CN110591997A - 一种提高木糖酸脱水酶活性的基因工程菌及其构建方法与应用 - Google Patents
一种提高木糖酸脱水酶活性的基因工程菌及其构建方法与应用 Download PDFInfo
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Abstract
本发明公开了一种提高木糖酸脱水酶活性的基因工程菌及其构建方法与应用，构建克隆表达铁硫簇插入蛋白SufA，并将构建好的基因转入宿主菌细胞内得基因工程菌，所述基因工程菌用于发酵生产D‑1,2,4‑丁三醇的中间产物2‑酮基‑3‑脱氧‑d‑木糖酸。通过提高了木糖酸脱水酶的活性，进而促进了木糖酸的消耗并提高了2‑酮基‑3‑脱氧‑d‑木糖酸的产量，最后提高了D‑1，2，4‑丁三醇的发酵生产。本发明方法简单，可靠性强，应用前景广。
Description
技术领域
本发明涉及2-酮基-3-脱氧-d-木糖酸的制备技术领域，具体涉及一种提高木糖酸脱水酶活性的基因工程菌及其构建方法与应用。
背景技术
SufA、HscB、ScdA蛋白属于涉及铁-硫簇生物合成的复杂蛋白质机器。它们被定义为支架蛋白，预组装的簇从其转移至靶载脂蛋白。
铁硫蛋白是最古老，高度保守的大分子之一。 它们在多种生物过程中起作用，包括铁稳态，电子转移，氧化还原和非氧化还原催化，固氮，基因表达调节和氧物种检测。尽管近年来生物[Fe-S]簇的化学反应性和光谱性质已被广泛表征，但其生物合成的机制尚处于探索的早期阶段。实际上，生物合成过程中，确定比例的铁和硫原子从其储存源中被动员并以受控方式组合以产生各种[Fe-S]簇，这需要复杂的蛋白质机制。
D-木糖酸脱水酶，系统命名是D-木糖酸氢-裂解酶，能催化木糖酸脱水反应。属于IlVD/EDD蛋白质家族，IlvD指支链氨基酸生物合成途径中的脱水酶，EDD指Entner-Doudoroff途径中的脱水酶，其代替一些细菌中的经典糖酵解。据报道，属于IlvD / EDD家族的酶在蛋白质结构中含有[2Fe-2S]或[4Fe-4S]簇，并且D-木糖酸脱水酶一直存在底物消耗缓慢的缺点，由于铁和硫原子无法在生物合成过程中从其储存源中被动员起来，因此在细胞反应过程中底物消耗缓慢添加一些有利于铁硫簇参与反应的操纵子系统的蛋白，以此来提高催化效率。
2-酮基-3-脱氧-d-木糖酸为生产D-1 ,2 ,4-丁三醇的中间产物，也是木糖酸脱水酶以木糖酸为底物时的产物，也是产D-1 ,2 ,4-丁三醇第三个酶的底物。通过提高了木糖酸脱水酶的活性，进而促进了木糖酸的消耗并提高了2-酮基-3-脱氧-d-木糖酸的产量，最后提高了D-1，2，4-丁三醇的发酵生产。
发明内容
针对现有技术的不足，本发明的目的在于提供一种提高木糖酸脱水酶活性的基因工程菌及其构建方法与应用，该工程菌构建方法简单，能够产生有利于铁硫簇参与反应的操纵子系统的蛋白，提高了木糖酸脱水酶的活性，并进一步提高了D-木糖酶脱水酶对底物的消耗，最后可用于产丁三醇的领域。
为解决上述技术问题，本发明采用的技术方案如下：
一种提高木糖酸脱水酶活性的基因工程菌，构建克隆表达铁硫簇插入蛋白SufA基因，并将构建好的基因转入宿主菌内得基因工程菌，所述基因工程菌用于发酵生产D-1 ,2 ,4-丁三醇的中间产物2-酮基-3-脱氧-d-木糖酸。
作为改进的是，所述的铁硫簇插入蛋白SufA基因来自大肠杆菌MG1655，且核苷酸序列如SEQ ID NO.1所示，所述的木糖酸脱水酶的核苷酸序列如SEQ ID NO.4所示。
作为优选，所述的宿主菌为大肠杆菌Trans 1 T1。
上述提高木糖酸脱水酶活性的基因工程菌的构建方法，包括如下步骤：步骤1，将木糖酸脱水酶基因的两端插入酶切位点NcoⅠ与Hind Ⅲ，然后将酶切后的木糖酸脱水酶基因克隆到质粒一中，得到重组质粒一；将铁硫簇插入蛋白SufA基因的两端插入酶切位点EcoRⅠ与KpnⅠ,然后将酶切后的铁硫簇插入蛋白SufA基因克隆到质粒二中；得到重组质粒二；步骤2，将重组质粒一和重组质粒二共同转入宿主菌内，即得到了基因工程菌E.coliT1-pCWJ-yjhG-pTRC99A-SufA。
作为改进的是，质粒一为pCWJ质粒，质粒二为pTRC99A质粒。
上述提高木糖酸脱水酶活性的基因工程菌在以D-木糖酸为底物产1,2，4-丁三醇上的应用。
上述应用，包括如下步骤：第一步，培养基因工程菌E.coliT1-pCWJ-yjhG-pTRC99A-SufA；第二步，收培养后的基因工程菌，接种到发酵培养基中，加入D-木糖酸和IPTG诱导反应体系发酵产D-1,2,4-丁三醇。
作为改进的是，所述的反应体系包括如下组分：D-木糖酸的添加量为20g/L，菌体OD600为60，置于33℃摇床反应17h。无操纵子系统底物木糖酸在17h内消耗了2g/L，而含有铁硫簇插入蛋白SufA基因的基因工程菌则在17h内分别消耗了6.12g/L，比对照组提高了约3倍。
有益效果：
与现有技术相比，本发明一种提高木糖酸脱水酶活性的基因工程菌及其构建方法与应用，该基因工程菌可以很好的表达木糖酸脱水酶，然后促进细胞可以合成Fe-S蛋白，进一步将其运输到最终产物，从而提高对底物D-木糖酸的降解，并且在17h内消耗了底物D-木糖酸6.12g/L，相对于现有的提高了约3倍。
从源头上提高了D-木糖酸的降解，增加了2-酮基-3-脱氧-d-木糖酸的产量，进而提高D-1，2，4-丁三醇的生产，具有良好的应用前景，且方法简单，易于施行。
附图说明
图1为重组质粒，（a）为pTRC99A-SufA、（b）为pTRC99A-HscB、（c）为pTRC99A-ScdA；
图2为pCWJ-yjhG的重组质粒图；
图3为不同表达菌株消耗底物木糖酸的量的对比；
图4为D-1,2,4-丁三醇的反应路线图。
具体实施方式
下面结合具体实施例对本发明作进一步描述。
实施例1 基因工程菌构建
分别将来源于大肠杆菌MG1655（常用菌株）的铁硫簇插入蛋白SufA基因、铁硫簇生物合成蛋白HscB基因、铁硫簇修复双铁蛋白ScdA基因构建到质粒中。
将来源于大肠杆菌的木糖酸脱水酶构建到质粒中，将两个构建好的质粒共同转入宿主细胞内得到重组的基因工程菌。
来源于大肠杆菌MG1655的铁硫簇插入蛋白SufA：SEQ ID NO.1，设计含有酶切位点的正向引物（SufA-EcoR I-F：CCGGAATTCCGGATGGACATGCATTC下划线为EcoR I酶切位点）和反向引物（SufA-Kpn I-R：CGGGGTACCCCGTTAGCTAAGTGCAG下划线为Kpn I酶切位点）,提取大肠杆菌MG1655的基因组，用上述引物按照94℃变性3min，20个循环，每个循环包括94℃变性60s，57℃退火60s,72℃延伸60s的程序进行PCR，然后用胶回收试剂盒（天根生化科技有限公司）回收PCR后的产物SufA，将回收后的产物与载体pTRC99A分别以EcoR I和Kpn I为酶切位点进行双酶切，将双酶切后的PCR产物SufA用纯化试剂盒（天根生化科技有限公司）进行纯化，得到纯化后的片段SufA，再利用胶回收试剂盒对双酶切后的载体pTRC99A进行回收，得到载体pTRC99A，最后将片段SufA与载体pTRC99A连接，并转化至大肠杆菌Trans 1T1中并送测序对比,得到pTRC99A-SufA，
设计含有酶切位点的正向引物（ScdA-Nco I-F：CATGCCATGGCATGATGAACGTTTTTAATC下划线为Nco I酶切位点）和反向引物（ScdA-BamH I-F：CGCGGATCCGCGTTAAACCTGCTTCG下划线为BamH I酶切位点）,设计含有酶切位点的正向引物（HscB-Nco I-F：CATGCCATGGCATGatgGATTACTTCAC下划线为Nco I酶切位点）和反向引物（HscB-BamH I-F：CGCGGATCCGCGttaTTCGGCCTCG下划线为BamH I酶切位点）。按照菌株pTRC99A-SufA的构建方法，将酶切后的片段ScdA与HscB与酶切后的载体pTRC99A连接，并转化至大肠杆菌Trans1T1中并送测序对比,得到pTRC99A-ScdA和pTRC99A-HscB。
含来自大肠大肠杆菌的木糖酸脱水酶YjhG的菌株构建：利用YjhG基因5’端和3’端引物引入酶切位点(NcoI和HindⅢ)，对YjhG基因和pCWJ质粒进行双酶切，然后将YjhG基因连接到pCWJ载体上；将连接液转入大肠杆菌Trans 1T1的感受态细胞（全式金生物技术有限公司）里，涂布在带有50mg/L氯霉抗性的LB平板，37℃过夜培养。挑取平板上生长的单菌落，转接到含有50mg/L氯霉素抗性的LB培养基里，然后提取质粒，再经限制性内切酶Spe I和Kpn I进行酶切验证，最后得到了重组质粒pCWJ-YjhG。
将质粒pCWJ-YjhG分别与质粒pTRC99A-SufA、pTRC99A-ScdA和pTRC99A-HscB共同转化到大肠杆菌Trans 1T1（购买于全式金生物技术有限公司）的感受态细胞中，得到基因工程菌E.coliT1-pCWJ-YjhG-pTRC99A-SufA、基因工程菌E.coliT1-pCWJ-yjhG-pTRC99A-ScdA和基因工程菌E.coliT1-pCWJ-yjhG-pTRC99A-HscB。
SufA核苷酸序列
ATGGACATGCATTCAGGAACCTTTAACCCACAAGATTTCGCCTGGCAAGGCTTAACGCTGACACCCGCAGCGGCGATACACATCCGTGAGCTGGTGGCAAAGCAGCCGGGTATGGTCGGCGTGCGCTTAGGCGTGAAGCAAACGGGCTGCGCGGGCTTTGGCTATGTGCTCGACAGTGTTAGCGAGCCGGACAAAGACGATCTGCTGTTTGAACACGACGGCGCGAAGCTGTTTGTCCCGCTGCAAGCGATGCCGTTTATTGATGGCACGGAAGTCGATTTCGTTCGTGAAGGACTTAATCAGATATTCAAATTTCACAACCCTAAAGCCCAGAATGAATGTGGCTGTGGCGAAAGCTTTGGGGTAtagatgTCTCGTAATACTGAAGCAACTGACGATGTCAAAACCTGGACCGGCGGCCCGCTGAATTATAAAGAAGGATTCTTCACCCAGTTAGCCACCGATGAGCTGGCAAAGGGGATAAACGAAGAGGTGGTGCGCGCAATTTCGGCGAAGCGTAATGAGCCGGAGTGGATGCTGGAGTTTCGTCTAAACGCCTATCGCGCATGGCTGGAGATGGAAGAACCGCACTGGTTGAAAGCGCACTACGACAAGCTGAATTATCAGGATTACAGCTACTACTCAGCACCATCGTGCGGTAATTGTGACGACACTTGCGCGTCTGAACCTGGCGCGGTGCAGCAAACTGGCGCGAACGCCTTTTTAAGTAAAGAGGTGGAGGCGGCGTTTGAGCAGTTGGGCGTTCCCGTGCGGGAAGGCAAAGAGGTGGCGGTGGATGCCATTTTCGACTCAGTTTCGGTTGCCACTACTTATCGCGAAAAACTGGCGGAGCAGGGAATTATTTTCTGTTCCTTTGGTGAGGCGATCCACGATCACCCGGAACTGGTGCGTAAATATCTCGGCACCGTGGTGCCGGGGAATGACAACTTCTTTGCCGCGCTTAATGCGGCGGTAGCCTCTGATGGTACGTTTATTTATGTGCCTAAAGGCGTGCGCTGCCCGATGGAACTTTCCACCTATTTTCGCATTAACGCAGAAAAAACCGGGCAGTTTGAGCGCACCATTCTGGTGGCCGACGAAGACAGCTACGTCAGCTACATTGAAGGCTGTTCCGCTCCGGTGCGTGACAGCTATCAGTTACACGCGGCAGTGGTGGAAGTCATCATCCATAAAAACGCCGAGGTGAAATATTCCACGGTACAAAACTGGTTTCCTGGCGATAACAACACCGGCGGTATTCTCAACTTCGTCACCAAGCGTGCTTTGTGCGAAGGCGAAAACAGCAAAATGTCATGGACGCAATCAGAAACCGGGTCAGCGATTACGTGGAAATATCCCAGCTGCATTTTGCGCGGCGATAACTCCATTGGTGAGTTTTACTCAGTGGCGCTGACCAGCGGTCATCAGCAAGCGGATACCGGCACCAAGATGATCCACATCGGTAAAAACACCAAATCGACCATTATCTCGAAAGGGATCTCTGCCGGACATAGTCAGAACAGTTATCGCGGCTTAGTGAAAATCATGCCGACGGCAACCAATGCGCGCAATTTCACTCAGTGCGACTCAATGCTGATTGGCGCTAATTGTGGGGCGCATACCTTCCCGTATGTTGAGTGTCGTAACAATAGTGCGCAACTGGAACACGAGGCAACGACATCACGTATTGGTGAAGATCAACTGTTTTACTGCCTGCAACGCGGGATCAGCGAAGAAGACGCCATCTCGATGATTGTTAACGGTTTCTGCAAAGACGTGTTCTCGGAGCTGCCGTTGGAATTTGCCGTTGAAGCACAAAAACTCCTCGCCATCAGTCTTGAACACAGCGTCGGAtaaatgTTAAGTATTAAAGATTTACACGTCAGCGTGGAAGATAAAGCTATCCTGCGCGGATTAAGCCTCGACGTTCATCCCGGCGAAGTTCACGCCATTATGGGGCCAAACGGTTCGGGCAAAAGTACCTTATCGGCAACGCTTGCCGGGCGAGAAGATTATGAAGTGACGGGCGGCACGGTTGAGTTCAAAGGCAAAGATTTGCTTGCGCTGTCGCCGGAAGATCGCGCGGGCGAAGGCATCTTTATGGCCTTCCAGTATCCGGTGGAGATTCCAGGTGTCAGTAACCAGTTTTTCCTGCAAACGGCACTTAATGCGGTGCGCAGCTATCGCGGCCAGGAAACGCTCGACCGCTTTGATTTTCAGGATTTGATGGAAGAGAAAATCGCTCTCCTGAAGATGCCGGAAGATTTATTAACCCGTTCGGTAAACGTTGGTTTTTCCGGCGGCGAGAAAAAGCGCAACGATATTTTGCAAATGGCGGTGCTGGAACCGGAGTTATGCATTCTTGATGAGTCGGACTCCGGGCTGGATATTGACGCATTAAAAGTGGTCGCCGATGGCGTGAACTCGCTGCGTGATGGCAAGCGCTCATTCATCATTGTTACGCACTACCAACGCATTCTCGACTACATCAAGCCTGATTACGTTCATGTGCTATATCAGGGACGAATTGTGAAATCCGGCGATTTCACGTTGGTCAAACAACTGGAGGAGCAGGGTTATGGCTGGCTTACCGAACAGCAGtaaatgGCTGGCTTACCGAACAGCAGTAACGCGCTGCAACAGTGGCATCACTTGTTTGAAGCTGAAGGGACAAAACGCTCCCCGCAAGCACAGCAGCATTTACAACAATTGCTGCGTACCGGACTGCCGACACGTAAACATGAAAACTGGAAATATACGCCGCTGGAAGGGCTGATCAATAGCCAGTTTGTCAGCATTGCGGGAGAGATATCCCCACAGCAGCGTGATGCCTTAGCGTTAACGTTAGACTCCGTGCGGCTGGTGTTTGTCGATGGGCGTTACGTGCCCGCACTGAGCGATGCAACTGAAGGCAGCGGATATGAAGTGAGCATTAACGACGACCGTCAGGGTTTACCCGACGCTATTCAGGCGGAAGTGTTTCTGCATTTGACGGAAAGCCTGGCACAAAGCGTGACGCATATCGCCGTGAAGCGCGGTCAACGGCCGGCAAAGCCATTGCTGTTAATGCATATCACCCAGGGCGTGGCAGGTGAAGAGGTGAACACTGCCCATTACCGACATCATCTGGATCTGGCGGAAGGTGCCGAAGCAACGGTGATCGAACATTTTGTCAGCCTGAATGATGCTCGTCATTTTACCGGGGCACGGTTCACTATCAACGTCGCAGCGAATGCCCACTTGCAGCATATCAAGCTGGCGTTTGAAAACCCGCTCAGTCACCACTTTGCTCATAACGATTTGTTGCTGGCTGAGGATGCCACCGCATTTAGCCACAGTTTCCTGCTGGGTGGCGCAGTGTTACGACACAACACCAGTACGCAACTCAATGGCGAAAACAGCACGCTGCGGATCAATAGCCTGGCGATGCCGGTGAAAAACGAGGTGTGTGATACCCGTACCTGGCTGGAACACAATAAAGGTTTTTGTAACAGCCGACAGTTGCACAAAACTATCGTCAGCGACAAAGGCCGCGCGGTATTTAACGGTTTGATCAACGTCGCGCAGCACGCCATCAAAACGGATGGTCAGATGACCAACAACAATCTGCTGATGGGCAAACTGGCGGAAGTGGATACGAAACCGCAGCTGGAAATCTATGCAGATGATGTGAAATGCAGCCACGGCGCGACGGTGGGGCGTATTGATGATGAACAGATATTCTATCTGCGCTCGCGCGGGATCAATCAGCAGGATGCCCAGCAGATGATCATTTACGCCTTCGCTGCCGAACTGACGGAAGCACTGCGTGATGAGGGGCTTAAACAGCAGGTGCTGGCCCGAATCGGTCAACGGCTGCCAGGAGGTGCAAGAtgaatgATTTTTTCCGTCGACAAAGTGCGGGCCGACTTTCCGGTGCTTTCGCGTGAGGTAAACGGTTTGCCGCTGGCTTATCTCGACAGCGCCGCCAGTGCGCAGAAACCGAGCCAGGTGATTGACGCCGAGGCCGAGTTTTATCGTCATGGCTACGCGGCGGTGCATCGTGGTATTCATACCTTAAGCGCCCAGGCGACCGAGAAAATGGAGAACGTGCGCAAGCGGGCATCGCTGTTTATTAATGCCCGTTCGGCGGAAGAGCTGGTGTTCGTCCGCGGCACGACGGAAGGGATCAATCTGGTCGCCAATAGCTGGGGCAACAGCAACGTGCGGGCGGGCGATAACATCATCATCAGTCAGATGGAGCACCACGCTAACATTGTTCCCTGGCAGATGCTTTGCGCACGCGTTGGCGCAGAGCTGCGTGTGATCCCGCTCAATCCCGATGGTACGTTGCAACTGGAGACGCTGCCTACGCTGTTTGATGAGAAAACTCGCCTGCTGGCAATTACTCATGTCTCCAACGTGCTTGGCACAGAAAATCCACTGGCGGAAATGATCACGCTTGCGCACCAGCATGGCGCAAAAGTGCTGGTGGATGGCGCTCAGGCGGTGATGCATCATCCGGTGGATGTTCAGGCGCTGGATTGCGACTTTTACGTGTTCTCCGGGCATAAACTGTATGGCCCCACCGGAATTGGCATTCTTTATGTGAAAGAAGCCTTGTTGCAGGAGATGCCGCCGTGGGAAGGGGGCGGTTCTATGATCGCCACCGTCAGCCTGAGTGAAGGCACTACCTGGACCAAAGCACCATGGCGGTTTGAAGCCGGTACACCCAATACCGGGGGCATCATTGGTCTTGGCGCGGCGCTGGAGTATGTTTCGGCGCTGGGGCTTAATAACATAGCCGAGTATGAACAGAATCTGATGCATTATGCGCTATCACAGCTGGAATCTGTACCGGATCTCACTCTCTATGGCCCACAAAACAGGCTTGGCGTTATTGCTTTTAATCTCGGTAAACACCACGCCTATGATGTTGGCAGTTTTCTCGATAATTACGGCATTGCTGTGCGTACCGGACATCACTGCGCAATGCCATTGATGGCCTATTACAACGTCCCTGCGATGTGTCGGGCGTCGCTGGCCATGTATAACACCCATGAAGAAGTGGATCGTCTGGTGACCGGCCTGCAACGTATTCACCGTTTGCTGGGAtaaatgGCTTTATTGCCGGATAAAGAAAAGTTGCTGCGTAATTTTTTACGCTGCGCCAACTGGGAAGAGAAATATCTCTACATTATTGAGCTGGGCCAGCGTCTGCCAGAATTACGCGACGAAGACAGAAGTCCACAAAATAGCATTCAGGGCTGTCAGAGTCAGGTGTGGATTGTCATGCGCCAGAATGCCCAGGGAATTATTGAATTACAGGGCGACAGCGATGCGGCGATTGTGAAAGGGCTTATTGCGGTCGTCTTTATTCTCTACGATCAGATGACGCCGCAGGATATTGTCAATTTCGATGTGCGTCCGTGGTTTGAAAAAATGGCGCTCACCCAACATCTCACCCCATCTCGTTCACAAGGTCTGGAAGCGATGATTCGCGCAATTCGCGCCAAAGCCGCTGCACTTAGCTAA
HscB核苷酸序列
ATGGATTACTTCACCCTCTTTGGCTTGCCTGCCCGCTATCAACTCGATACCCAGGCGCTGAGCCTGCGTTTTCAGGATCTACAACGTCAGTATCATCCTGATAAATTCGCCAGCGGAAGCCAGGCGGAACAACTCGCCGCCGTACAGCAATCTGCAACCATTAACCAGGCCTGGCAAACGCTGCGTCATCCGTTAATGCGCGCGGAATATTTGCTTTCTTTGCACGGCTTTGATCTCGCCAGCGAGCAGCATACTGTGCGCGACACCGCGTTCCTGATGGAACAGTTGGAGCTGCGCGAAGAGCTGGACGAGATCGAACAGGCGAAAGATGAAGCGCGGCTGGAAAGCTTTATCAAACGTGTGAAAAAGATGTTTGATACCCGCCATCAGTTGATGGTTGAACAGTTAGACAACGAGACGTGGGACGCGGCGGCGGATACCGTGCGTAAGCTGCGTTTTCTCGATAAACTGCGAAGCAGTGCCGAACAACTCGAAGAAAAACTGCTCGATTTTtaaatgGCCTTATTACAAATTAGTGAACCTGGTTTGAGTGCTGCGCCGCATCAGCGTCGTCTGGCGGCCGGTATTGACCTGGGCACAACCAACTCGCTGGTGGCGACAGTGCGCAGCGGTCAGGCCGAAACGTTAGCCGATCATGAAGGCCGTCACCTGCTGCCATCTGTTGTTCACTATCAACAGCAAGGGCATTCGGTGGGTTATGACGCGCGTACTAATGCAGCGCTCGATACCGCCAACACAATTAGTTCTGTTAAACGCCTGATGGGACGCTCGCTGGCTGATATCCAGCAACGCTATCCGCATCTGCCTTATCAATTCCAGGCCAGCGAAAACGGCCTGCCGATGATTGAAACGGCGGCGGGGCTGCTGAACCCGGTGCGCGTTTCTGCGGACATCCTCAAAGCACTGGCGGCGCGGGCAACTGAAGCCCTGGCAGGCGAGCTGGATGGTGTAGTTATCACCGTTCCGGCGTACTTTGACGATGCCCAGCGTCAGGGCACCAAAGACGCGGCGCGTCTGGCGGGCCTTCACGTCCTGCGCTTACTTAACGAACCGACCGCTGCGGCTATCGCCTACGGGCTGGATTCCGGTCAGGAAGGCGTGATCGCCGTTTATGACCTCGGTGGCGGGACGTTTGATATTTCCATTCTGCGCTTAAGTCGCGGCGTGTTTGAAGTGCTGGCAACCGGCGGTGATTCCGCGCTCGGCGGCGATGATTTCGACCATCTGCTGGCGGATTACATTCGCGAGCAGGCGGGCATTCCTGATCGTAGCGATAACCGCGTTCAGCGTGAACTGCTGGATGCCGCCATTGCAGCCAAAATCGCGCTGAGCGATGCGGACTCCGTGACCGTTAACGTTGCGGGCTGGCAGGGCGAAATCAGCCGTGAACAATTCAATGAACTGATCGCGCCACTGGTAAAACGAACCTTACTGGCTTGTCGTCGCGCGCTGAAAGACGCGGGTGTAGAAGCTGATGAAGTGCTGGAAGTGGTGATGGTGGGCGGTTCTACTCGCGTGCCGCTGGTGCGTGAACGGGTAGGCGAATTTTTCGGTCGTCCACCGCTGACTTCCATCGACCCGGATAAAGTCGTCGCTATTGGCGCGGCGATTCAGGCGGATATTCTGGTGGGTAACAAGCCAGACAGCGAAATGCTGTTGCTTGATGTGATCCCACTGTCGCTGGGCCTCGAAACGATGGGCGGCCTGGTGGAGAAAGTGATTCCGCGTAATACCACTATTCCGGTGGCCCGCGCTCAGGATTTCACCACCTTTAAAGATGGTCAGACGGCGATGTCTATCCATGTAATGCAGGGTGAGCGCGAACTGGTGCAGGACTGCCGCTCACTGGCGCGTTTTGCGCTGCGTGGTATTCCGGCGCTACCGGCTGGCGGTGCGCATATTCGCGTGACGTTCCAGGTCGATGCCGACGGTCTTTTGAGCGTGACGGCGATGGAGAAATCCACCGGCGTTGAGGCGTCTATTCAGGTCAAACCGTCTTACGGTCTGACCGATAGCGAAATCGCTTCGATGATCAAAGACTCAATGAGCTATGCCGAGCAGGACGTAAAAGCCCGAATGCTGGCAGAACAAAAAGTAGAAGCGGCGCGTGTGCTGGAAAGTCTGCACGGCGCGCTGGCTGCTGATGCCGCGCTGTTAAGCGCCGCAGAACGTCAGGTCATTGACGATGCTGCCGCTCACCTGAGTGAAGTGGCGCAGGGCGATGATGTTGACGCCATCGAACAAGCGATTAAAAACGTAGACAAACAAACCCAGGATTTCGCCGCTCGCCGCATGGACCAGTCGGTTCGTCGTGCGCTGAAAGGCCATTCCGTGGACGAGGTTtaaatgCCAAAGATTGTTATTTTGCCTCATCAGGATCTCTGCCCTGATGGCGCTGTTCTGGAAGCTAATAGCGGTGAAACCATTCTCGACGCAGCTCTGCGTAACGGTATCGAGATTGAACACGCCTGTGAAAAATCCTGTGCTTGCACCACCTGCCACTGCATCGTTCGTGAAGGTTTTGACTCACTGCCGGAAAGCTCAGAGCAGGAAGACGACATGCTGGACAAAGCCTGGGGACTGGAGCCGGAAAGCCGTTTAAGCTGCCAGGCGCGCGTTACCGACGAAGATTTAGTAGTCGAAATCCCGCGTTACACTATCAACCATGCGCGTGAGCATtaaatgGGACTTAAGTGGACCGATAGCCGCGAAATTGGCGAAGCACTGTACGATGCGTATCCCGATCTTGATCCGAAAACGGTTCGATTCACCGATATGCATCAGTGGATTTGCGATCTGGAAGATTTCGACGACGACCCGCAGGCATCCAACGAGAAAATCCTCGAAGCGATTTTGTTAGTCTGGCTGGACGAGGCCGAATAA
sScdA基因的核苷酸序列
ATGAACGTTTTTAATCCCGCGCAGTTTCGCGCCCAGTTTCCCGCACTACAGGATGCGGGCGTCTATCTCGACAGCGCCGCGACCGCGCTTAAACCTGAAGCCGTGGTTGAAGCCACCCAACAGTTTTACAGTCTGAGCGCCGGAAACGTCCATCGCAGCCAGTTTGCCGAAGCCCAACGCCTGACCGCGCGTTATGAAGCTGCACGAGAGAAAGTGGCGCAATTACTGAATGCACCGGATGATAAAACTATCGTCTGGACGCGCGGCACCACTGAATCCATCAACATGGTGGCACAATGCTATGCGCGTCCGCGTCTGCAACCGGGCGATGAGATTATTGTCAGCGTGGCAGAACACCACGCCAACCTCGTCCCCTGGCTGATGGTCGCCCAACAAACTGGAGCCAAAGTGGTGAAATTGCCGCTTAATGCGCAGCGACTGCCGGATGTCGATTTGTTGCCAGAACTGATTACTCCCCGTAGTCGGATTCTGGCGTTGGGTCAGATGTCGAACGTTACTGGCGGTTGCCCGGATCTGGCGCGAGCGATTACCTTTGCTCATTCAGCCGGGATGGTGGTGATGGTTGATGGTGCTCAGGGGGCAGTGCATTTCCCCGCGGATGTTCAGCAACTGGATATTGATTTCTATGCTTTTTCAGGTCACAAACTGTATGGCCCGACAGGTATCGGCGTGCTGTATGGTAAATCAGAACTGCTGGAGGCGATGTCGCCCTGGCTGGGCGGCGGCAAAATGGTTCACGAAGTGAGTTTTGACGGCTTCACGACTCAATCTGCGCCGTGGAAACTGGAAGCTGGAACGCCAAATGTCGCTGGTGTCATAGGATTAAGCGCGGCGCTGGAATGGCTGGCAGATTACGATATCAACCAGGCCGAAAGCTGGAGCCGTAGCTTAGCAACGCTGGCGGAAGATGCGCTGGCGAAACGTCCCGGCTTTCGTTCATTCCGCTGCCAGGATTCCAGCCTGCTGGCCTTTGATTTTGCTGGCGTTCATCATAGCGATATGGTGACGCTGCTGGCGGAGTACGGTATTGCCCTGCGGGCCGGGCAGCATTGCGCTCAGCCGCTACTGGCAGAATTAGGCGTAACCGGCACACTGCGCGCCTCTTTTGCGCCATATAATACAAAGAGTGATGTGGATGCGCTGGTGAATGCCGTTGACCGCGCGCTGGAATTATTGGTGGATtaaatgACAAACCCGCAATTCGCCGGACATCCGTTCGGCACAACCGTAACCGCAGAAACGTTACGCAATACCTTCGCACCGTTGACGCAATGGGAAGATAAATATCGCCAGTTGATCATGCTGGGGAAACAGCTTCCGGCATTGCCAGACGAGTTAAAAGCGCAGGCTAAAGAGATTGCCGGATGCGAAAACCGCGTCTGGCTGGGATATACAGTGGCTGAAAACGGCAAAATGCATTTCTTTGGCGACAGCGAAGGGCGCATTGTGCGCGGCCTGCTGGCGGTGTTGTTGACTGCCGTTGAGGGGAAAACCGCCGCCGAGTTGCAGGCACAGTCACCACTGGCATTGTTTGATGAGCTGGGATTACGTGCGCAGCTTAGCGCCTCACGCAGCCAGGGGTTAAATGCGTTAAGCGAGGCGATTATCGCTGCGACGAAGCAGGTTTAA
YjhG核苷酸序列
ATGGAAATGTCTGTTCGCAATATTTTTGCTGACGAGAGCCACGATATTTACACCGTCAGAACGCACGCCGATGGCCCGGACGGCGAACTCCCATTAACCGCAGAGATGCTTATCAACCGCCCGAGCGGGGATCTGTTCGGTATGACCATGAATGCCGGAATGGGTTGGTCTCCGGACGAGCTGGATCGGGACGGTATTTTACTGCTCAGTACACTCGGTGGCTTACGCGGCGCAGACGGTAAACCCGTGGCGCTGGCGTTGCACCAGGGGCATTACGAACTGGACATCCAGATGAAAGCGGCGGCCGAGGTTATTAAAGCCAACCATGCCCTGCCCTATGCCGTGTACGTCTCCGATCCTTGTGACGGGCGTACTCAGGGTACAACGGGGATGTTTGATTCGCTACCATACCGAAATGACGCATCGATGGTAATGCGCCGCCTTATTCGCTCTCTGCCCGACGCGAAAGCAGTTATTGGTGTGGCGAGTTGCGATAAGGGGCTTCCGGCCACCATGATGGCACTCGCCGCGCAGCACAACATCGCAACCGTGCTGGTCCCCGGCGGCGCGACGCTGCCCGCAAAGGATGGAGAAGACAACGGCAAGGTGCAAACCATTGGCGCACGCTTCGCCAATGGCGAATTATCTCTACAGGACGCACGCCGTGCGGGCTGTAAAGCCTGTGCCTCTTCCGGCGGCGGCTGTCAATTTTTGGGCACTGCCGGGACATCTCAGGTGGTGGCCGAAGGATTGGGACTGGCAATCCCACATTCAGCCCTGGCCCCTTCCGGTGAGCCTGTGTGGCGGGAGATCGCCAGAGCTTCCGCGCGAGCTGCGCTGAACCTGAGTCAAAAAGGCATCACCACCCGGGAAATTCTCACCGATAAAGCGATAGAGAATGCGATGACGGTCCATGCCGCGTTCGGTGGTTCAACAAACCTGCTGTTACACATCCCGGCAATTGCTCACCAGGCAGGTTGCCATATCCCGACCGTTGATGACTGGATCCGCATCAACAAGCGCGTGCCCCGACTGGTGAGCGTACTGCCTAATGGCCCGGTTTATCATCCAACGGTCAATGCCTTTATGGCAGGTGGTGTGCCGGAAGTCATGTTGCATCTGCGCAGCCTCGGATTGTTGCATGAAGACGTTATGACGGTTACCGGCAGCACGCTGAAAGAAAACCTCGACTGGTGGGAGCACTCCGAACGGCGTCAGCGGTTCAAGCAACTCCTGCTCGATCAGGAACAAATCAACGCTGACGAAGTGATCATGTCTCCGCAGCAAGCAAAAGCGCGCGGATTAACCTCAACTATCACCTTCCCGGTGGGCAATATTGCGCCAGAAGGTTCGGTGATCAAATCCACCGCCATTGACCCCTCGATGATTGATGAGCAAGGTATCTATTACCATAAAGGTGTGGCGAAGGTTTATCTGTCCGAGAAAAGTGCGATTTACGATATCAAACATGACAAGATCAAGGCGGGCGATATTCTGGTCATTATTGGCGTTGGACCTTCAGGTACAGGGATGGAAGAAACCTACCAGGTTACCAGTGCCCTGAAGCATCTGTCATACGGTAAGCATGTTTCGTTAATCACCGATGCACGTTTCTCGGGCGTTTCTACTGGCGCGTGCATCGGCCATGTGGGGCCAGAAGCGCTGGCCGGAGGCCCCATCGGTAAATTACGCACCGGGGATTTAATTGAAATTAAAATTGATTGTCGCGAGCTTCACGGCGAAGTCAATTTCCTCGGAACCCGTAGCGATGAACAATTACCTTCACAGGAGGAGGCAACTGCAATATTAAATGCCAGACCCAGCCATCAGGATTTACTTCCCGATCCTGAATTGCCAGATGATACCCGGCTATGGGCAATGCTTCAGGCCGTGAGTGGTGGGACATGGACCGGTTGTATTTATGATGTAAACAAAATTGGCGCGGCTTTGCGCGATTTTATGAATAAAAACTGA
实施例2 基因工程菌E.coliT1-pCWJ-yjhG-pTRC99A-SufA的发酵
在平板上面挑取实施例1中的3个单菌落分别接入到5ml LB种子培养基中摇8-10h，将种子液以1%v/v的量接入到100ml发酵培养基中，待OD 600 到0.6加入IPTG进行诱导，33℃200rpm下培养11h，离心收菌，用菌体重悬使OD600=60，底物木糖酸为20g/L,无操纵子系统底物木糖酸在17h内消耗了2g/L，而含有SufA、HscB、ScdA基因的基因工程菌则在17h内分别消耗了6.12g/L、3.8g/L、1.45g/L。
种子培养基：蛋白胨10g/L、酵母粉5g/L、氯化钠10g/L
发酵培养基：蛋白胨10g/L、酵母粉5g/L、氯化钠5g/L、氯化铵2g/L、磷酸二氢钾3g/L。
D-木糖酸的检测条件为：Agilent Technologies1290高效液相色谱；Bio-
Rad HPX-87H IonExclusion Column(300mm×7.8mm)有机酸柱；流动相为5mmol/L H 2SO 4 ；流速0.6mL/min，柱温55℃，进样量20μL，视差检测器。
序列表
<110> 南京工业大学
<120> 一种提高木糖酸脱水酶活性的基因工程菌及其构建方法与应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccggaattcc ggatggacat gcattc 26
<210> 2
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cggggtaccc cgttagctaa gtgcag 26
<210> 3
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
catgccatgg catgatgaac gtttttaatc 30
<210> 4
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cgcggatccg cgttaaacct gcttcg 26
<210> 5
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
catgccatgg catgatggat tacttcac 28
<210> 6
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cgcggatccg cgttattcgg cctcg 25
<210> 7
<211> 5514
<212> DNA
<213> 基因序列(Gene sequence)
<400> 7
atggacatgc attcaggaac ctttaaccca caagatttcg cctggcaagg cttaacgctg 60
acacccgcag cggcgataca catccgtgag ctggtggcaa agcagccggg tatggtcggc 120
gtgcgcttag gcgtgaagca aacgggctgc gcgggctttg gctatgtgct cgacagtgtt 180
agcgagccgg acaaagacga tctgctgttt gaacacgacg gcgcgaagct gtttgtcccg 240
ctgcaagcga tgccgtttat tgatggcacg gaagtcgatt tcgttcgtga aggacttaat 300
cagatattca aatttcacaa ccctaaagcc cagaatgaat gtggctgtgg cgaaagcttt 360
ggggtataga tgtctcgtaa tactgaagca actgacgatg tcaaaacctg gaccggcggc 420
ccgctgaatt ataaagaagg attcttcacc cagttagcca ccgatgagct ggcaaagggg 480
ataaacgaag aggtggtgcg cgcaatttcg gcgaagcgta atgagccgga gtggatgctg 540
gagtttcgtc taaacgccta tcgcgcatgg ctggagatgg aagaaccgca ctggttgaaa 600
gcgcactacg acaagctgaa ttatcaggat tacagctact actcagcacc atcgtgcggt 660
aattgtgacg acacttgcgc gtctgaacct ggcgcggtgc agcaaactgg cgcgaacgcc 720
tttttaagta aagaggtgga ggcggcgttt gagcagttgg gcgttcccgt gcgggaaggc 780
aaagaggtgg cggtggatgc cattttcgac tcagtttcgg ttgccactac ttatcgcgaa 840
aaactggcgg agcagggaat tattttctgt tcctttggtg aggcgatcca cgatcacccg 900
gaactggtgc gtaaatatct cggcaccgtg gtgccgggga atgacaactt ctttgccgcg 960
cttaatgcgg cggtagcctc tgatggtacg tttatttatg tgcctaaagg cgtgcgctgc 1020
ccgatggaac tttccaccta ttttcgcatt aacgcagaaa aaaccgggca gtttgagcgc 1080
accattctgg tggccgacga agacagctac gtcagctaca ttgaaggctg ttccgctccg 1140
gtgcgtgaca gctatcagtt acacgcggca gtggtggaag tcatcatcca taaaaacgcc 1200
gaggtgaaat attccacggt acaaaactgg tttcctggcg ataacaacac cggcggtatt 1260
ctcaacttcg tcaccaagcg tgctttgtgc gaaggcgaaa acagcaaaat gtcatggacg 1320
caatcagaaa ccgggtcagc gattacgtgg aaatatccca gctgcatttt gcgcggcgat 1380
aactccattg gtgagtttta ctcagtggcg ctgaccagcg gtcatcagca agcggatacc 1440
ggcaccaaga tgatccacat cggtaaaaac accaaatcga ccattatctc gaaagggatc 1500
tctgccggac atagtcagaa cagttatcgc ggcttagtga aaatcatgcc gacggcaacc 1560
aatgcgcgca atttcactca gtgcgactca atgctgattg gcgctaattg tggggcgcat 1620
accttcccgt atgttgagtg tcgtaacaat agtgcgcaac tggaacacga ggcaacgaca 1680
tcacgtattg gtgaagatca actgttttac tgcctgcaac gcgggatcag cgaagaagac 1740
gccatctcga tgattgttaa cggtttctgc aaagacgtgt tctcggagct gccgttggaa 1800
tttgccgttg aagcacaaaa actcctcgcc atcagtcttg aacacagcgt cggataaatg 1860
ttaagtatta aagatttaca cgtcagcgtg gaagataaag ctatcctgcg cggattaagc 1920
ctcgacgttc atcccggcga agttcacgcc attatggggc caaacggttc gggcaaaagt 1980
accttatcgg caacgcttgc cgggcgagaa gattatgaag tgacgggcgg cacggttgag 2040
ttcaaaggca aagatttgct tgcgctgtcg ccggaagatc gcgcgggcga aggcatcttt 2100
atggccttcc agtatccggt ggagattcca ggtgtcagta accagttttt cctgcaaacg 2160
gcacttaatg cggtgcgcag ctatcgcggc caggaaacgc tcgaccgctt tgattttcag 2220
gatttgatgg aagagaaaat cgctctcctg aagatgccgg aagatttatt aacccgttcg 2280
gtaaacgttg gtttttccgg cggcgagaaa aagcgcaacg atattttgca aatggcggtg 2340
ctggaaccgg agttatgcat tcttgatgag tcggactccg ggctggatat tgacgcatta 2400
aaagtggtcg ccgatggcgt gaactcgctg cgtgatggca agcgctcatt catcattgtt 2460
acgcactacc aacgcattct cgactacatc aagcctgatt acgttcatgt gctatatcag 2520
ggacgaattg tgaaatccgg cgatttcacg ttggtcaaac aactggagga gcagggttat 2580
ggctggctta ccgaacagca gtaaatggct ggcttaccga acagcagtaa cgcgctgcaa 2640
cagtggcatc acttgtttga agctgaaggg acaaaacgct ccccgcaagc acagcagcat 2700
ttacaacaat tgctgcgtac cggactgccg acacgtaaac atgaaaactg gaaatatacg 2760
ccgctggaag ggctgatcaa tagccagttt gtcagcattg cgggagagat atccccacag 2820
cagcgtgatg ccttagcgtt aacgttagac tccgtgcggc tggtgtttgt cgatgggcgt 2880
tacgtgcccg cactgagcga tgcaactgaa ggcagcggat atgaagtgag cattaacgac 2940
gaccgtcagg gtttacccga cgctattcag gcggaagtgt ttctgcattt gacggaaagc 3000
ctggcacaaa gcgtgacgca tatcgccgtg aagcgcggtc aacggccggc aaagccattg 3060
ctgttaatgc atatcaccca gggcgtggca ggtgaagagg tgaacactgc ccattaccga 3120
catcatctgg atctggcgga aggtgccgaa gcaacggtga tcgaacattt tgtcagcctg 3180
aatgatgctc gtcattttac cggggcacgg ttcactatca acgtcgcagc gaatgcccac 3240
ttgcagcata tcaagctggc gtttgaaaac ccgctcagtc accactttgc tcataacgat 3300
ttgttgctgg ctgaggatgc caccgcattt agccacagtt tcctgctggg tggcgcagtg 3360
ttacgacaca acaccagtac gcaactcaat ggcgaaaaca gcacgctgcg gatcaatagc 3420
ctggcgatgc cggtgaaaaa cgaggtgtgt gatacccgta cctggctgga acacaataaa 3480
ggtttttgta acagccgaca gttgcacaaa actatcgtca gcgacaaagg ccgcgcggta 3540
tttaacggtt tgatcaacgt cgcgcagcac gccatcaaaa cggatggtca gatgaccaac 3600
aacaatctgc tgatgggcaa actggcggaa gtggatacga aaccgcagct ggaaatctat 3660
gcagatgatg tgaaatgcag ccacggcgcg acggtggggc gtattgatga tgaacagata 3720
ttctatctgc gctcgcgcgg gatcaatcag caggatgccc agcagatgat catttacgcc 3780
ttcgctgccg aactgacgga agcactgcgt gatgaggggc ttaaacagca ggtgctggcc 3840
cgaatcggtc aacggctgcc aggaggtgca agatgaatga ttttttccgt cgacaaagtg 3900
cgggccgact ttccggtgct ttcgcgtgag gtaaacggtt tgccgctggc ttatctcgac 3960
agcgccgcca gtgcgcagaa accgagccag gtgattgacg ccgaggccga gttttatcgt 4020
catggctacg cggcggtgca tcgtggtatt cataccttaa gcgcccaggc gaccgagaaa 4080
atggagaacg tgcgcaagcg ggcatcgctg tttattaatg cccgttcggc ggaagagctg 4140
gtgttcgtcc gcggcacgac ggaagggatc aatctggtcg ccaatagctg gggcaacagc 4200
aacgtgcggg cgggcgataa catcatcatc agtcagatgg agcaccacgc taacattgtt 4260
ccctggcaga tgctttgcgc acgcgttggc gcagagctgc gtgtgatccc gctcaatccc 4320
gatggtacgt tgcaactgga gacgctgcct acgctgtttg atgagaaaac tcgcctgctg 4380
gcaattactc atgtctccaa cgtgcttggc acagaaaatc cactggcgga aatgatcacg 4440
cttgcgcacc agcatggcgc aaaagtgctg gtggatggcg ctcaggcggt gatgcatcat 4500
ccggtggatg ttcaggcgct ggattgcgac ttttacgtgt tctccgggca taaactgtat 4560
ggccccaccg gaattggcat tctttatgtg aaagaagcct tgttgcagga gatgccgccg 4620
tgggaagggg gcggttctat gatcgccacc gtcagcctga gtgaaggcac tacctggacc 4680
aaagcaccat ggcggtttga agccggtaca cccaataccg ggggcatcat tggtcttggc 4740
gcggcgctgg agtatgtttc ggcgctgggg cttaataaca tagccgagta tgaacagaat 4800
ctgatgcatt atgcgctatc acagctggaa tctgtaccgg atctcactct ctatggccca 4860
caaaacaggc ttggcgttat tgcttttaat ctcggtaaac accacgccta tgatgttggc 4920
agttttctcg ataattacgg cattgctgtg cgtaccggac atcactgcgc aatgccattg 4980
atggcctatt acaacgtccc tgcgatgtgt cgggcgtcgc tggccatgta taacacccat 5040
gaagaagtgg atcgtctggt gaccggcctg caacgtattc accgtttgct gggataaatg 5100
gctttattgc cggataaaga aaagttgctg cgtaattttt tacgctgcgc caactgggaa 5160
gagaaatatc tctacattat tgagctgggc cagcgtctgc cagaattacg cgacgaagac 5220
agaagtccac aaaatagcat tcagggctgt cagagtcagg tgtggattgt catgcgccag 5280
aatgcccagg gaattattga attacagggc gacagcgatg cggcgattgt gaaagggctt 5340
attgcggtcg tctttattct ctacgatcag atgacgccgc aggatattgt caatttcgat 5400
gtgcgtccgt ggtttgaaaa aatggcgctc acccaacatc tcaccccatc tcgttcacaa 5460
ggtctggaag cgatgattcg cgcaattcgc gccaaagccg ctgcacttag ctaa 5514
<210> 8
<211> 2904
<212> DNA
<213> 基因序列(Gene sequence)
<400> 8
atggattact tcaccctctt tggcttgcct gcccgctatc aactcgatac ccaggcgctg 60
agcctgcgtt ttcaggatct acaacgtcag tatcatcctg ataaattcgc cagcggaagc 120
caggcggaac aactcgccgc cgtacagcaa tctgcaacca ttaaccaggc ctggcaaacg 180
ctgcgtcatc cgttaatgcg cgcggaatat ttgctttctt tgcacggctt tgatctcgcc 240
agcgagcagc atactgtgcg cgacaccgcg ttcctgatgg aacagttgga gctgcgcgaa 300
gagctggacg agatcgaaca ggcgaaagat gaagcgcggc tggaaagctt tatcaaacgt 360
gtgaaaaaga tgtttgatac ccgccatcag ttgatggttg aacagttaga caacgagacg 420
tgggacgcgg cggcggatac cgtgcgtaag ctgcgttttc tcgataaact gcgaagcagt 480
gccgaacaac tcgaagaaaa actgctcgat ttttaaatgg ccttattaca aattagtgaa 540
cctggtttga gtgctgcgcc gcatcagcgt cgtctggcgg ccggtattga cctgggcaca 600
accaactcgc tggtggcgac agtgcgcagc ggtcaggccg aaacgttagc cgatcatgaa 660
ggccgtcacc tgctgccatc tgttgttcac tatcaacagc aagggcattc ggtgggttat 720
gacgcgcgta ctaatgcagc gctcgatacc gccaacacaa ttagttctgt taaacgcctg 780
atgggacgct cgctggctga tatccagcaa cgctatccgc atctgcctta tcaattccag 840
gccagcgaaa acggcctgcc gatgattgaa acggcggcgg ggctgctgaa cccggtgcgc 900
gtttctgcgg acatcctcaa agcactggcg gcgcgggcaa ctgaagccct ggcaggcgag 960
ctggatggtg tagttatcac cgttccggcg tactttgacg atgcccagcg tcagggcacc 1020
aaagacgcgg cgcgtctggc gggccttcac gtcctgcgct tacttaacga accgaccgct 1080
gcggctatcg cctacgggct ggattccggt caggaaggcg tgatcgccgt ttatgacctc 1140
ggtggcggga cgtttgatat ttccattctg cgcttaagtc gcggcgtgtt tgaagtgctg 1200
gcaaccggcg gtgattccgc gctcggcggc gatgatttcg accatctgct ggcggattac 1260
attcgcgagc aggcgggcat tcctgatcgt agcgataacc gcgttcagcg tgaactgctg 1320
gatgccgcca ttgcagccaa aatcgcgctg agcgatgcgg actccgtgac cgttaacgtt 1380
gcgggctggc agggcgaaat cagccgtgaa caattcaatg aactgatcgc gccactggta 1440
aaacgaacct tactggcttg tcgtcgcgcg ctgaaagacg cgggtgtaga agctgatgaa 1500
gtgctggaag tggtgatggt gggcggttct actcgcgtgc cgctggtgcg tgaacgggta 1560
ggcgaatttt tcggtcgtcc accgctgact tccatcgacc cggataaagt cgtcgctatt 1620
ggcgcggcga ttcaggcgga tattctggtg ggtaacaagc cagacagcga aatgctgttg 1680
cttgatgtga tcccactgtc gctgggcctc gaaacgatgg gcggcctggt ggagaaagtg 1740
attccgcgta ataccactat tccggtggcc cgcgctcagg atttcaccac ctttaaagat 1800
ggtcagacgg cgatgtctat ccatgtaatg cagggtgagc gcgaactggt gcaggactgc 1860
cgctcactgg cgcgttttgc gctgcgtggt attccggcgc taccggctgg cggtgcgcat 1920
attcgcgtga cgttccaggt cgatgccgac ggtcttttga gcgtgacggc gatggagaaa 1980
tccaccggcg ttgaggcgtc tattcaggtc aaaccgtctt acggtctgac cgatagcgaa 2040
atcgcttcga tgatcaaaga ctcaatgagc tatgccgagc aggacgtaaa agcccgaatg 2100
ctggcagaac aaaaagtaga agcggcgcgt gtgctggaaa gtctgcacgg cgcgctggct 2160
gctgatgccg cgctgttaag cgccgcagaa cgtcaggtca ttgacgatgc tgccgctcac 2220
ctgagtgaag tggcgcaggg cgatgatgtt gacgccatcg aacaagcgat taaaaacgta 2280
gacaaacaaa cccaggattt cgccgctcgc cgcatggacc agtcggttcg tcgtgcgctg 2340
aaaggccatt ccgtggacga ggtttaaatg ccaaagattg ttattttgcc tcatcaggat 2400
ctctgccctg atggcgctgt tctggaagct aatagcggtg aaaccattct cgacgcagct 2460
ctgcgtaacg gtatcgagat tgaacacgcc tgtgaaaaat cctgtgcttg caccacctgc 2520
cactgcatcg ttcgtgaagg ttttgactca ctgccggaaa gctcagagca ggaagacgac 2580
atgctggaca aagcctgggg actggagccg gaaagccgtt taagctgcca ggcgcgcgtt 2640
accgacgaag atttagtagt cgaaatcccg cgttacacta tcaaccatgc gcgtgagcat 2700
taaatgggac ttaagtggac cgatagccgc gaaattggcg aagcactgta cgatgcgtat 2760
cccgatcttg atccgaaaac ggttcgattc accgatatgc atcagtggat ttgcgatctg 2820
gaagatttcg acgacgaccc gcaggcatcc aacgagaaaa tcctcgaagc gattttgtta 2880
gtctggctgg acgaggccga ataa 2904
<210> 9
<211> 1650
<212> DNA
<213> 基因序列(Gene sequence)
<400> 9
atgaacgttt ttaatcccgc gcagtttcgc gcccagtttc ccgcactaca ggatgcgggc 60
gtctatctcg acagcgccgc gaccgcgctt aaacctgaag ccgtggttga agccacccaa 120
cagttttaca gtctgagcgc cggaaacgtc catcgcagcc agtttgccga agcccaacgc 180
ctgaccgcgc gttatgaagc tgcacgagag aaagtggcgc aattactgaa tgcaccggat 240
gataaaacta tcgtctggac gcgcggcacc actgaatcca tcaacatggt ggcacaatgc 300
tatgcgcgtc cgcgtctgca accgggcgat gagattattg tcagcgtggc agaacaccac 360
gccaacctcg tcccctggct gatggtcgcc caacaaactg gagccaaagt ggtgaaattg 420
ccgcttaatg cgcagcgact gccggatgtc gatttgttgc cagaactgat tactccccgt 480
agtcggattc tggcgttggg tcagatgtcg aacgttactg gcggttgccc ggatctggcg 540
cgagcgatta cctttgctca ttcagccggg atggtggtga tggttgatgg tgctcagggg 600
gcagtgcatt tccccgcgga tgttcagcaa ctggatattg atttctatgc tttttcaggt 660
cacaaactgt atggcccgac aggtatcggc gtgctgtatg gtaaatcaga actgctggag 720
gcgatgtcgc cctggctggg cggcggcaaa atggttcacg aagtgagttt tgacggcttc 780
acgactcaat ctgcgccgtg gaaactggaa gctggaacgc caaatgtcgc tggtgtcata 840
ggattaagcg cggcgctgga atggctggca gattacgata tcaaccaggc cgaaagctgg 900
agccgtagct tagcaacgct ggcggaagat gcgctggcga aacgtcccgg ctttcgttca 960
ttccgctgcc aggattccag cctgctggcc tttgattttg ctggcgttca tcatagcgat 1020
atggtgacgc tgctggcgga gtacggtatt gccctgcggg ccgggcagca ttgcgctcag 1080
ccgctactgg cagaattagg cgtaaccggc acactgcgcg cctcttttgc gccatataat 1140
acaaagagtg atgtggatgc gctggtgaat gccgttgacc gcgcgctgga attattggtg 1200
gattaaatga caaacccgca attcgccgga catccgttcg gcacaaccgt aaccgcagaa 1260
acgttacgca ataccttcgc accgttgacg caatgggaag ataaatatcg ccagttgatc 1320
atgctgggga aacagcttcc ggcattgcca gacgagttaa aagcgcaggc taaagagatt 1380
gccggatgcg aaaaccgcgt ctggctggga tatacagtgg ctgaaaacgg caaaatgcat 1440
ttctttggcg acagcgaagg gcgcattgtg cgcggcctgc tggcggtgtt gttgactgcc 1500
gttgagggga aaaccgccgc cgagttgcag gcacagtcac cactggcatt gtttgatgag 1560
ctgggattac gtgcgcagct tagcgcctca cgcagccagg ggttaaatgc gttaagcgag 1620
gcgattatcg ctgcgacgaa gcaggtttaa 1650
<210> 10
<211> 1974
<212> DNA
<213> 基因序列(Gene sequence)
<400> 10
atggaaatgt ctgttcgcaa tatttttgct gacgagagcc acgatattta caccgtcaga 60
acgcacgccg atggcccgga cggcgaactc ccattaaccg cagagatgct tatcaaccgc 120
ccgagcgggg atctgttcgg tatgaccatg aatgccggaa tgggttggtc tccggacgag 180
ctggatcggg acggtatttt actgctcagt acactcggtg gcttacgcgg cgcagacggt 240
aaacccgtgg cgctggcgtt gcaccagggg cattacgaac tggacatcca gatgaaagcg 300
gcggccgagg ttattaaagc caaccatgcc ctgccctatg ccgtgtacgt ctccgatcct 360
tgtgacgggc gtactcaggg tacaacgggg atgtttgatt cgctaccata ccgaaatgac 420
gcatcgatgg taatgcgccg ccttattcgc tctctgcccg acgcgaaagc agttattggt 480
gtggcgagtt gcgataaggg gcttccggcc accatgatgg cactcgccgc gcagcacaac 540
atcgcaaccg tgctggtccc cggcggcgcg acgctgcccg caaaggatgg agaagacaac 600
ggcaaggtgc aaaccattgg cgcacgcttc gccaatggcg aattatctct acaggacgca 660
cgccgtgcgg gctgtaaagc ctgtgcctct tccggcggcg gctgtcaatt tttgggcact 720
gccgggacat ctcaggtggt ggccgaagga ttgggactgg caatcccaca ttcagccctg 780
gccccttccg gtgagcctgt gtggcgggag atcgccagag cttccgcgcg agctgcgctg 840
aacctgagtc aaaaaggcat caccacccgg gaaattctca ccgataaagc gatagagaat 900
gcgatgacgg tccatgccgc gttcggtggt tcaacaaacc tgctgttaca catcccggca 960
attgctcacc aggcaggttg ccatatcccg accgttgatg actggatccg catcaacaag 1020
cgcgtgcccc gactggtgag cgtactgcct aatggcccgg tttatcatcc aacggtcaat 1080
gcctttatgg caggtggtgt gccggaagtc atgttgcatc tgcgcagcct cggattgttg 1140
catgaagacg ttatgacggt taccggcagc acgctgaaag aaaacctcga ctggtgggag 1200
cactccgaac ggcgtcagcg gttcaagcaa ctcctgctcg atcaggaaca aatcaacgct 1260
gacgaagtga tcatgtctcc gcagcaagca aaagcgcgcg gattaacctc aactatcacc 1320
ttcccggtgg gcaatattgc gccagaaggt tcggtgatca aatccaccgc cattgacccc 1380
tcgatgattg atgagcaagg tatctattac cataaaggtg tggcgaaggt ttatctgtcc 1440
gagaaaagtg cgatttacga tatcaaacat gacaagatca aggcgggcga tattctggtc 1500
attattggcg ttggaccttc aggtacaggg atggaagaaa cctaccaggt taccagtgcc 1560
ctgaagcatc tgtcatacgg taagcatgtt tcgttaatca ccgatgcacg tttctcgggc 1620
gtttctactg gcgcgtgcat cggccatgtg gggccagaag cgctggccgg aggccccatc 1680
ggtaaattac gcaccgggga tttaattgaa attaaaattg attgtcgcga gcttcacggc 1740
gaagtcaatt tcctcggaac ccgtagcgat gaacaattac cttcacagga ggaggcaact 1800
gcaatattaa atgccagacc cagccatcag gatttacttc ccgatcctga attgccagat 1860
gatacccggc tatgggcaat gcttcaggcc gtgagtggtg ggacatggac cggttgtatt 1920
tatgatgtaa acaaaattgg cgcggctttg cgcgatttta tgaataaaaa ctga 1974
Claims (8)
1.一种提高木糖酸脱水酶活性的基因工程菌，其特征在于，构建克隆表达铁硫簇插入蛋白SufA基因， 并将构建好的基因转入宿主菌内得基因工程菌，所述基因工程菌用于发酵生产D-1 ,2 ,4-丁三醇的中间产物2-酮基-3-脱氧-d-木糖酸。
2.根据权利要求1所述提高木糖酸脱水酶活性的基因工程菌，其特征在于，所述的铁硫簇插入蛋白SufA基因来自大肠杆菌MG1655，且核苷酸序列如SEQ ID NO.1所示，所述的木糖酸脱水酶的核苷酸序列如SEQ ID NO.4所示。
3.根据权利要求1所述的提高木糖酸脱水酶活性的基因工程菌，其特征在于，所述的宿主菌为大肠杆菌Trans 1 T1。
4.权利要求1所述的提高木糖酸脱水酶活性的基因工程菌的构建方法，其特征在于，包括如下步骤：步骤1，将木糖酸脱水酶基因的两端插入酶切位点NcoⅠ与Hind Ⅲ，然后将酶切后的木糖酸脱水酶基因克隆到质粒一中，得到重组质粒一；将铁硫簇插入蛋白SufA基因的两端插入酶切位点EcoRⅠ与KpnⅠ,然后将酶切后的铁硫簇插入蛋白SufA基因克隆到质粒二中；得到重组质粒二；步骤2，将重组质粒一和重组质粒二共同转入宿主菌内，即得到了基因工程菌E.coliT1-pCWJ-yjhG-pTRC99A-SufA。
5.根据权利要求4所述的提高木糖酸脱水酶活性的基因工程菌的构建方法：其特征在于，质粒一为pCWJ质粒，质粒二为pTRC99A质粒。
6.基于权利要求1所述的提高木糖酸脱水酶活性的基因工程菌在以D-木糖酸为底物产1,2，4-丁三醇上的应用。
7.根据权利要求6所述的应用，其特征在于，包括如下步骤：第一步，培养基因工程菌E.coliT1-pCWJ-yjhG-pTRC99A-SufA；第二步，收培养后的基因工程菌，接种到发酵培养基中，加入D-木糖酸和IPTG诱导反应体系发酵产D-1,2,4-丁三醇。
8.根据权利要求7所述的应用，其特征在于，所述的反应体系包括如下组分：D-木糖酸的添加量为20g/L，菌体OD600为60，置于33℃摇床反应17h。
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