CN114107285B - 一种利用烷烃传感器进化产烃酶生产长链烷烃的方法 - Google Patents
一种利用烷烃传感器进化产烃酶生产长链烷烃的方法 Download PDFInfo
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Abstract
一种利用烷烃传感器进化产烃酶生产长链烷烃的方法,涉及酶工程酶蛋白定向进化技术领域,利用烷烃诱导型传感器对产烃基因进行进化和筛选,所利用的烷烃传感器是野生型烷烃诱导型启动子的突变子,基于该传感器对产烃基因进行进化和筛选,得到进化后的产烃突变子基因,最后利用该产烃突变子基因发酵生产长链烷烃。本发明首次提出了利用烷烃诱导型传感器对产烃基因进行进化和筛选,所利用的烷烃传感器是野生型烷烃诱导型启动子的突变子,基于该传感器对产烃基因进行进化和筛选,经过四轮的进化筛选,得到进化后的产烃突变子,相较于野生型基因的产烃量,本发明进化后的产烃基因,其发酵后烷烃的产量显著提高。
Description
技术领域
本发明涉及酶工程酶蛋白定向进化技术领域,具体是涉及一种利用烷烃传感器进化产烃酶生产长链烷烃的方法。
背景技术
烷烃,即饱和链烃(Saturated chain hydrocarbon),是碳氢化合物中的一种饱和烃,其整体构造仅由碳和氢两种元素所构成,因此,它是最简单的一种有机化合物。烷烃广泛存在于自然界,在昆虫、鸟、哺乳动物、植物等高等真核生物中,这些由新陈代谢产生的、疏水性的烷烃分子具有抗干燥、防水、神经保护、信号传递的功能(WaruiDM,LiN,NrgaardH,etal.Detection of formate,rather than carbon monoxide,as the stoichiometriccoproduct inconversion of fatty aldehydes to alkanes by a cyanobacterialaldehydedecarbonylase.Journal of the American ChemicalSociety,2011,133(10):3316-3319)。同时,烷烃还是汽油、柴油、航空燃料等化石燃料的主要组成部分,在整个能源系统中占有至关重要的地位(Schirmer A,udeMA,LiX,etal.Microbial biosynthesisofalkanes.Science,2010,329(5991):559-562)。商业化的烷烃是以石油为原料通过分馏得到的。利用分馏法生产烷烃会不可避免的消耗大量石油资源,并且生产过程会对环境造成污染。目前,随着全球经济快速增长对能源需求的激增,石油等化石燃料资源不断减少,而伴随化石燃料燃烧造成的环境问题日益激增,可再生生物燃料的发展逐渐引起了人们的重视。
生物能源作为一种可再生的能源,对其进一步的研究可缓解甚至最终消除能源危机。理想的生物燃料替代品必须具备高能量密度、低吸湿性、低挥发性,并且与现存发动机设备和运输设施相兼容等性能(L.P.Wackett,Biomss to fuels via microbialtransformations,Current Opinion in Chemical Biology 12(2008)187-193)。烷烃作为汽油、柴油、航空煤油的最主要成分,是十分理想的能源替代品。烷烃在自然界中广泛存在,很多生物包括植物、藻类、真菌都能够产烃。在较早的研究中,油料农作物、林木及油藻等被作为重点研究和开发对象,但都面临高成本,低产量的问题。
目前,人们普遍认为微生物细胞中合成烷烃是依赖脂肪酸合成途径,它以脂肪酸合成途径的中间代谢产物脂肪酰-酰基载体蛋白(Acylcarrierprotein,ACP)作为合成烷烃的直接原料。但是,细胞中脂肪酸的合成途径在转录水平和蛋白水平上都被严格地调控,故在自然条件下微生物细胞中无法实现大量积累其合成途径的中间代谢产物(Fujita Y,Matsuoka H,HirookaK.Regulation of fatty acidmetabolism in bacteria.MolecularMicrobiology,2007,66(4)):829-839)。随着合成生物学和代谢工程的发展,人们利用遗传学、酶学及代谢工程手段通过改造微生物代谢途径提高微生物脂肪烃的产量获得了许多进展(X.Tan,L.Yao,Q.Gao,et al.Photosynthesis driven conversion of carbon dioxideto fatty alcohols and hydrocarbons in cyanobacteria,Metabolic Engineering 13(2011)169-176)。2010年,Schirmer等成功鉴定了蓝藻中脂肪烃的合成途径,并且将来源于点形念珠藻PCC73102的npun_R1711和来源于聚球藻PCC7942的orf1594进行组合,在敲除fadE的大肠杆菌中进行表达,脂肪烃产量达到300mg/L,并且产生的脂肪烃80%是分泌到细胞外,这将有利于脂肪烃产物的提取(A.Schirmer,M.A.Rude,X.LI,E.Popova,S.B.delCardayre,Microbial biosynthesis of alkane,Science 329(2010)559-562)。Sang Yup Le等2019年通过敲除带有酰基CoA脱氢酶和alkane-1单加氧酶的大肠杆菌菌株,过表达脂肪酶,折叠酶,酰基CoA合成酶和异源酰基CoA还原酶,酰基ACP还原酶和醛脱甲酰加氧酶,脂肪烃的产量达到脂肪烃的产量达到了5.2g/L(Kim H M,Tong U C,Choi SY,etal.Engineering of an oleaginous bacterium for the production of fatty acidsand fuels[J].Nature Chemical Biology,2019,15(7)721-729),是到目前为止报道的最高产烃量。
现阶段微生物烷烃产量的提高主要通过改造微生物代谢途径来实现的,这种进化方法虽然目的明确、可行性高,但是对进化设计思路要求极高并且无法对缺乏了解的目标物进行改造,使用受到了很大的限制,并且所获得的突变菌株烷烃生产效率仍无法满足现代化商业应用的要求。定向进化技术不需要准确了解待进化物的分子机制及结构功能关系,而是通过引入随机突变和重组,人为制造出原本不存在的多样性突变子,并且按照特定的需要施以选择压力,筛选出具有期望特征的突变子,实现分子水平的模拟进化,进化更具有针对性且拥有更好的应用价值.
近年来利用生物传感器检测和高通量筛选细胞内重要中间代谢产物被广泛应用。这种细胞内代谢物水平的监测对于理解代谢通量的分布非常重要,而全细胞生物传感器对关键代谢物的监测,正是检测重要代谢途径中碳流量的理想选择,进而可以指导在代谢工程中对微生物进行优化和改良。然而,由于缺乏相关代谢物的生物传感器,极大限制其应用。
发明内容
本发明的目的在于克服现有技术的不足,提供一种利用烷烃传感器进化产烃酶生产长链烷烃的方法,以解决现有技术的基于野生型产烃基因所存在的产烃量低的问题。
为了实现上述目的,本发明所采用的技术方案为:一种利用烷烃传感器进化产烃酶生产长链烷烃的方法,利用烷烃诱导型传感器对产烃基因进行进化和筛选,所利用的烷烃传感器是野生型烷烃诱导型启动子的突变子,基于该传感器对产烃基因进行进化和筛选,得到进化后的产烃突变子基因,最后利用该产烃突变子基因发酵生产长链烷烃;所述烷烃诱导型传感器为含有pUC19-ep3alks-EGFP载体的Top10工程菌,其中ep3alks具有如SEQID NO:9所示的核苷酸序列;所述进化后的产烃突变子基因含有p15A-m4aar-ado质粒,其中m4aar-ado具有如SEQ ID NO:18所示的核苷酸序列。
本发明首先开发了一种对中长链烷烃有特异反应的基因编码的全细胞生物传感器。并成功将该生物传感器应用在了产烃基因的筛选研究中,它是通过对烷烃操纵子的alkS调节蛋白构建随机突变库并进行高通量筛选而开发的。该生物传感器已成功应用于分析和设计烷烃生产途径中的代谢通量,并成功从产烃突变体库筛选获得了高产烷烃菌株。另外,本发明通过定向进化技术,构建了产烃基因aar-ado的突变体库,并通过烷烃生物传感器进行高通量筛选,实现了产烃酶的进化,获得了高产烷烃菌株,为提高烷烃的产量提供了一种新的研究方法。
与现有技术相比,本发明的有益效果表现在:
1、本发明首次提出了利用烷烃诱导型传感器对产烃基因进行进化和筛选。
2、相较于野生型基因的产烃量,本发明将烷烃的产量提高了约3倍,且烷烃产量较之已有技术提升效果显著。
附图说明
图1分别为pUC19-Alks-EGFP(A)、pUC19-AID-EGFP(B)的质粒图谱。
图2分别为野生型细菌生物传感器(A)和进化后的细菌生物传感器(B)对中长链烷烃的响应情况。
图3为p15A-aar-ado(A)、p15A-AID(B)的质粒图谱。
图4为利用烷烃响应生物传感器检测产烃质粒p15A-aar-ado和空白对照质粒p15A-AID荧光响应情况。
图5为四轮进化获得的突变子的荧光响应情况。
图6为四轮进化获得的突变子发酵制备烷烃产量情况。
具体实施方式
下面对本发明的实施例作详细说明,本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,以便本领域的技术人员更了解本发明,但本发明的保护范围不限于下述的实施例。
其中,LB培养基的制备方法为:
LB液体培养基:10g/L胰蛋白胨,5g/L酵母提取物,10g/L氯化钠;
LB固体培养基:每升LB液体培养基中加入琼脂15g;
氨苄青霉素抗性的LB固体培养基:将配置好的LB固体培养基加热完全溶解,待温度降至55℃左右加入总重量1‰的氨苄青霉素。
实施例1
中长链烷烃诱导型生物传感器的制备,步骤如下:
1)、野生型烷烃诱导型操纵子基因的获得:
以质粒pCOM8-Alks为模板,以SEQ ID NO:1和SEQ ID NO:2所述的引物做PCR扩增,获得含有烷烃结合位点ABS,启动子Palks和调节蛋白alkS基因的野生型烷烃诱导型操纵子alks。
2)、野生型烷烃诱导型重组载体的构建
利用EcoRI和XhoI双酶切pUC19载体和步骤1)扩增获得的野生型烷烃诱导型操纵子,T4连接酶连接,使野生型烷烃诱导型操纵子替代pUC19载体中原本的lac启动子,获得pUC19-Alks载体;再在pUC19-Alks载体的调节蛋白alkS基因的下游引入绿色荧光蛋白EGFP基因,获得野生型烷烃诱导型重组载体pUC19-Alks-EGFP,所述pUC19-Alks-EGFP的质粒图谱如图1A所示,所述EGFP基因是以SEQ ID NO:3和SEQ ID NO:4为引物,以质粒pPRars-GFP为模板克隆获得的。
利用EcoRI和XhoI双酶切pUC19载体同步构建pUC19-AID-EGFP空白对照质粒,用无功能序列AID(activation-induced cytidine deaminase)替换pUC19-Alks-EGFP质粒中的烷烃诱导型操纵子,所述pUC19-AID-EGFP的质粒图谱如图1B所示,所述AID序列是以SEQ IDNO:5和SEQ ID NO:6为引物,以pCI-mAID为模板克隆获得的。
3)、定向进化获得烷烃诱导型生物传感器
以pUC19-Alks-EGFPP质粒为模板,对烷烃诱导型操纵子的烷烃结合位点ABS,启动子Palks和调节蛋白alkS基因进行易错PCR,获得随机突变体文库,对随机突变体文库进行流式高通量筛选。
利用EcoRI和XhoI进行双酶切,用获得的随机突变体替换pUC19-AID-EGFP中的AID基因,构建重组突变体文库,其中连接体系中,插入片段和载体的摩尔比为4:1,或在每100ul连接体系中加入50ng载体以及200ng片段,连接反应条件为22℃连接30min;连接产物经电转化,导入Top10感受态细胞,获得流式筛选文库,进行流式高通量筛选;流式筛选文库构建时,库容量达到2x107个克隆,以保证能有足够突变基因型以供筛选;所述易错PCR的引物如SEQ ID NO:7和SEQ ID NO:8所示。
易错PCR的反应体系如下表所示:
易错PCR的反应程序为:94℃预变性5min,94℃变性30s,62℃退火45s,72℃延伸2.5min,25个循环后,再在72℃下继续延伸10min后,置于4℃下保存备用。
经过三轮流式高通量筛选,最终获得进化后的含有pUC19-ep3alks-EGFP载体的Top10工程菌,即为进化后的细菌生物传感器,其中,ep3alks具有如SEQ ID NO:9所示的核苷酸序列,该ep3alks序列也可以通过人工合成的方式获得。
该细菌生物传感器对不同链长的烷烃诱导试验,步骤如下:
1)将进化后的细菌生物传感器pUC19-ep3alks-EGFP接种于含氨苄青霉素抗性的LB固体培养基平板上,37℃培养过夜;同时,接种一份野生型烷烃诱导型传感器pUC19-AID-EGFP,作对照。
2)分别挑取野生型和进化后的传感器单菌落,接种于1mL含有氨苄青霉素抗性的LB液体培养基中,在37℃,200rpm下过夜培养,获得检测菌液。
3)将检测菌液用上述含有氨苄青霉素抗性的LB液体培养基稀释50倍,获得稀释菌液,继续培养至对数期。
4)准备链长为C8-C17等一系列烷烃标准品,标准品购自西格玛奥德里奇(上海)贸易有限公司。
5)对数期菌液加入终浓度为100μM的烷烃标准品,作为诱导组;同步取对数期菌液加入等量去离子水,作为空白对照;在37℃,200rpm下培养1h,获得诱导菌液;
6)将诱导后的菌液置于离心机中5000rpm离心3min,弃上清。
7)1×M9缓冲液重悬后再次离心,反复漂洗3次最后使用1×PBS重悬,流式细胞仪检测荧光表达。
获得的响应情况如图2所示,由图2A可以看出,野生型烷烃响应生物传感器仅对癸烷及以下链长的烷烃有所响应,且对癸烷的响应较低。而进化后的烷烃响应生物传感器(图2B)不仅有更广泛的检测碳谱,能够对癸烷以上链长的烷烃产生响应,而且荧光响应有了更大的提高。
实施例2
一种利用烷烃传感器进化产烃酶生产长链烷烃的方法,利用烷烃诱导型传感器对产烃基因进行进化和筛选,所利用的烷烃传感器是野生型烷烃诱导型启动子的突变子,基于该传感器对产烃基因进行进化和筛选,得到进化后的产烃突变子基因,最后利用该产烃突变子基因发酵生产长链烷烃。具体步骤如下:
1)、野生型产烃基因的获得:
以质粒pAL112为模板,以SEQ ID NO:10,SEQ ID NO:11,SEQ ID NO:12和SEQ IDNO:13所述的引物做overlapPCR扩增,获得含启动子Ptrc和野生型产烃基因aar-ado。
2)、野生型产烃重组载体的构建
利用XbaI和XhoI双酶切pSB3K3-merR-GFP载体和步骤1)扩增获得的野生型产烃基因,T4连接酶连接,使野生型产烃基因替代pSB3K3-merR-GFP载体中原本的启动子和merR-GFP基因,获得野生型产烃重组载体p15A-aar-ado;质粒图谱如图3A。
同时利用EcoRI和XhoI双酶切pSB3K3-merR-GFP载体同步构建p15A-AID空白对照质粒,质粒图谱如图3B,用无功能序列AID(activation-induced cytidine deaminase)替换p15A-aar-ado质粒中的产烃基因,所述AID序列是以SEQ ID NO:14和SEQ ID NO:15为引物,以pCI-mAID为模板克隆获得的。
3)、烷烃传感器检测野生型重组载体p15A-aar-ado产烃的可行性分析
将烷烃传感器质粒pUC19-ep3alks-EGFP(实施例1制备)分别与野生型产烃质粒p15A-aar-ado和空白对照质粒p15A-AID共同电转至大肠杆菌Top10,涂板培养获得实验组的双质粒产烃单克隆和对照组的双质粒不产烃单克隆。
分别挑取实验组和对照组单克隆,接种于1mL含有氨苄青霉素和卡那霉素的双抗性的LB液体培养基中,在37℃,200rpm下过夜培养,获得检测菌液。
将检测菌液用上述含有氨苄青霉素和卡那霉素双抗性的LB液体培养基稀释50倍,获得稀释菌液,继续培养至对数期。
对数期实验组和对照组菌液分别加入终浓度为0.5mM的IPTG(异丙基-β-D-硫代半乳糖苷),进行诱导产烷烃,在37℃,200rpm下培养24h,期间取样获得诱导菌液。
将诱导后的菌液置于离心机中5000rpm离心3min,弃上清;1×M9缓冲液重悬后再次离心,反复漂洗3次最后使用1×PBS重悬,流式细胞仪检测荧光表达。
如图4所示,在利用烷烃响应生物传感器同时检测产烃质粒p15A-aar-ado和空白对照质粒p15A-AID时,可以明显看出较大的荧光响应差异,随着发酵时间的增加,空白对照质粒几乎无荧光响应增强(微弱的增强可以看做是传感器的背景荧光泄露),而产烃质粒随着发酵时间的延长,荧光响应逐步增强,也表明有中长链烷烃的产生,同时也证明了用烷烃传感器检测细胞内烷烃代谢的可行性,为后续对产烃基因的进化和筛选提供了可靠的保障。
4)、定向进化产烃基因,提高产烃量
以p15A-aar-ado质粒为模板,对产烃基因aar-ado基因进行易错PCR,获得随机突变体文库,对随机突变体文库进行流式高通量筛选。
利用XbaI和XhoI进行双酶切,用获得的随机突变体替换p15A-AID中的AID基因,构建重组突变体文库,其中连接体系中,插入片段和载体的摩尔比为4:1,或在每100ul连接体系中加入50ng载体以及100ng片段,连接反应条件为22℃连接30min;连接产物经电转化,导入Top10感受态细胞,获得流式筛选文库,进行流式高通量筛选;所述易错PCR的引物如SEQID NO:16和SEQ ID NO:17所示。
经过四轮流式高通量筛选,最终获得进化后的含有p15A-m4aar-ado载体的产烃工程菌,其中,m4aar-ado具有如SEQ ID NO:18所示的核苷酸序列。
易错PCR的反应体系如下表所示:
易错PCR的反应程序为:94℃预变性5min,94℃变性30s,62℃退火45s,72℃延伸2.5min,25个循环后,再在72℃下继续延伸10min后,置于4℃下保存备用。
实施例3
烷烃响应生物传感器快速定性分析产烃情况:
1)将实施例2四轮进化获得的产烃基因突变子,分别与烷烃传感器共转化至Top10感受态中,涂布于含氨苄青霉素和卡那霉素的双抗性的LB固体培养基平板上,37℃培养过夜;同时共转一份野生型产烃基因和传感器质粒做对照。
2)分别挑取含有传感器质粒和各轮产烃突变子单菌落,接种于1mL含有氨苄青霉素和卡那霉素的双抗性的LB液体培养基中,在37℃,200rpm下过夜培养,获得检测菌液。
3)将检测菌液用上述含有氨苄青霉素和卡那霉素的抗性的LB液体培养基稀释50倍,获得稀释菌液,继续培养至对数期。
4)对数期实验组和对照组菌液分别加入终浓度为0.5mM的IPTG,进行诱导产烷烃,在37℃,200rpm下培养24h,期间取样获得诱导菌液;
5)将诱导后的菌液置于离心机中5000rpm离心3min,弃上清。1×M9缓冲液重悬后再次离心,反复漂洗3次最后使用1×PBS重悬,流式细胞仪检测荧光表达。
获得的响应情况如图5所示,由图5可以看出,野生型荧光强度最低,表明产烃量最低。而经过定向进化和传感器高通量筛选获得的各轮突变子的荧光响应逐轮增加,经过四轮进化获得的突变子的荧光响应最强,即通过该组进化产烃酶发酵制备烷烃的产量最高。
实施例4
GC-MS快速定量分析产烃情况:
1)将实施例2四轮进化获得的产烃基因突变子,电转化至BL31感受态中,涂布于卡那霉素抗性的LB固体培养基平板上,37℃培养过夜;同时电转一份野生型产烃基因质粒做对照。
2)将检测菌液用上述含有氨苄青霉素抗性的改良M9液体培养基稀释50倍,获得稀释菌液,继续培养至对数期。
3)对数期菌液加入终浓度为0.5mM的IPTG,在37℃,200rpm下培养60h,获得检测菌液。
4)收集检测菌液于超声破碎仪超声破碎60min(功率300w,8s on,12s off),离心收集上清。
5)取2ml含有烷烃的上述培养基上清加入2mL含有7μg/mL二十碳烷烃作为内标的乙酸乙酯溶液,混合均匀后,于5000g离心10分钟,收集上层溶液,进行GC-MS分析。
检测结果如图6所示,结果表明,各轮进化后的产烃基因,发酵后其烷烃产量由8.93mg/L提高到39.21mg/L。
以上内容仅仅是对本发明的构思所作的举例和说明,所属本技术领域的技术人员对所描述的具体实施例做各种各样的修改或补充或采用类似的方式替代,只要不偏离发明的构思或者超越本权利要求书所定义的范围,均应属于本发明的保护范围。
序列表
<110> 安徽大学
皖南医学院
<120> 一种利用烷烃传感器进化产烃酶生产长链烷烃的方法
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
catgcactcg agttagataa ttccttgacg ctcagc 36
<210> 2
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cctttctcct ctttaaatgg aattctccaa t 31
<210> 3
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tataaggagg aaggatccat gagtaaagga gaagaac 37
<210> 4
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tcagctaatt gaatccttat ttgtatagtt catccat 37
<210> 5
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atcgccgaat tcatgtatcc atatgatg 28
<210> 6
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
aagccgctcg agtcaaaatc ccaacatac 29
<210> 7
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gctagccatg cactcgagtt agataattcc ttgacgc 37
<210> 8
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
agtggacctt tctcctcttt aaatggaatt ctccaa 36
<210> 9
<211> 3089
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tccaattttt attaaattag tcgctacgag atttaagacg taattttatg cctaactgag 60
aaagttaagc cgcccactct cactctcgac atcttaaacc tgagctaatc ggacgcttgc 120
gccaactaca cctacgggta gtttttgctc cgtcgtctgc tggaaaaaca cgagctggcc 180
gcaagcatgc caggtaccgc gagctactcg cgacggctga aagcaccgaa atgagcgagc 240
tatctggtcg attttgaccc ggtgcccgtc ttcaaaatcg gcgaaggccg aagtcggcca 300
gaaatagcgg cctacttcag accttcccta gtaaatattt tgcaccaccg atcatgccga 360
ctacacttaa gtgtagtttt aatatttaac accgtaacct atggtgagaa tttccagtca 420
gctggcgcta gaattgcata atgaaaataa taataaataa tgatttcccg gtcgctaagg 480
tcggagcgga tcaaattacg actctagtaa gtgccaaagt tcatagttgc atatatcggc 540
caagattgag tatcgcggat ggagccgctc ccagagtatg cctttacaga gccccacctg 600
gatatgggaa aaccgttgct cttgcgttcg agtggctacg ccacagaaca gccggacgtc 660
ctgcagtgtg gctttcttta agagccagtt cttacagtga atttgatatc tgcgcagaga 720
ttattgagca gcttgaaact ttcgaaatgg taaaattcag ccgtgtgaga gagggtgtga 780
gcaagcctgc gctcttgcga gaccttgcat ctagtctttg gcagagcacc tcgaataacg 840
agatagaaac gctagtttgt ttggataata ttaatcatga cttagacttg ccgttgttgc 900
acgcacttat ggagtttatg ttaaatacac caaaaaatat caggtttgca gttgcaggca 960
atacaataaa agggttctcg cagcttaaac ttgcaggcgc tatgcgggag tacaccgaga 1020
aagacttggc ctttagcgca gaagaggcgg tggcgttagc ggaggcagag tctgttcttg 1080
gagttcctga agaacagata gagaccttgg tgcaagaagt tgaggggtgg cctgctcttg 1140
tagttttttt gttaaagcgt gagttgccgg ccaagcatat ttcagcagta gttgaagtag 1200
acaattactt tagggatgaa atatttgagg cgattcccga gcgctatcgt gtttttcttg 1260
caaattcttc attgctcgat ttcgtgacgc ctgatcaata caattatgta ttcaaatgcg 1320
tcaatggggt cacatgtatt aagtatttaa gcactaatta catgttgctt cgccatgtga 1380
gcggtgagcc agcgcagttt acactgcatc cagtactgcg taattttcta cgagaaatta 1440
cttggactga aaatcctgct aaaagatcct acctgcttaa gcgtgcagct ttctggcatt 1500
ggcgtagagg tgaataccag tatgcaatac gaatatccct acgggcgaat gactgtcgct 1560
gggcagtcag catgtctgag agaataattt tagatttgtc atttcgtcag ggcgaaatag 1620
atgcgctgag acagtggctg ttagagctgc cgaagcaggc ctggcacaaa aaacccatag 1680
tgcttattag ttacgcgtgg gtattgtatt tcagtcagca aggcgcgcga gcagagaagt 1740
taattaaaga cttatcttca caatccgata aaaaaaataa atggcaagaa aaggaatggc 1800
tgcagcttgt gcttgcaata ggtaaagcaa cgaaagatga aatgcttacg agtgaggagc 1860
tctgtaataa gtggattagt ttatttgggg attcaaacgc agttggaaaa ggggccgcgc 1920
taacctgttt ggctttaatt tttgccagtg agtatagatt tgcagagttg gagagggtgc 1980
tggctcaggc ccaagccgtg aataaatttg caaaacaaaa ttttgctttt ggttggctgt 2040
atgtcgcgag gtttcaacaa gccctagcaa gcggagaaat gggctgggcg aggcagatta 2100
taactcaagc gcgcacagac tgtcgcgcgc agatgatgga atccgagttt acttcgaaaa 2160
tgtttgacgc tctagagctt gagttacatt atgaattgcg ctgcttggac acctcagaag 2220
aaaagctctc caaaatttta gagttcattt ccaatcacgg ggtgacagac gtgttttttt 2280
ccgtatgccg tgctgtgtca gcttggcggc ttggaaggag tgacctaaat ggctccattg 2340
agatattgga gtgggcgaag gcgcatgcgg ttgaaaaaaa tctaccaaga ttggaagtta 2400
tgagccaaat tgagatctat cagcgcttag tctgtcaagg cataacgggc ataaataatt 2460
taaaaactct tgaagatcat aagattttct ccggacagca ctcagccccc ctaaaagcac 2520
gcctgctgct tgttcaatca ctagtgcttt cccgagatcg gaactttcat agtgccgcgc 2580
acagtgcgtt attggctatt cagcaagccc gtaaaattaa cgcgggccag ctggaagtcc 2640
gtggattatt gtgtttggcc ggagcgcagg caggtgccgg tgatttaaaa aaggctcagc 2700
ttaacattgt ttatgcagtg gagatagcaa aacagcttca atgctttcaa acagttcttg 2760
atgaagtatg tttaattgag cgaataatac cggcttcatg tgaagccttc acagcagtta 2820
atttagatca agcgattggg gcttttagtc ttccgcgaat agttgagatt ggaaagtccg 2880
cagagaataa agctgacgct ttattgacac ggaagcagat tgctgtcttg aggcatgtaa 2940
aagaggggtg ctcaaacaaa caaatagcaa gaaatatgta tgtcaccgaa gatgctataa 3000
agtggcacat gaggaaaata tttgccacct tgaatgtagt gaatcgcacg caagcaacaa 3060
ttgaagctga gcgtcaagga attatctaa 3089
<210> 10
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gctctagatt aagcacctat gagtccgtag gc 32
<210> 11
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ggtacccatc tctttcacac aggaaacaga ccgaattcat ggcacagcag cttac 55
<210> 12
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggtctgtttc ctgtgtgaaa gagatgggta cctcaaattg ccaatgccaa gggttg 56
<210> 13
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ccgctcgagg ccgacatcat aacggttctg gc 32
<210> 14
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
cctggcctcg agatggacag ccttctgatg aagc 34
<210> 15
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gctctagatc aaaatcccaa catacgaaat gc 32
<210> 16
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
cttgagagcc ttcaacctct agattaagca cc 32
<210> 17
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
caggaaacag accagatcta tggcattcg 29
<210> 18
<211> 1769
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ttaagcacct atgagtccgt aggctgacta gcgcataata acgcgagtcg aataaccaat 60
gttgctcaat gcttcaccat actgaatcat gaagtcttct accaaagcat ctttttccat 120
tgccattgtg tgggcatcac cttttacttg gttgagaatt ttccagacga tgggtaggtt 180
ccggagattt gcaagttcaa gttcagctct ggatactgca aagtgttctt ccaaccaaac 240
ttctccaaaa ttgaggtggc tgtattcttc tttaaatact ccttcagtaa ttttacgggc 300
gaaatcggcg gcaacgggga tgtatatgtt atatgctgtg atcgcaaaac attcaataat 360
taaagactga atcaacagac aagtaaccac tttcccttct gcggcagctg tttgaaaatt 420
ttggtgtagg ccggagaaaa actctttggc aaactgcaaa tctggggtaa cagctaaatt 480
gcgcccacaa gctacaaatc ctttcttatg gcggctttcc atcttggata ggcgaatcaa 540
ttcatcgtta gattctggca gcagttgggc tagtgtgatg taattttcaa gggcatcttg 600
ttccccttca atcacgatcg cattaatccg gctacaagca tctttgtatg tttcgctctt 660
gaaatctaat tcttcagatt ggtctgtaag ctgctgtgcc atgaattcgg tctgtttcct 720
gtgtgaaaga gatgggtacc tcaaattgcc aatgcctagg gttggaagcc gtggcgcacc 780
gatgcctcac cgatcgcttc catcttctcg atcgtgattt ggttgcggcc ccaggagaag 840
ttagtatgcc agccttcaaa ttccaagagc atcgcctcgg caaagcaggc aaacatctgg 900
cgctcgggcc gcgccatctc tgcagcggac atgatctgcc agtcgatgtc gaagcaatgt 960
tcaactaccc cgccattgag gacatagatg ccctcacctt ggactttgct gcccaagctt 1020
ttggggtagc ccccgttgat taggacgcag ggttgcttca gggttgctgg gtcgatcact 1080
acgccctgag gcatactgac gacccacacg ataaagtcag cttccggcag agcggcttcc 1140
aagggcagaa tcttgcccct gccgtgttca gcctgcaggt tatccaaacg ctcctgattg 1200
cgcgccgtcg ggatcaaatc accgacaccc agtttgaggt cgagccagcg gcagacagcg 1260
ctaccgatat cgccagtcgc gccgacaacc gctactgtcg cttgggtaat gtcgatgcct 1320
agcgttttag cagcagcttc cacctgtcta cagattacgt aggccgtgtg agtattgccg 1380
gtggtgaacc gttcaaactc caaggtagtg tcgcgcactt gccgcaaact ggccaaatcg 1440
aaattctcga aaataatcga ggtaaagccc cacaaggccg agatgtcgat gccgtgtttt 1500
tgggcatggg acatggcatt gagaactttg cgcgtggctg ttttgaagcg gcgcgctgcc 1560
agcatttccg gcaagaaaca cgattcgatg tagcgaccgt gaatctcctt gcctgtggca 1620
ctggtgactg tgatttcatc aacgatttga gggggagcgc tactccaaaa ctacaaccct 1680
tgatcggcgt attcgtcgta gcccatcctg cgagaaacgt cgcgggcctg ctccaaactg 1740
gtgagatgac cgataagacc gaatgccat 1769
Claims (1)
1.一种烷烃诱导型传感器在进化生产长链烷烃的产烃酶的应用,其特征在于,所述烷烃诱导型传感器为含有pUC19-ep3alks-EGFP载体的Top10工程菌,其中ep3alks的核苷酸序列如SEQ ID NO:9所示。
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