所有菌株構築體中。一般來說，本發明尤其包括新苯丙酮酸脫羧酶的兩個特定實施例，其可在第一實施例中為對應於基因庫寄存編號l26240的獲自巴西固氮螺菌(a.brasilense)或與其至少80百分比(％)同源的胺基酸序列的表達。在第二實施例中，本發明包括先前定義的基因修飾苯丙酮酸脫羧酶，但具有另外的有意基因工程改造以在序列內插入特定胺基酸的一個、兩個或三個修飾，其同樣用於以在許多情況下在進行多種生物合成(其中苯丙酮酸脫羧酶參與)中有利的方式修改苯丙酮酸脫羧酶的催化效率。確切地說，野生型或核酸修飾的巴西固氮螺菌苯丙酮酸脫羧酶可與異丙基蘋果酸合成酶、異丙基蘋果酸異構酶和異丙基蘋果酸去氫酶組合以產生醇、羧酸或烷烴。如將在本文中顯示，相比於所選宿主微生物的野生型酶具有改進的特性的新苯丙酮酸脫羧酶是通過以描述於本文中的多種方式中的一種進行基因修飾而產生；或為由巴西固氮螺菌苯丙酮酸脫羧酶至少80％同源且包括相同修飾的胺基酸序列表示的酶；經由重組、工程改造或組合重組和工程改造方法的技術製造其的方法；使用野生型或新苯丙酮酸脫羧酶製造c6-c10醇、羧酸和烷烴的方法；以及可表達或過度表達此酶且可用於產生c6-c10醇、羧酸和烷烴的基因修飾微生物有機體。在本文使用所述術語時，同源性是指序列中列出的胺基酸在其給定位置的80％或更大的一致或功能對應。新苯丙酮酸脫羧酶可在某些特定實施例中用作或表示為以下代謝路徑的一部分：經由合成代謝(例如伍德-永達爾(wood-ljundahl))或分解代謝(例如糖酵解或磷酸戊糖路徑)途徑產生乙醯基co-a，且最終參與c7-c112-酮酸的轉化以形成少一個碳的對應c6-10醛。在一些實施例中，c6-c10醛可進一步反應以形成c6-c10醇、羧酸或烷烴。本發明的基因修飾苯丙酮酸脫羧酶由於描述於本文中的其胺基酸序列中的特異性改變而提供對各種底物的特異性中的一些顯著差異，且特異性的此改變提供就產物產率和減少或消除非所需和/或競爭副產物來說的重要優點。本發明包括巴西固氮螺菌苯丙酮酸脫羧酶的多種改變的胺基酸序列，其已鑑別為相比於seqid4中顯示的對應於基因庫寄存編號l26240的野生型巴西固氮螺菌胺基酸序列展現改進的c7-c112-酮酸的脫羧。野生型序列內的六個位點已鑑別為對於獲得所述改進至關重要。這些為met-380、phe-385、met-461、phe-465、phe-532、gln-536和其組合。在每一改變中作出變化，其中纈氨酸、亮氨酸、丙氨酸、甘氨酸或異亮氨酸在野生型胺基酸的鑑別位點處經取代，其中取代從單位點(即，構成三個鹼基對單一胺基酸)取代到多種定義為優選地2到3個鑑別位點的「組合」的多位點(2到5個位點)取代改變。seqid3-82顯示包括指定的取代中的一或多者的產生的許多變化形式的胺基酸序列。取代可概述如下：(1)用纈氨酸取代met-380；或(2)用纈氨酸、亮氨酸或異亮氨酸取代phe-385；或(3)用纈氨酸、亮氨酸、丙氨酸或半胱氨酸取代met-461；或(4)用纈氨酸或亮氨酸取代phe-465；或(5)用甘氨酸、丙氨酸、纈氨酸或亮氨酸取代phe-532；或(6)用纈氨酸、亮氨酸、異亮氨酸、丙氨酸或甘氨酸取代gln-536；或(7)如(1)-(6)中所述的三種取代的任何組合；所屬領域的技術人員應了解，本發明的基因修飾苯丙酮酸脫羧酶可在體內(即，通過基因修飾微生物)或體外使用。鑑於此，如本文所用的術語「經基因修飾」或「經修飾」是指具有刻意改變的胺基酸序列，即「非野生型」胺基酸序列的本發明苯丙酮酸脫羧酶的群組，或具有關於(至少)在本文中描述和定義為發明性的特定經修飾脫羧酶已刻意改變的基因組的微生物(取決於作為形容詞的任一術語的放置)。此類改變可經由重組技術實現，其中一或多種基因從第二不同微生物轉移至目標微生物中；或工程改造技術，其中目標微生物內的核酸一般經由定點突變誘發改變，導致至少一種核酸轉化為不同核酸並且因此修飾一或多種酶。在當今dna合成技術的情況下，重組技術也可使用完全合成dna實現，所述dna使用常規方法轉移至目標微生物。也可採用以上方法中的任一種的組合。本發明進一步包括一種製備c6-c10醛、c6-c10羧酸、c6-c10烷烴和c6-10醇(如己醇、庚醇和/或1-辛醇)的方法，其經由起始底物與包括本發明的基因修飾苯丙酮酸脫羧酶中的一或多種的一系列酶之間的接觸以使用另外的酶和步驟將底物最終轉化為所需c6-c10醛、醇、羧酸或烷烴。此方法可在非天然存在，即基因工程改造的細胞的所描述實施例中的一個中，即在非天然存在的微生物中以生物合成方式進行；或c6-c10醇、羧酸或烷烴的生產可經由體外方法進行，通常從不包括微生物的起始點開始。為了獲得本發明的經修飾苯丙酮酸脫羧酶的群組，在一個實施例中需要與下文所描述類似地執行方案。一般來說，實例顯示涉及改變給定密碼子中的一或多個核酸鹼基以改變核酸鹼基為一部分的酶的工程改造的基因修飾。這可僅用於產生用於例如體外分析目的的改變的酶。相比之下，宿主微生物的基因組可優選地經改變以用於較大規模生產株。可如所屬領域的技術人員一般理解地進行被設計用於體外酶生產的以下方法。一般來說，適合的資料庫(如基因庫(genbank))用於獲得用於野生型酶的基因編碼，接著識別適合於修飾的密碼子。此識別可用作蛋白質工程的技術已知方法的基礎，其中計算機分子建模鑑別可有效地採用酶/底物界面的修飾的結構位置並且使得能夠區別所述結構位置。接著使用分子生物學技術進行給定所需修飾，其中經由定點突變誘發進行一或多個核酸鹼基的改變。變異型酶必須接著經受純化以分離出非目標蛋白質，留下將展現高於野生型的催化效率的純化酶。這可根據最適合於給定特定酶的方法經恰當地體外分析。顯示具有所需催化效率水準的分析的酶進而確認為所需基因修飾的產物，且可用於體外生產方法，如將c7-c112-酮酸體外轉化為少一個碳的對應c6-c10醛(例如將2-酮基壬酸酯轉化為辛醛，或將2-酮基辛酸酯轉化為庚醛)，其可接著在一個實施例中通過與適當野生型或非野生型醇脫氫酶接觸而還原，以形成對應c6-c10醇。如上文所提到，可在體內或體外進行本發明。體內方法可對於商業規模生產為優選的，而在一些情況下，體外方法可適合於商業規模生產。通常，體外方法可尤其便於實驗室和一般研究目的，以便進行酶分析。舉例來說，可製備適用於酶促產物大規模或商業規模發酵生產，如在某些特定實施例中，c6-c10醇或c6-c10醇的組合的所需微生物有機體。此類製備可如下進行：通過使用重組技術將來自第一微生物的編碼所需改進的酶的dna或dna片段插入至第二「宿主」微生物的基因組中，已知或相信所述第二「宿主」微生物具有一或多個所需代謝路徑和/其它所需特徵，如耐抑制發酵能力。一般來說，體內方法採用此類微生物野生型代謝路徑在不同步數中首先將適合的含碳底物轉化為丙酮酸酯，且接著將丙酮酸酯轉化為2-酮基丁酸酯，或者，2-酮基異戊酸酯。舉例來說，在一個實施例中，適合的含碳底物，如c5或c6糖(例如葡萄糖、蔗糖、戊糖或其組合)可經由分解代謝或合成代謝路徑中的一個，如糖酵解或磷酸戊糖路徑直接轉化為丙酮酸鹽。此後，丙酮酸鹽可經由pc(丙酮酸羧化酶)；aat(天冬氨酸轉氨酶)；thrabc(thra，其為雙官能天冬氨酸激酶/高絲氨酸去氫酶；thrb，其為高絲氨酸激酶；thrc，其為蘇氨酸合成酶；且asd，其為天冬氨酸半醛去氫酶)首先轉化為l-蘇氨酸。l-蘇氨酸接著經由ilva(蘇氨酸脫水酶)轉化為2-酮基丁酸酯。在一替代實施例中，丙酮酸鹽可經由亮氨酸生物合成中的ilvbn/ilvgm、ilvc和ilvd的活性轉化為2-酮基異戊酸酯。另外參見zhang,k.；sawaya,m.r.等人,同上。從這個角度，涉及拉長2-酮酸的亮氨酸生物合成路徑內的野生型或基因修飾形式的三種酶中的一或多者經操作以將2-酮基丁酸酯或2-酮基異戊酸酯轉化為c7-c112-酮酸。在不提及任何特定微生物的情況下，這些酶一般稱為異丙基蘋果酸合成酶、異丙基蘋果酸異構酶和異丙基蘋果酸去氫酶。但是，特定在大腸桿菌中，其分別稱為leua(基因庫寄存編號nc000913.3，基因標識：947465)、leub(基因庫寄存編號nc000913.3，基因標識：944798)和leucd(基因庫寄存編號nc000913.3，基因標識945076和基因標識：945642)。此鏈延長的一個實例顯示於圖1中，其中2-酮基丁酸酯經由稱為「+1路徑」的多個步驟轉化為c7-c112-酮酸2-酮基壬酸酯。在某些特定實施例中，涉及延伸2-酮酸的的亮氨酸生物合成路徑的野生型酶可尤其通過包括至少一種外源酶、酶複合物或其組合而修飾以在發生鏈延長時將2-酮基丁酸酯首先轉化為2-酮基戊酸酯，接著轉化為2-酮基己酸酯，接著轉化為2-酮基庚酸酯且必要時繼續轉化為另一延長的2-酮酸直至轉化為2-酮基十一酸酯，即所需c7-c112-酮酸。但是，任選地可能僅修飾酶、酶複合物或其組合中的一個或兩個以便獲得可接受或所需產量的c7-c112-酮酸。這些酶可包括如上文所提及的leua、leub和/或leucd。尤其適用於路徑的此部分的修飾的為2014年12月10日提交的同在申請中的國際專利申請序號pct/us14/69438(代理人案號75413-wo-pct)的公開內容，所述專利申請要求2013年12月12日提交的美國臨時專利申請第61/915,040號(代理人案號75413-us-psp)的權益，其均以全文引用的方式併入本文中。在某些實施例中，至少選擇經修飾的異丙基蘋果酸去氫酶變異體(其為大腸桿菌中的leub基因的產物)，或在其它實施例中，至少包括經修飾的leua(leua')和leub'變異體，其優選地但未必如引用的專利申請中的一個或兩個中所述。也優選地採用leua'、leub'和經修飾的leucd(leucd')酶/酶複合物的其它組合。同樣，應注意「leu」+字母(a、b、cd)名稱為大腸桿菌中的異丙基蘋果酸合成酶、異丙基蘋果酸異構酶和異丙基蘋果酸去氫酶的亮氨酸路徑酶的特定名稱，而其它生物體的亮氨酸路徑中的相同或等效酶可具有不同名稱。最後，本發明的基因修飾苯丙酮酸脫羧酶可在此特定實施例中用以將c7-c112-酮酸轉化為比底物2-酮酸少一個碳的醛。在各種實施例中，所得c6-c10醛可發現多種用途，作為產物本身或作為用於生產包括c6-c10醇的產物的起始或中間產物。可經由c6-c10醛通過適當野生型或基因修飾醇脫氫酶的轉化實現c6-c10醇的製備，但其它產物，如c6-c10烷烴也可經由脂肪醛脫羧酶的作用或表達製備，或c6-10羧酸可通過醛去氫酶的作用或表達製備。參見例如choi,y.j.；lee,s.y.「短鏈烷烴的微生物生產(microbialproductionofshort-chainalkanes)」,《自然(nature)》,2013,502:571-574。因此，c6-10醛在工業上高度適用作用於製備多種其它產物的極佳中間產物。因此，預期本發明的基因修飾苯丙酮酸脫羧酶家族將可適用於多種行業。此類行業可包括(例如)在燃料、塑料、食品、包裝、化妝品、香料、製藥、清潔材料、汙染控制、香料、藥物和許多其它行業中的用途。儘管存在多種完全屬於本發明的權利要求的範圍內的可能的胺基酸序列，但應注意，通過序列標識號(seqid)鑑別為選自seqid34、36、38、40、42、46、62、68和76的某些胺基酸序列就對c7-c112-酮酸脫羧基來說是尤其適合和優選的。實例1設計對於2-酮基壬酸脫羧具有較高催化效率的巴西固氮螺菌苯丙酮酸脫羧酶(abppdc)變異體abppdc與3-脫氮-硫胺素二磷酸酯和5-苯基-2-氧代戊酸(pdb標識碼2q5q)的三元複合物的晶體結構模型用於鑑別加襯abppdc的活性位點內的2-酮酸結合袋的殘基。參見例如versees,w.；spaepen,s.；wood,m.d.；leeper,f.j.；vanderleyden,j.；steyaert,j.「二磷酸硫胺素依賴性脫羧酶中的異位底物活化的分子機制(molecularmechanismofallostericsubstrateactivationinathiaminediphosphate-dependentdecarboxylase)」,《生物化學雜誌(j.biol.chem.)》,2007,282:35269-35278。將命名為met-380、met-461、phe-385、phe-465、gln-536和phe-532的胺基酸位點基於其與5-苯基-2-氧代戊酸的關係選擇用於取代實驗。如表1中所列地進行一或多個位點的取代且製備變異體。酶f532v以纈氨酸置換abppdc中的phe-532，而酶f532l以亮氨酸置換phe-532。酶f385l/m461v以亮氨酸置換phe-385且以纈氨酸置換met-461。表1中的其餘的巴西固氮螺菌苯丙酮酸脫羧酶(abppdc)變異體是根據胺基酸(第一個字母，其中「f」表示「苯丙氨酸[phe]」；「m」表示「甲硫氨酸」[met]；且「q」表示「穀氨醯胺」[gln])、其在胺基酸序列中的位置(編號)和用作替換的胺基酸(最後一個字母，其中「l」表示「亮氨酸」；「v」表示「纈氨酸」；「a」表示「丙氨酸」；「c」表示「半胱氨酸」；「i」表示「異亮氨酸」；且「g」表示「甘氨酸」)命名。經修飾abppdc變異體中的每一個經表達和純化，且接著測試針對三種底物的活性，所述底物為2-酮基己酸酯(2-kh)、2-酮基辛酸酯(2-ko)和2-酮基壬酸酯(2-kn)。將預期2-kh、2-ko和2-kn在通過abppdc脫羧時分別形成戊醛、庚醛和辛醛。使用下文描述的高通量酶分析在兩個步驟中進行abppdc變異體的評估。起初，測試所有變異體針對單一高濃度(2mm)的2-kh和2-kn的活性(如表1中所示)。在初始評估之後，對於選擇數目的變異體進行詳細動力學分析以測定最大速率(kcat)、產生半最大速率(k0.5，酶在michaelis-menten動力學之後的km的等效物)的底物濃度和酶針對2-ko和2-kn的催化效率(kcat/k0.5)(如表2中所示)。對於2-kn具有較高特異性(較高kcat/k0.5)的abppdc變異體將在細胞內部產生衍生自其的辛醛和化學物質中有效。表1.abppdc變異體的序列清單和活性seqid4為巴西固氮螺菌苯丙酮酸脫羧酶(基因庫寄存編號l26240)的胺基酸序列。seqid6-82為設計和表示於本發明中的蛋白質的序列。本發明中表示的所有蛋白質在n端具有13個以組氨酸標籤形式添加的額外胺基酸(顯示於seqid2中)。實例2a.巴西固氮螺菌苯丙酮酸脫羧酶(abppdc)和其工程改造變異體在大腸桿菌中的異源表達為了評估表1中列出的野生型abppdc和其變異體的底物特異性，所有蛋白質的基因分別表達於大腸桿菌細胞中且蛋白質產物與細胞分離。從ncbi資料庫下載巴西固氮螺菌苯丙酮酸脫羧酶(基因庫寄存編號l26240)的基因序列。將包括六個(6)組氨酸(his)的13個額外胺基酸的密碼子添加到abppdc基因序列的met-1密碼子的上遊。此類修飾允許具有13個額外胺基酸的組氨酸標記的abppdc在n端上的表達。額外胺基酸以使用ni-nta色譜在單一步驟中純化蛋白質的輔助的形式附接。具有13個額外胺基酸的整個abppdc序列(seqid2)經化學合成且接著克隆到syntheticgenomics,inc.(sandiego,ca)的t7聚合酶促進劑下遊的prsfduet-1載體(emdbiosciences)中。最終載體在裝運之前由syntheticgenomics,inc.定序。使用newenglandbiolab的q5定點突變誘發試劑盒(目錄號e0554s)化學合成或產生表1中列出的abppdc變異體的基因且克隆至prsfduet-1載體中。含有abppdc或abppdc變異體基因的prsfduet-1載體如下所述地轉化成大腸桿菌、abppdc或其變異體，接著表達且最終純化。接著使用獲自emdbiosciences的感受態bl21(de3)細胞進行大腸桿菌表達研究。根據試劑盒說明書進行轉化且涉及混合50微升(μl)等分試樣的感受態細胞與1μl載體。使用康黴素作為生長培養基中的標記物選擇含有abppdc表達載體的細胞。接著在含有50微克/毫升(μg/ml)康黴素的盧裡亞-貝爾塔尼(luria-bertani；lb)培養液瓊脂板上選擇含有abppdc或abppdc變異體表達載體的大腸桿菌轉化體。在37攝氏度(℃)下培育所述板16小時(h)。通過將單一群落的轉化體轉移至50毫升(ml)含有50μg/ml康黴素的lb培養基中且在37℃下在振蕩的情況下以220轉/分(rpm)培育過夜而開始起子培養。次日，將7ml起子培養物接種至800mlterrificbroth(tb)中且在37℃下培育培養物直至培養物達到0.5的600納米下的光學密度(od600nm)。添加1mm的最終濃度下的異丙基β-d-1-硫代哌喃半乳糖苷(iptg)以誘發abppdc或abppdc變異體基因的表達且將培養物轉移至15℃恆溫箱後維持16小時(h)。在16h結束時，培養物在8000轉/分(rpm)下離心以使細胞粒化。將細胞團分成兩個等分試樣且儲存於-80℃下過夜，隨後純化。獲自400ml表達培養物的大腸桿菌細胞團懸浮於含有1μg/mldna酶(thermofisherscientific,inc.,rockford,il)、1μg/ml溶菌酶(thermofisherscientific,inc.,rockford,il)、1毫摩爾(mm)二硫蘇糖醇和蛋白酶抑制劑混合液(rpicorp.,mountprospect,il)的b-per試劑(thermofisherscientific,inc.,rockford,il)中。將懸浮液在室溫下平緩地搖動30分鐘(min)且在15,000倍重力(×g)下離心20min以使細胞碎片粒化。分離上清液且與5mlco-nta樹脂(thermofisherscientific,inc.,rockford,il)一起培育，所述樹脂已用平衡緩衝液(50mm磷酸鈉，ph8.0，含有300mm氯化鈉、20mm咪唑、50μl蛋白酶抑制劑混合液和15％甘油)預平衡。在4℃下的1h的培育期之後，用5倍體積的平衡緩衝液洗滌酶結合樹脂。abppdc或其變異體用過含有200mm咪唑的平衡緩衝液從co-nta樹脂洗脫。洗脫的蛋白質相對於磷酸鹽緩衝鹽水滲析且以20％甘油溶液的形式儲存於-20℃下。b.測定abppdc和abppdc變異體的底物特異性使用如在實例1中詳細描述的方法進行abppdc變異體的底物特異性的評估。進行高通量abppdc偶合酶分析以評估abppdc變異體的底物特異性。分析涉及使用醇脫氫酶(adh6，基因庫寄存編號np014051.3)減少產生自abppdc介導的2-酮酸脫羧的醛。abppdc催化反應的初始速度測定自在醛的adh6催化的還原期間發生的還原煙醯胺腺嘌呤二核苷酸磷酸(nadph)的氧化的速率。htp篩選分析涉及用含0.5mm二磷酸硫胺素、0.35mmnadph、4.7微克(μg)酵母菌adh6(基因庫寄存編號np014051.3)和0.3毫克/毫升(mg/ml)牛血清白蛋白(bsa)的abppdc分析緩衝液(50mm3-(n-嗎啉基)丙磺酸，ph6.8，含有2.5mm氯化鎂(mgcl2))在30℃下培育2mm2-kh或2mm2-kn。反應通過在30℃下添加含有0.5μg至3.5μg稀釋於含有1mg/mlbsa的abppdc分析緩衝液中的abppdc變異體的工作酶儲備液而開始。含有200μl反應混合物的所述板在2500×g下離心15秒且在30℃下預平衡的biotektm板讀取器上在340nm處以分光光度法追蹤反應混合物的吸收率變化。使用340nm處的nadph消耗速率和nadph的消光係數(6.22mm-1cm-1)計算酶反應的初始速度。所有變異體的活性以存在於反應混合物中的酶的量標準化且表示為納摩爾/分鐘/毫克(nmol.min-1.mg-1)。使用購自thermofisherscientific,inc.的來自piercebiotechnologyinc.的660nm總蛋白質分析試劑盒，使用bsa作為標準物測定用於使活性標準化的蛋白質濃度。2-酮基辛酸酯(2-ko)和2-酮基壬酸酯(2-kn)通過abppdc和其變異體的脫羧的動力學參數也使用相同htpabppdc偶合酶分析測定，除了2-ko或2-kn的濃度從0變為4mm。對於展現底物活化的abppdc變異體，如從相對於底物濃度曲線的初始速度的s形曲線顯而易見，通過使用非線性回歸將數據擬合至希爾方程式(hillequation)(表2的圖例中示出)獲得2-酮基酸脫羧的動力學參數(kcat、k0.5和kcat/k0.5)。對於遵循正態飽和動力學的變異體，通過使用非線性回歸將初始速度擬合至michaelis-menten方程式而動力學參數(kcat、km和kcat/km)。使用graphpadprismtm軟體進行非線性回歸。表2列出通過abppdc和其變異體的2-ko和2-kn脫羧的動力學參數。使用piercebiotechnologyinc.tm660nm總蛋白質分析試劑盒且使用bsa作為標準物來測定反應混合物中的酶的量。預期使abppdc的底物特異性變窄會改進特定醛和其下遊產物的積聚。一般來說，abppdc偏好更龐大2-酮酸，如5-苯基-2-酮基戊酸酯和苯基丙酮酸，如通過關於那些底物的高催化效率證明(參見例如spaepen,s.；versees,w.；gocke,d.；pohl,m.；steyaert,j.；vanderleyden,j.「參與巴西固氮螺菌的生長素生產的苯丙酮酸脫羧酶的表徵(characterizationofphenylpyruvatedecarboxylase,involvedinauxinproductionofazospirillumbrasilense),」《細菌學雜誌(j.bacteriol.)》,2007,189:7626-7633)。對表1中列出的abppdc和變異體篩選針對作為底物的2mm2-酮基己酸酯(2-kh)和2mm2-酮基壬酸酯(2-kn)的活性。篩選展現野生型abppdc催化2-kn的脫羧，但在分析條件下展現針對2-kh的不佳活性。所有abppdc變異體也催化2-kn的脫羧，且展現針對2-kh的相對低活性(表1)。gln-536經丙氨酸、纈氨酸、異亮氨酸或亮氨酸的取代相比於野生型酶增加2-kn脫羧基活性，但也改進針對作為底物的2-kh的活性。這些結果表明表1中列出的所有abppdc變異體可以活性形式表達於異源系統中。此外，其全部具有相比於2-kh顯著較高的針對2-kn的活性，表明本文所述的abppdc和變異體偏好>c62-酮酸。對所有酶進行詳細穩態動力學分析以測定對2-酮基辛酸酯(2-ko)和2-酮基壬酸酯(2-kn)脫羧的最大速率和催化效率。兩種底物均展現如從表2顯而易見的雙曲線和非雙曲線動力學。對於顯示非雙曲線動力學的abppdc變異體，將2-ko和2-kn的脫羧的速度擬合至hill方程式(表2圖例)且如表2中所示地計算最大速率和催化效率(kcat/k0.5)。大於1的希爾係數表明存在通過2-ko和2-kn的底物活化。已在abppdc和其它脫羧酶的情況下報導底物活化。另外參見spaepen,s.,同上。如從表2顯而易見，胺基酸取代以不同方式影響捕獲2-ko和2-kn用於催化中的變異體的催化效率。對於一些變異體，例如f532v，2-kn和2-ko的脫羧的催化效率分別為相比於野生型abppdc的180％和45％。這表明f532v取代增加2-kn的底物特異性，同時減少2-ko的底物特異性。通過採用變異體的催化效率的比率計算相比於2-ko的abppdc變異體對於2-kn的偏好且顯示於表2中。如表2中所證明，abppdc和f532v的特異性分別為1.8和5.6，指示其對2-kn脫羧的催化效率高於對2-ko脫羧的效率1.8倍和5.6倍。這也指示相比於abppdc，f532v變異體在相比於2-ko偏好2-k中的特異性高3倍。類似地，相比於2-ko的f385l對於2-kn的偏好比abppdc高5倍。此數據表明相比於較短2-酮酸(例如2-ko)，f385l和f532v取代改進較長2-酮酸(例如2-kn)的底物特異性。因此，當使用「+1路徑」延長2-酮酸時，f385l和f532v變異體將改進基於較長(c7-c10)醛的產物的積聚(圖1)。類似地，m461l、f532l、q536g、q536l、f532v/q536v、m380v/m461v、f532a/q536v、f532v/q536a、f385l/q536v、m461v/f532v和m461v/f532v/q536v變異體對於2-kn的特異性相比於每一變異體對於2-ko的特異性分別為3.3、4.3、4.8、2.7、3.6、2.7、6.8、4.6、4.3、5.4和2.1。這表明所有這些變異體在捕獲2-kn用於催化中比abppdc更具特異性。除abppdc變異體對於2-kn的特異性以外，辛醛和衍生自其的生物化學品的最大積聚也將取決於相對於abppdc變異體的2-kn產生路徑的相對效率。舉例來說，當產生2-kn中的工程改造的2-酮酸鏈延長路徑(涉及三種酶，異丙基蘋果酸合成酶、異丙基蘋果酸異構酶和異丙基蘋果酸去氫酶)的效率相比於產生2-ko相對較低時，將由於2-ko通過abppdc變異體的脫羧以及細胞內部基於辛醛的化學物質的積聚的減少而導致庚醛形成。在這些情形下，如f385l的abppdc變異體將基於其相對高特異性(9.1)，以及其減小的作為2-kn脫羧催化劑的效率(表2)而為優選的脫羧酶。結果也顯示經疏水性胺基酸(即，甘氨酸、丙氨酸、纈氨酸、亮氨酸或異亮氨酸)取代gln-536改進abppdc的催化效率和如表2中所示的其它特異性增強取代。q536v變異體分別在對2-ko和2-kn脫羧中比野生型酶高效8倍和5.7倍(表2)。類似地，m461v/f532v/q536v變異體分別在對2-ko和2-kn脫羧中比m461v/f532v變異體高效27倍和10倍(表2)。m461v/f532v/q536v變異體為分別在對2-ko和2-kn脫羧中比野生型酶高效約17倍和20倍的酶(表2)。m461v/f532v/q536v變異體的較高催化效率允許2-ko在比野生型酶低17倍的胞內含量下的有效脫羧且促進細胞內部的庚醛衍生性化學品，如庚醇(經由與醇脫氫酶的共表達)或庚酸鹽(經由醛去氫酶的共表達)的積聚。也改進脫羧的催化效率的gln-536的其它取代(如經甘氨酸、丙氨酸、亮氨酸或異亮氨酸)也將改進特異性增強取代的催化效率。這通過q536a取代展現，其在添加到f532v變異體(對於2-ko，kcat/k0.5＝4.8mm-1min-1且對於2-kn，kcat/k0.5＝27mm-1min-1)中時產生針對2-ko和2-kn的催化效率分別高72％和40％的f532v/q536a變異體(對於2-ko，kcat/k0.5＝8.3mm-1min-1和對於2-kn，kcatk0.5＝38mm-1min-1)。總體來說，結果表明abppdc和其基因修飾變異體的表達允許c7-c11(且在此實例中尤其為c7-c9)2-酮酸在體內的有效脫羧，且進而允許例如衍生自醛的化學物質，如己醛、庚醛和/或辛醛在細胞內部的積聚。此外，單獨或組合形式的f532、f385、q536、m380、m461、f465的修飾可產生在細胞內部展現例如類似地衍生的化學物質的尤其改進的積聚的微生物。表2.abppdc和其變異體的動力學表徵**使用本文中描述的htp偶合分析測定初始速度研究。除了通過§指示的所有酶的初始速度使用graphpadprismtm軟體擬合至希爾方程式(v為給定底物濃度s下的初始速度)。kcat、k0.5、h和kcat/k0.5分別為最大速度、最大速度的一半處的底物濃度、希爾係數和催化效率。結果為2-3個獨立實驗的平均值±標準誤差。通過取用關於2-kn與2-ko的變異體的催化效率比(kcat/k0.5)計算abppdc變異體的特異性。1應用的命名規則為第一個字母指示已改變的胺基酸殘基。f＝苯丙氨酸[phe]；q＝穀氨醯胺[gln]；m＝甲硫氨酸[met]。數字指示胺基酸序列中的位置(顯示的相應為位置380、385、461、465、532和536)。最後一個字母指示在所述位置處經取代的胺基酸殘基。g＝甘氨酸；a＝丙氨酸；i＝異亮氨酸；v＝纈氨酸；l＝亮氨酸。§這些變異體的初始速度擬合至經典michaelis-menton方程式。實例3用巴西固氮螺菌苯丙酮酸脫羧酶(abppdc)的f385l變異體(seqid.8)體外合成c5-c9醇如下進行用f385l變異體體外合成直鏈醇：用含0.5mm二磷酸硫胺素、2.5mmnad+、0.2毫克/毫升(0.2mg/ml)牛血清白蛋白(bsa)、5mm乙醯基輔酶a、0.036mg/ml大腸桿菌異丙基蘋果酸合成酶的h97a/s139g/n167g/p169a/g462d變異體(由marcheschi,r.j.等人.「用於碳鏈延長的合成遞歸「+1」路徑(asyntheticrecursive「+1」pathwayforcarbonchainelongation)」《acs化學生物學(acschem.biol.)》7:689-697,2012)、0.16mg/ml異丙基蘋果酸異構酶的leuc亞單元(基因庫寄存編號nc000913.3，基因標識：945076)和0.21mg/ml異丙基蘋果酸異構酶的leud亞單元(基因庫寄存編號nc000913.3，基因標識：945642)、0.264mg/ml大腸桿菌異丙基蘋果酸去氫酶(leub；基因庫寄存編號nc_000913.3，基因標識：944798)、0.192mg/ml異丙基蘋果酸去氫酶的l96g/v198a變異體(報導於wo2015089127a1中)、0.025mg/ml釀酒酵母醇脫氫酶(adh6，基因庫寄存編號np_014051.3)和0.0054mg/mlf385l變異體(seqid8)的體外合成緩衝液(50mm2-[4-(2-羥乙基)哌嗪-1-基]乙磺酸，ph7.5，含有30mm氯化鉀(kcl)和5mm氯化鎂(mgcl2))培育0.5mm2-酮基丁酸酯(2-kb)。反應通過添加2-酮基丁酸酯至反應混合物的其餘部分而起始。相同體積的分析級甲苯(用於hplc的chromosolvplustm，≥99％，產品目錄編號650579)覆加在反應混合物的頂部上且在30℃下培育溶液。在30℃下培育2.5小時之後，添加nadph至水層直到1mm的最終濃度。在30℃下培育6小時之後，添加額外nadph至水層直到2mm的最終濃度。在30℃下再培育反應物18小時，接著通過在-20℃下冷凍30分鐘而停止。去除甲苯層的一部分且使用裝備有火焰電離檢測器(fid)的氣相色譜儀分析。通過在以上反應混合物中用0.5mm2-酮基異戊酸酯(2-kiv)或0.5mm3-甲基-2-酮基戊酸酯(3m-2kp)置換2-酮基丁酸酯且如上文所述地進行實驗而進行f385l變異體的分支鏈醇的體外合成。使用裝備有火焰電離檢測器(fid)、g1513a型號自動注射器和gc自動取樣器控制器的hewlettpackard(hp)6890系列氣相色譜儀對醇進行定量。使用agilentj&wdb-ffap毛細管gc柱(30m×0.320mmid×0.25μm膜厚度；產品目錄編號123-3232，agilenttechnologies,inc.,wilmington,de19808)分離分析物。初始gc烘箱溫度為40℃，其保持1.50分鐘，接著以40℃/分鐘梯度增加至235℃。此梯度給出6.38分鐘的總運行時間。柱流動速率為4.0ml/min，其中氦氣作為載氣。注入體積為1μl。注射器和檢測器的溫度設定為225℃。產生自這些體外合成反應的醇效價顯示於圖2、3和4中。結果指示f385l變異體與大腸桿菌異丙基蘋果酸合成酶(leua)、大腸桿菌異丙基蘋果酸異構酶(leucd)、野生型和經修飾大腸桿菌異丙基蘋果酸去氫酶(leub)以及醇脫氫酶(adh6)的h97a/s139g/n167g/-p169a/g462d變異體組合，在與2-酮基丁酸酯、2-酮基異戊酸酯或3-甲基-2-酮基戊酸酯一起培育後產生延長的c5-c9醇。此外，結果展示f385l變異體對於較長直鏈醇的特異性，其中1-辛醇表示在與2-酮基丁酸酯一起培育後產生的總醇的大致60％。在分支鏈2-酮酸(2-酮基異戊酸酯(kiv)和3-甲基-2-酮基戊酸酯(3m-2-kp))的情況下，在與2-酮基異戊酸酯一起培育後產生大致等效量的5-甲基-1-己醇和6-甲基-1-庚醇，且在與3-甲基-2-酮基戊酸酯一起培育後產生大致等效量的3-甲基-1-戊醇和5-甲基-1-庚醇。這些結果展示f385l變異體接受直鏈以及分支鏈2-酮酸作為底物且可產生對應直鏈和分支鏈醛，其可隨後轉化為其它產物，如醇、羧酸或烷烴。實例4使用野生型abppdc和其變異體以及「+1路徑」酶在大腸桿菌的工程改造菌株中體內產生c5-c8醇大腸桿菌(e.coli)mg1655經工程改造以促進長鏈直鏈醇產生且使得能夠從t7啟動子進行基因表達。為了改進直鏈醇生產，ilvbn和ilvih經由λred介導的同源重組不活化，如datsenko,ka,wanner,bl,「大腸桿菌k-12中的染色體基因使用pcr產物的單步不活化(one-stepinactivationofchromosomalgenesinescherichiacolik-12usingpcrproducts),」《美國國家科學院院刊(proc.natl.acad.sci.u.s.a.)》2000,97(12),6640-6645所描述。ilvbn和ilvih基因參與分支鏈胺基酸產生，因此這些基因的不活化消除分支鏈醇的產生。參與2-酮基丁酸酯生產的ilva基因通過經由λred介導的同源重組用強力組成性啟動子和強力核糖體結合位點置換其天然促進劑和核糖體結合位點而上調，如datsenko和wanner,同上所描述。為了使得能夠從t7啟動子進行基因表達，使用λde3溶原化試劑盒(emdmillipore目錄編號69734)將de3溶源整合到mg1655中。所得菌株基因型為mg1655(de3)δilvbnδilvihilvaup。經由八種蛋白質的表達在經工程化大腸桿菌菌株中產生c5-c8醇：(1)大腸桿菌異丙基蘋果酸合成酶(leua)；(2)工程改造異丙基蘋果酸合成酶(由marcheschi等人《acs化學生物學(acschem.biol.)》2012,7,689-697)；(3)和(4)大腸桿菌異丙基蘋果酸異構酶(leucd)的兩個次單元；(5)異丙基蘋果酸去氫酶(leub)；(6)大腸桿菌異丙基蘋果酸去氫酶的l96g/v198a變異體(如2014年12月10日提交的同在申請中的國際專利申請序號pct/us14/69438(代理人案號75413-wo-pct)中較詳細描述，所述專利申請要求2013年12月12日提交的美國臨時專利申請第61/915,040號(代理人案號75413-us-psp)的權益，兩者均以全文引用的方式併入本文中)；(7)abppdc或其變異體；和(8)釀酒酵母醇脫氫酶(adh6)。總計產生十一個菌株。產生僅含有野生型abppdc的一個菌株。作為陰性對照，也產生不具有ppdc的菌株。也產生含有abppdc變異體f532v、f358l、f385v、f532vq536v、m461c、m461v、f385vm461c和f385lm461v的八個菌株。最後，產生含有野生型雷特氏乳球菌(lactococcuslactis)酮基異戊酸酯脫羧酶(kivd；基因寄存編號aj746364)作為比較，因為先前操作已顯示kivd能夠產生與「+1路徑」酶組合的長鏈醇。參見例如marcheschi等人,同上。允許使用四種相容質體同時表達八種基因的novagenduetvector系統(emdmillipore目錄編號71146、71341、71340和71147)用於表達上文所提及的基因。四種duet載體中的每一者用t7啟動子下遊的八種基因中的兩種克隆，且四種duet載體轉化成經工程化大腸桿菌菌株。帶有所有質體的重組菌株關於使用抗生素(25微克/毫升(μg/ml)下的安比西林(ampicillin)、17μg/ml下的氯胺苯醇、25μg/ml下的大觀黴素(spectinomycin)和15μg/ml下的康黴素)而選擇且使用所屬領域的技術人員已知的方法以聚合酶鏈反應(pcr)確認。在每一固體和液體培養步驟添加抗生素以確保質體的維持。在轉型、板選擇和pcr確認之後，菌株起初在37℃下生長的盧裡亞-貝爾塔尼(lb)瓊脂板上培養。單一瓊脂板群落用於接種250ml搖瓶中的50mllb培養基，其使用設定於200rpm下的恆溫箱振蕩器在37℃下好氧地培養。在lb搖瓶中培養12-16小時之後，在1％v/v下接種血清瓶以評估醇生產。使用根據表1中示出的濃度的去離子水製備血清瓶發酵培養基。培養基經過濾滅菌，且將20ml培養基添加至丁基橡膠塞住的125ml血清瓶中。在培養基添加之前，血清瓶通過使用sterisamscocenturysv-160hprevac滅菌器在125℃下高壓滅菌30分鐘而預滅菌。表1.用於展示來自經重組工程改造以含有「+1路徑」以及巴西固氮螺菌脫羧酶(abppdc)或其變異體的大腸桿菌的醇生產的培養基組成。組分濃度(g/l)mops緩衝液26.2甘油20胰蛋白腖10酵母提取物5泛酸鈣1.19na2hpo40.105(nh4)2so40.661nh4cl1.6在接種之後，在37℃下在200rpm於恆溫箱振蕩器中振蕩的情況下培養血清瓶培養物。在接種之後大致三小時，使用0.1mm異丙基β-d-1-硫代半乳糖苷(iptg)誘導培養物以確保所有基因的表達。在誘導之後24小時收穫發酵物用於分析。在發酵結束時，血清瓶通過置於冷凍機中20-30分鐘而緊接著冷卻至4℃。將血清瓶去瓶蓋，且將發酵培養液快速倒入含有1ml飽和氯化鈉溶液和2ml分析級甲苯(用於hplc的chromosolvplustm，≥99.9％，產品目錄編號650579)的50ml錐形管中。培養液-氯化鈉-甲苯混合物經渦旋30秒。接著將甲苯萃取物的300μl等分試樣提交至如實例3中所述地使用gc/fid進行分析。血清瓶的平均醇分布顯示於圖5中。結果指示與「+1路徑」基因和adh6組合的野生型巴西固氮螺菌(abppdc)的表達導致用於產生介於戊醇至辛醇範圍內的直鏈醇的功能路徑。未在無abppdc的菌株中檢測到c5-c8醇，證實此基因的存在對長鏈醇產生至關重要。此外，結果展示含有野生型abppdc的菌株比含有kivd的菌株累積基本上更多的己醇、庚醇和辛醇。未在kivd菌株中檢測到己醇、庚醇或辛醇產生，但abppdcwt菌株分別產生>2mg/l、>3mg/l和>0.1mg/l的己醇、庚醇和辛醇。醇生產的大致50％為庚醇和辛醇，其為相比於主要導致戊醇和己醇生產的其它脫羧酶的情況下的前述成果的顯著改進(marcheschi等人《acs化學生物學》2012,7:689-697)。因此，使用abppdc脫羧酶似乎將醇生產轉化至較長鏈長度，其結果與實例1和2內包含的體外數據一致。圖5中的額外數據表明所有abppdc變異體具有產生c5-c8醇的能力。三種abppdc變異體m461c、m461v和f385l/m461v展示相比於野生型abppdc的c5-c8醇產生的顯著改進。abppdc變異體m461c產生>3mg/l己醇和>5mg/l庚醇，表示相比於野生型abppdc的大於40％改進。最印象深刻地，abppdc變異體m461v顯示相比於野生型abppdc的戊醇、己醇和庚醇產生的大於2倍改進。m461v變異體也顯示相對於野生型abppdc的就辛醇效價來說的30％改進。含有m461v變異體的此菌株產生具有約9mg/l己醇和約8.5mg/l庚醇的最高效價。變異體f532v、f385l、f532vq536v、m461c、m461v和f385lm461v全部顯示相比於kivd和abppdc野生型基因的1-辛醇效價的改進。最後，abppdc變異體f385l/m461v顯示相比於abppdc野生型的己醇和庚醇產生的約60％改進。序列表陶氏環球技術有限責任公司p·c·桑格哈尼c·c·斯託爾斯e·c·史樂s·a·格林沃爾特基因修飾苯丙酮酸脫羧酶、其製備方法和用途77272-wo-pcttobeassigned2015-12-0362/089,9122014-12-1082patentinversion3.5139dna人工序列表達的abppdc變異體的n端處的his-tag的13胺基酸序列cds(1)..(39)1atgggcagcagccatcatcatcatcatcacagcagcggc39metglyserserhishishishishishissersergly1510213prt人工序列合成構築體2metglyserserhishishishishishissersergly151031638dna人工序列野生型巴西固氮螺菌苯丙酮酸脫羧酶（abppdc）cds(1)..(1638)3atgaagctggccgaagccctgctgcgcgcgctgaaggatcgcggcgca48metlysleualaglualaleuleuargalaleulysaspargglyala151015caggccatgttcgggattccgggtgatttcgccctgcccttcttcaag96glnalametpheglyileproglyaspphealaleuprophephelys202530gtggcggaggaaacgcagatcctgccgctccacacgctgagccacgag144valalaglugluthrglnileleuproleuhisthrleuserhisglu354045ccggcggtgggcttcgcggcggacgcggcggcgcgttacagttcgacg192proalavalglyphealaalaaspalaalaalaargtyrserserthr505560ctgggggtggcggcggtcacctacggagcgggcgccttcaacatggtg240leuglyvalalaalavalthrtyrglyalaglyalapheasnmetval65707580aacgcggtggccggcgcctacgccgagaagtcgccggtggtcgtcatc288asnalavalalaglyalatyralaglulysserprovalvalvalile859095tccggcgcgccgggcacgacggagggcaacgccggcctgctgctgcac336serglyalaproglythrthrgluglyasnalaglyleuleuleuhis100105110caccagggccgcacgctggacacgcagttccaggtgttcaaggagatc384hisglnglyargthrleuaspthrglnpheglnvalphelysgluile115120125accgtcgcccaggcgcggctggacgacccggccaaggccccggcggag432thrvalalaglnalaargleuaspaspproalalysalaproalaglu130135140atcgcccgcgtgctgggggccgcccgcgccctgtcgcgcccggtctat480ilealaargvalleuglyalaalaargalaleuserargprovaltyr145150155160ctggaaattccccgcaacatggtcaacgccgaggtcgagccggtgggc528leugluileproargasnmetvalasnalagluvalgluprovalgly165170175gacgacccggcttggccggtggaccgcgacgcgctggccgcctgcgcg576aspaspproalatrpprovalaspargaspalaleualaalacysala180185190gacgaggtgctggcggccatgcgctcggccacgtcgccggtgctgatg624aspgluvalleualaalametargseralathrserprovalleumet195200205gtctgcgtcgaggtccgccgctacgggctggaggccaaggtggcggag672valcysvalgluvalargargtyrglyleuglualalysvalalaglu210215220ctggcgcagcggctgggcgtgccggtggtgaccaccttcatggggcgc720leualaglnargleuglyvalprovalvalthrthrphemetglyarg225230235240ggcctgctggccgacgcgccgaccccgccgctcggcacctacatcggc768glyleuleualaaspalaprothrproproleuglythrtyrilegly245250255gtcgccggcgacgcggagatcacccggctggtcgaggagtcggacggg816valalaglyaspalagluilethrargleuvalglugluseraspgly260265270ctgttcctgctcggcgcgatcctcagcgacaccaacttcgcggtgtcc864leupheleuleuglyalaileleuseraspthrasnphealavalser275280285cagcgcaagatcgacctgcgcaagaccatccacgccttcgaccgggcg912glnarglysileaspleuarglysthrilehisalapheaspargala290295300gtgacgctgggctatcacacctacgccgacatcccgctggccgggctg960valthrleuglytyrhisthrtyralaaspileproleualaglyleu305310315320gtggacgccctcttggaagggctgccgccgtccgaccggacgacccgc1008valaspalaleuleugluglyleuproproseraspargthrthrarg325330335ggcaaggagccccacgcctacccgaccggccttcaggcggacggcgag1056glylysgluprohisalatyrprothrglyleuglnalaaspglyglu340345350ccgatcgccccgatggacatcgcccgcgccgtcaacgaccgcgtccgc1104proilealaprometaspilealaargalavalasnaspargvalarg355360365gccgggcaggaaccgctgctgatcgcggcggacatgggcgactgcctg1152alaglyglngluproleuleuilealaalaaspmetglyaspcysleu370375380ttcaccgcgatggacatgatcgacgccgggctgatggcgccgggctat1200phethralametaspmetileaspalaglyleumetalaproglytyr385390395400tacgcgggcatgggcttcggggtgccggcgggcatcggggcgcagtgc1248tyralaglymetglypheglyvalproalaglyileglyalaglncys405410415gtgtcgggcggcaagcgcatcctgaccgtggtcggcgacggcgccttc1296valserglyglylysargileleuthrvalvalglyaspglyalaphe420425430cagatgaccgggtgggagcttggcaactgccgacggctgggcatcgac1344glnmetthrglytrpgluleuglyasncysargargleuglyileasp435440445cccatcgtgatcctgttcaacaacgccagttgggagatgctgcgcacc1392proilevalileleupheasnasnalasertrpglumetleuargthr450455460ttccagcccgaatcggccttcaacgacctggacgactggcgcttcgcc1440pheglnprogluseralapheasnaspleuaspasptrpargpheala465470475480gacatggcggcgggcatgggcggcgacggcgtccgcgtgcgcacacgg1488aspmetalaalaglymetglyglyaspglyvalargvalargthrarg485490495gcggagctgaaggcggcgctggacaaggccttcgccacgcgcgggcgc1536alagluleulysalaalaleuasplysalaphealathrargglyarg500505510ttccagctgatcgaggcgatgatcccgcgcggcgtgctgtccgacacg1584pheglnleuileglualametileproargglyvalleuseraspthr515520525ctggcccgcttcgtccaggggcagaagcgcctgcacgccgcgccccgg1632leualaargphevalglnglyglnlysargleuhisalaalaproarg530535540gagtaa1638glu5454545prt人工序列合成構築體4metlysleualaglualaleuleuargalaleulysaspargglyala151015glnalametpheglyileproglyaspphealaleuprophephelys202530valalaglugluthrglnileleuproleuhisthrleuserhisglu354045proalavalglyphealaalaaspalaalaalaargtyrserserthr505560leuglyvalalaalavalthrtyrglyalaglyalapheasnmetval65707580asnalavalalaglyalatyralaglulysserprovalvalvalile859095serglyalaproglythrthrgluglyasnalaglyleuleuleuhis100105110hisglnglyargthrleuaspthrglnpheglnvalphelysgluile115120125thrvalalaglnalaargleuaspaspproalalysalaproalaglu130135140ilealaargvalleuglyalaalaargalaleuserargprovaltyr145150155160leugluileproargasnmetvalasnalagluvalgluprovalgly165170175aspaspproalatrpprovalaspargaspalaleualaalacysala180185190aspgluvalleualaalametargseralathrserprovalleumet195200205valcysvalgluvalargargtyrglyleuglualalysvalalaglu210215220leualaglnargleuglyvalprovalvalthrthrphemetglyarg225230235240glyleuleualaaspalaprothrproproleuglythrtyrilegly245250255valalaglyaspalagluilethrargleuvalglugluseraspgly260265270leupheleuleuglyalaileleuseraspthrasnphealavalser275280285glnarglysileaspleuarglysthrilehisalapheaspargala290295300valthrleuglytyrhisthrtyralaaspileproleualaglyleu305310315320valaspalaleuleugluglyleuproproseraspargthrthrarg325330335glylysgluprohisalatyrprothrglyleuglnalaaspglyglu340345350proilealaprometaspilealaargalavalasnaspargvalarg355360365alaglyglngluproleuleuilealaalaaspmetglyaspcysleu370375380phethralametaspmetileaspalaglyleumetalaproglytyr385390395400tyralaglymetglypheglyvalproalaglyileglyalaglncys405410415valserglyglylysargileleuthrvalvalglyaspglyalaphe420425430glnmetthrglytrpgluleuglyasncysargargleuglyileasp435440445proilevalileleupheasnasnalasertrpglumetleuargthr450455460pheglnprogluseralapheasnaspleuaspasptrpargpheala465470475480aspmetalaalaglymetglyglyaspglyvalargvalargthrarg485490495alagluleulysalaalaleuasplysalaphealathrargglyarg500505510pheglnleuileglualametileproargglyvalleuseraspthr515520525leualaargphevalglnglyglnlysargleuhisalaalaproarg530535540glu54551638dna人工序列m380vcds(1)..(1638)5atgaagctggccgaagccctgctgcgcgcgctgaaggatcgcggcgca48metlysleualaglualaleuleuargalaleulysaspargglyala151015caggccatgttcgggattccgggtgatttcgccctgcccttcttcaag96glnalametpheglyileproglyaspphealaleuprophephelys202530gtggcggaggaaacgcagatcctgccgctccacacgctgagccacgag144valalaglugluthrglnileleuproleuhisthrleuserhisglu354045ccggcggtgggcttcgcggcggacgcggcggcgcgttacagttcgacg192proalavalglyphealaalaaspalaalaalaargtyrserserthr505560ctgggggtggcggcggtcacctacggagcgggcgccttcaacatggtg240leuglyvalalaalavalthrtyrglyalaglyalapheasnmetval65707580aacgcggtggccggcgcctacgccgagaagtcgccggtggtcgtcatc288asnalavalalaglyalatyralaglulysserprovalvalvalile859095tccggcgcgccgggcacgacggagggcaacgccggcctgctgctgcac336serglyalaproglythrthrgluglyasnalaglyleuleuleuhis100105110caccagggccgcacgctggacacgcagttccaggtgttcaaggagatc384hisglnglyargthrleuaspthrglnpheglnvalphelysgluile115120125accgtcgcccaggcgcggctggacgacccggccaaggccccggcggag432thrvalalaglnalaargleuaspaspproalalysalaproalaglu130135140atcgcccgcgtgctgggggccgcccgcgccctgtcgcgcccggtctat480ilealaargvalleuglyalaalaargalaleuserargprovaltyr145150155160ctggaaattccccgcaacatggtcaacgccgaggtcgagccggtgggc528leugluileproargasnmetvalasnalagluvalgluprovalgly165170175gacgacccggcttggccggtggaccgcgacgcgctggccgcctgcgcg576aspaspproalatrpprovalaspargaspalaleualaalacysala180185190gacgaggtgctggcggccatgcgctcggccacgtcgccggtgctgatg624aspgluvalleualaalametargseralathrserprovalleumet195200205gtctgcgtcgaggtccgccgctacgggctggaggccaaggtggcggag672valcysvalgluvalargargtyrglyleuglualalysvalalaglu210215220ctggcgcagcggctgggcgtgccggtggtgaccaccttcatggggcgc720leualaglnargleuglyvalprovalvalthrthrphemetglyarg225230235240ggcctgctggccgacgcgccgaccccgccgctcggcacctacatcggc768glyleuleualaaspalaprothrproproleuglythrtyrilegly245250255gtcgccggcgacgcggagatcacccggctggtcgaggagtcggacggg816valalaglyaspalagluilethrargleuvalglugluseraspgly260265270ctgttcctgctcggcgcgatcctcagcgacaccaacttcgcggtgtcc864leupheleuleuglyalaileleuseraspthrasnphealavalser275280285cagcgcaagatcgacctgcgcaagaccatccacgccttcgaccgggcg912glnarglysileaspleuarglysthrilehisalapheaspargala290295300gtgacgctgggctatcacacctacgccgacatcccgctggccgggctg960valthrleuglytyrhisthrtyralaaspileproleualaglyleu305310315320gtggacgccctcttggaagggctgccgccgtccgaccggacgacccgc1008valaspalaleuleugluglyleuproproseraspargthrthrarg325330335ggcaaggagccccacgcctacccgaccggccttcaggcggacggcgag1056glylysgluprohisalatyrprothrglyleuglnalaaspglyglu340345350ccgatcgccccgatggacatcgcccgcgccgtcaacgaccgcgtccgc1104proilealaprometaspilealaargalavalasnaspargvalarg355360365gccgggcaggaaccgctgctgatcgcggcggacgtgggcgactgcctg1152alaglyglngluproleuleuilealaalaaspvalglyaspcysleu370375380ttcaccgcgatggacatgatcgacgccgggctgatggcgccgggctat1200phethralametaspmetileaspalaglyleumetalaproglytyr385390395400tacgcgggcatgggcttcggggtgccggcgggcatcggggcgcagtgc1248tyralaglymetglypheglyvalproalaglyileglyalaglncys405410415gtgtcgggcggcaagcgcatcctgaccgtggtcggcgacggcgccttc1296valserglyglylysargileleuthrvalvalglyaspglyalaphe420425430cagatgaccgggtgggagcttggcaactgccgacggctgggcatcgac1344glnmetthrglytrpgluleuglyasncysargargleuglyileasp435440445cccatcgtgatcctgttcaacaacgccagttgggagatgctgcgcacc1392proilevalileleupheasnasnalasertrpglumetleuargthr450455460ttccagcccgaatcggccttcaacgacctggacgactggcgcttcgcc1440pheglnprogluseralapheasnaspleuaspasptrpargpheala465470475480gacatggcggcgggcatgggcggcgacggcgtccgcgtgcgcacacgg1488aspmetalaalaglymetglyglyaspglyvalargvalargthrarg485490495gcggagctgaaggcggcgctggacaaggccttcgccacgcgcgggcgc1536alagluleulysalaalaleuasplysalaphealathrargglyarg500505510ttccagctgatcgaggcgatgatcccgcgcggcgtgctgtccgacacg1584pheglnleuileglualametileproargglyvalleuseraspthr515520525ctggcccgcttcgtccaggggcagaagcgcctgcacgccgcgccccgg1632leualaargphevalglnglyglnlysargleuhisalaalaproarg530535540gagtaa1638glu5456545prt人工序列合成構築體6metlysleualaglualaleuleuargalaleulysaspargglyala151015glnalametpheglyileproglyaspphealaleuprophephelys202530valalaglugluthrglnileleuproleuhisthrleuserhisglu354045proalavalglyphealaalaaspalaalaalaargtyrserserthr505560leuglyvalalaalavalthrtyrglyalaglyalapheasnmetval65707580asnalavalalaglyalatyralaglulysserprovalvalvalile859095serglyalaproglythrthrgluglyasnalaglyleuleuleuhis100105110hisglnglyargthrleuaspthrglnpheglnvalphelysgluile115120125thrvalalaglnalaargleuaspaspproalalysalaproalaglu130135140i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alaalaproarg530535540gagtaa1638glu54522545prt人工序列合成構築體22metlysleualaglualaleuleuargalaleulysaspargglyala151015glnalametpheglyileproglyaspphealaleuprophephelys202530valalaglugluthrglnileleuproleuhisthrleuserhisglu354045proalavalglyphealaalaaspalaalaalaargtyrserserthr505560leuglyvalalaalavalthrtyrglyalaglyalapheasnmetval65707580asnalavalalaglyalatyralaglulysserprovalvalvalile859095serglyalaproglythrthrgluglyasnalaglyleuleuleuhis100105110hisglnglyargthrleuaspthrglnpheglnvalphelysgluile115120125thrvalalaglnalaargleuaspaspproalalysalaproalaglu130135140ilealaargvalleuglyalaalaargalaleuserargprovaltyr145150155160leugluileproargasnmetvalasnalagluvalgluprovalgly165170175aspaspproalatrpprovalaspargaspalaleualaalacysala180185190aspgluvalleualaalametargseralathrserprovalleumet195200205valcysvalgluvalargargtyrglyleuglualalysvalalaglu210215220leualaglnargleuglyvalprovalvalthrthrphemetglyarg225230235240glyleuleualaaspalaprothrproproleuglythrtyrilegly245250255valalaglyaspalagluilethrargleuvalglugluseraspgly260265270leupheleuleuglyalaileleuseraspthrasnphealavalser275280285glnarglysileaspleuarglysthrilehisalapheaspargala290295300valthrleuglytyrhisthrtyralaaspileproleualaglyleu305310315320valaspalaleuleugluglyleuproproseraspargthrthrarg325330335glylysgluprohisalatyrprothrglyleuglnalaaspglyglu340345350proilealaprometaspilealaargalavalasnaspargvalarg355360365alaglyglngluproleuleuilealaalaaspmetglyaspcysleu370375380phethralametaspmetileaspalaglyleumetalaproglytyr385390395400tyralaglymetglypheglyvalproalaglyileglyalaglncys405410415valserglyglylysargileleuthrvalvalglyaspglyalaphe420425430glnmetthrglytrpgluleuglyasncysargargleuglyileasp435440445proilevalileleupheasnasnalasertrpglumetleuargthr450455460leuglnprogluseralapheasnaspleuaspasptrpargpheala465470475480aspmetalaalaglymetglyglyaspglyvalargvalargthrarg485490495alagluleulysalaalaleuasplysalaphealathrargglyarg500505510pheglnleuileglualametileproargglyvalleuseraspthr515520525leualaargphevalglnglyglnlysargleuhisalaalaproarg530535540glu545231638dna人工序列f532acds(1)..(1638)23atgaagctggccgaagccctgctgcgcgcgctgaaggatcgcggcgca48metlysleualaglualaleuleuargalaleulysaspargglyala151015caggccatgttcgggattccgggtgatttcgccctgcccttcttcaag96glnalametpheglyileproglyaspphealaleuprophephelys202530gtggcggaggaaacgcagatcctgccgctccacacgctgagccacgag144valalaglugluthrglnileleuproleuhisthrleuserhisglu354045ccggcggtgggcttcgcggcggacgcggcggcgcgttacagttcgacg192proalavalglyphealaalaaspalaalaalaargtyrserserthr505560ctgggggtggcggcggtcacctacggagcgggcgccttcaacatggtg240leuglyvalalaalavalthrtyrglyalaglyalapheasnmetval65707580aacgcggtggccggcgcctacgccgagaagtcgccggtggtcgtcatc288asnalavalalaglyalatyralaglulysserprovalvalvalile859095tccggcgcgccgggcacgacggagggcaacgccggcctgctgctgcac336serglyalaproglythrthrgluglyasnalaglyleuleuleuhis100105110caccagggccgcacgctggacacgcagttccaggtgttcaaggagatc384hisglnglyargthrleuaspthrglnpheglnvalphelysgluile115120125accgtcgcccaggcgcggctggacgacccggccaaggccccggcggag432thrvalalaglnalaargleuaspaspproalalysalaproalaglu130135140atcgcccgcgtgctgggggccgcccgcgccctgtcgcgcccggtctat480ilealaargvalleuglyalaalaargalaleuserargprovaltyr145150155160ctggaaattccccgcaacatggtcaacgccgaggtcgagccggtgggc528leugluileproargasnmetvalasnalagluvalgluprovalgly165170175gacgacccggcttggccggtggaccgcgacgcgctggccgcctgcgcg576aspaspproalatrpprovalaspargaspalaleualaalacysala180185190gacgaggtgctggcggccatgcgctcggccacgtcgccggtgctgatg624aspgluvalleualaalametargseralathrserprovalleumet195200205gtctgcgtcgaggtccgccgctacgggctggaggccaaggtggcggag672valcysvalgluvalargargtyrglyleuglualalysvalalaglu210215220ctggcgcagcggctgggcgtgccggtggtgaccaccttcatggggcgc720leualaglnargleuglyvalprovalvalthrthrphemetglyarg225230235240ggcctgctggccgacgcgccgaccccgccgctcggcacctacatcggc768glyleuleualaaspalaprothrproproleuglythrtyrilegly245250255gtcgccggcgacgcggagatcacccggctggtcgaggagtcggacggg816valalaglyaspalagluilethrargleuvalglugluseraspgly260265270ctgttcctgctcggcgcgatcctcagcgacaccaacttcgcggtgtcc864leupheleuleuglyalaileleuseraspthrasnphealavalser275280285cagcgcaagatcgacctgcgcaagaccatccacgccttcgaccgggcg912glnarglysileaspleuarglysthrilehisalapheaspargala290295300gtgacgctgggctatcacacctacgccgacatcccgctggccgggctg960valthrleuglytyrhisthrtyralaaspileproleualaglyleu305310315320gtggacgccctcttggaagggctgccgccgtccgaccggacgacccgc1008valaspalaleuleugluglyleuproproseraspargthrthrarg325330335ggcaaggagccccacgcctacccgaccggccttcaggcggacggcgag1056glylysgluprohisalatyrprothrglyleuglnalaaspglyglu340345350ccgatcgccccgatggacatcgcccgcgccgtcaacgaccgcgtccgc1104proilealaprometaspilealaargalavalasnaspargvalarg355360365gccgggcaggaaccgctgctgatcgcggcggacatgggcgactgcctg1152alaglyglngluproleuleuilealaalaaspmetglyaspcysleu370375380ttcaccgcgatggacatgatcgacgccgggctgatggcgccgggctat1200phethralametaspmetileaspalaglyleumetalaproglytyr385390395400tacgcgggcatgggcttcggggtgccggcgggcatcggggcgcagtgc1248tyralaglymetglypheglyvalproalaglyileglyalaglncys405410415gtgtcgggcggcaagcgcatcctgaccgtggtcggcgacggcgccttc1296valserglyglylysargileleuthrvalvalglyaspglyalaphe420425430cagatgaccgggtgggagcttggcaactgccgacggctgggcatcgac13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