CN113481136B - 重组嗜盐单胞菌及构建方法及催化柠檬酸制备衣康酸的应用 - Google Patents
重组嗜盐单胞菌及构建方法及催化柠檬酸制备衣康酸的应用 Download PDFInfo
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Abstract
本发明公开了重组嗜盐单胞菌及构建方法及催化柠檬酸制备衣康酸的应用，一种重组嗜盐单胞菌的构建方法，其特征是包括如下步骤：在野生型嗜盐单胞菌Halomonas sp.TD01基因组上整合Mmp1RNA聚合酶表达单元，并将其icd基因起始密码子ATG替换为TTG，得到Halomonas sp.TD2.0；向其中依次导入高拷贝表达载体pN59‑PMmp1‑RBS‑cadA‑acn和低拷贝表达载体pN85‑PMmp1‑RBS‑GroESL‑PMmp1‑RBS‑acn；得到重组嗜盐单胞菌Halomonas sp.IA02；该菌细胞催化可高效制备衣康酸。本发明周期短、简便、产率高。
Description
技术领域
本发明属于生物工程技术领域，具体涉及一种重组嗜盐单胞菌及构建方法及细胞催化柠檬酸制备衣康酸的应用。
背景技术
衣康酸(Itaconic acid，IA)，一种C5不饱和二元酸，是美国能源部公布的十二种最具价值的生物基平台化合物之一，广泛应用于合成树脂、塑料、合成纤维和制药领域等。目前主要通过土曲霉(Aspergillus terreus)发酵法生产，最高衣康酸产量可达160g/L。微生物体中从柠檬酸出发合成衣康酸有两个关键酶参与，概括见图1，即TCA循环中间产物柠檬酸经顺乌头酸酶(Aconitase，ACN)催化生成顺乌头酸，进一步在顺乌头酸脱羧酶(Cis-aconitate decarboxylase，CAD)作用下脱羧生成为IA。其他真菌如玉蜀黍黑粉菌(Ustilago maydis，IA 最高产量205.6g/L)、拟酵母属(Pseudozyma sp.，最高产量74.7g/L)、假丝酵母属(Candida sp.，最高产量35g/L)等也具有天然合成衣康酸的能力。此外，异源真菌宿主也被改造用于发酵生产衣康酸，如黑曲霉(Aspergillus niger，最高产量42.7g/L)和解脂耶氏酵母(Yarrowia lipolytica，最高产量22.03g/L)等。但真菌发酵法存在着糖耗高、发酵周期长、发酵过程难控和对氧敏感等缺点，选择生长快、培养条件宽松、易控的菌株作为宿主非常必要，所以后续又改造了大肠杆菌(Escherichia coli，最高产量47g/L)、谷氨酸棒状杆菌(Corynebacterium glutamicum，最高产量7.8g/L)等细菌为宿主发酵合成衣康酸。但与土曲霉等真菌相比，细菌宿主发酵产生的衣康酸在效价和产率方面仍处于较低水平，没有竞争力。
2017年，一种生物催化策略——全细胞催化法，被Kim等人应用于衣康酸的生产中。选用易培养、生长快速的大肠杆菌全细胞作为生物催化剂，以廉价柠檬酸为底物高效合成衣康酸。通过引入和增强衣康酸合成关键酶ACN和CAD构建工程菌株，利用其全细胞转化系统，反应19h可催化500mM柠檬酸生成319.8mM衣康酸(41.61g/L)，转化率为63.96.0％，产率为2.19g/L/h。该方法简单、方便、高效且成本低，远优于传统发酵制备法，但反应体系中底物仍有大量剩余，不能完全转化，衣康酸产量和效率远不及大肠杆菌全细胞催化的其他产品(如尸胺、丙烯酰胺等)。这可能与衣康酸合成酶表达选用的异源宿主不适合、酶表达量少及酶活稳定性低等有关，进一步地筛选其他宿主和优化衣康酸合成酶表达是解决细胞催化高效制备衣康酸的关键问题。
嗜盐单胞菌(Halomonas sp.)TD01是一株中度嗜盐嗜碱细菌，具有耐高盐(NaCl，5％～6％) 和高碱(pH，9～11)、生长速度快、细胞干重大等生理特点。相比于普通模式微生物，如大肠杆菌和谷氨酸棒状杆菌等，嗜盐单胞菌具有以下优势：1)可不灭菌、开放式培养，降低了灭菌能耗和工艺复杂性；2)可用海水进行发酵，节约淡水资源，符合可持续发展要求；3) 染菌风险小可连续发酵，提高生产效率；4)可利用非糖底物(如厨余垃圾)，降低原料成本； 5)可通过快速低渗处理破碎细胞，降低产物纯化成本；6)可用塑料、陶瓷甚至水泥材料制作发酵罐体和管道，降低设备投资。此外，已完成了嗜盐单胞菌的全基因组测序和建立起了分子操作技术，可进行分子设计、编辑遗传信息，实现对生命性状和代谢网络的改造。基于自身的优势和对其研究的深入，嗜盐单胞菌TD01已成为“下一代工业生物技术”的重要底盘细胞，被广泛地改造用于生产各类高附加值化学品、蛋白质及生物材料，如PHA、蛋白(PhaP 和PhaR)和小分子化合物(5-ALA，L-苏氨酸、四氢嘧啶、3-HP)等。
经检索，目前未见有改造嗜盐单胞菌及用于细胞催化柠檬酸生产衣康酸的相关报道。
发明内容
本发明的目的是克服现有技术的不足，提供一种重组嗜盐单胞菌。
本发明的第二个目的是提供一种重组嗜盐单胞菌的构建方法。
本发明的第三个目的是提供一种重组嗜盐单胞菌催化柠檬酸制备衣康酸的应用。
本发明的技术方案概述如下：
一种重组嗜盐单胞菌的构建方法，包括如下步骤：
(1)在野生型嗜盐单胞菌Halomonas sp.TD01保藏登记号为CGMCCNo.4353基因组上整合Mmp1RNA聚合酶表达单元，得到Halomonas sp.TD1.0；
再将Halomonas sp.TD1.0基因组上异柠檬酸脱氢酶编码基因icd的起始密码子ATG替换为TTG，得到Halomonas sp.TD2.0；
所述异柠檬酸脱氢酶编码基因icd的核苷酸序列如SEQ ID NO.1所示；
(2)在高拷贝表达载体pN59多克隆位点区域插入PMmp1-RBS-cadA-acn，得到高拷贝表达载体pN59-PMmp1-RBS-cadA-acn；
所述高拷贝表达载体pN59的核苷酸序列如SEQ ID NO.2所示；
所述PMmp1为IPTG诱导型启动子，其核苷酸序列如SEQ ID NO.3所示；
所述RBS为大肠杆菌Escherichia coliMG1655强核糖体结合位点，其核苷酸序列如SEQ ID NO.4所示；
所述cadA为土曲霉Aspergillus terreus来源的经密码子优化得到的顺乌头酸脱羧酶基因，其核苷酸序列如SEQ ID NO.5所示；
所述acn为谷氨酸棒状杆菌Corynebacterium glutamicum ATCC13032来源的顺乌头酸酶基因，其核苷酸序列如SEQ ID NO.6所示；
(3)在低拷贝表达载体pN85多克隆位点区域插入PMmp1-RBS-GroESL，得到表达载体pN85-PMmp1-RBS-GroESL；
在表达载体pN85-PMmp1-RBS-GroESL的GroESL基因后再插入PMmp1-RBS-acn，得到低拷贝表达载体pN85-PMmp1-RBS-GroESL-PMmp1-RBS-acn；
所述低拷贝表达载体pN85的核苷酸序列如SEQ ID NO.7所示；
所述GroESL是嗜盐单胞菌Halomonassp.TD01来源的分子伴侣基因，其核苷酸序列如 SEQ ID NO.8所示；
(4)向步骤(1)获得的Halomonas sp.TD2.0中导入高拷贝表达载体 pN59-PMmp1-RBS-cadA-acn，得到Halomonas sp.IA01；
(5)向步骤(4)获得的Halomonas sp.IA01中导入低拷贝表达载体 pN85-PMmp1-RBS-GroESL-PMmp1-RBS-acn，得到重组嗜盐单胞菌Halomonas sp.IA02。
上述构建方法构建得重组嗜盐单胞菌。
上述重组嗜盐单胞菌催化柠檬酸制备衣康酸的应用。
一种重组嗜盐单胞菌催化柠檬酸制备衣康酸的应用，包括如下步骤：
(1)将重组嗜盐单胞菌进行诱导培养；
(2)将步骤(1)获得的培养液，于4000-8000rpm，4-10℃离心10-20min，PB高渗缓冲液洗涤2-3次，用PB高渗缓冲液重悬，测定OD600nm，收集获得重组嗜盐单胞菌；
所述PB高渗缓冲液配置为0.2M Na2HPO4水溶液和0.2MNaH2PO4水溶液以1:2.17体积比混合，添加NaCl使终浓度60g/L；
(3)按比例，向容器中加入底物柠檬酸，使终浓度为500-600mM，步骤(2)获得的重组嗜盐单胞菌，使终浓度为30OD600nm/mL，Trixon X-100使体积分数为0.1％-0.6％，余量为pH 是6.0-6.5的0.01M PB缓冲液，混合均匀后在30-35℃反应，得到衣康酸。
本发明的优点：
本发明的重组嗜盐单胞菌催化柠檬酸制备衣康酸法，与传统真菌发酵法相比，具有反应周期短、条件温和、过程简单易操控、效率高、无其他副产物便于纯化。本发明的重组嗜盐菌Halomonas sp.IA02细胞催化柠檬酸生成IA，转化率为81.8％-95.2％。本发明的重组嗜盐单胞菌构建和细胞催化方法简单、高效，有望成为衣康酸生产的另一种替代方法，适合工业化生产。
附图说明
图1为柠檬酸出发合成衣康酸的反应示意图；
图2为重组嗜盐单胞菌Halomonas sp.IA02催化柠檬酸生产衣康酸图；
具体实施方式
基于嗜盐单胞菌宿主的优势和应用前景，本发明改造嗜盐单胞菌用于催化柠檬酸制备衣康酸。通过引入顺乌头酸酶ACN和顺乌头酸脱羧酶CAD，同时为增强蛋白的可溶性表达和协调衣康酸合成关键酶的表达水平，又过表达了分子伴侣蛋白GroESL和另一拷贝的顺乌头酸酶ACN构建重组嗜盐单胞菌。利用重组嗜盐单胞菌催化生产衣康酸，通过诱导培养优化和催化条件优化，实现了重组菌细胞催化柠檬酸高效制备衣康酸。
下面结合实施例对本发明做进一步说明，下述实施例是为了使本领域的技术人员能够更好地理解本发明，但对本发明不作任何限制。
所用限制性内切酶、DNA聚合酶等分子生物学试剂购买自Thermo公司 (http://www.thermoscientificbio.com/fermentas)。
所用PCR仪和电转仪购自Bio-Rad(http://www.bio-rad.com/)。
所用其他生化试剂从生工生物工程(上海)股份有限公司(http:// www.sangon.com/)购买。
LB液体培养基：胰蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L，蒸馏水定容至1L，(固体培养基需添加2％琼脂)，需要时添加抗性氯霉素(25mg/L)或/和壮观霉素(100mg/L)。
20LB固体培养基：胰蛋白胨10g/L，酵母提取物5g/L，氯化钠20g/L，蒸馏水定容至1L，加2％琼脂。
60LB液体培养基包括：胰蛋白胨10g/L，酵母提取物5g/L，氯化钠60g/L，蒸馏水定容至1L，5M NaOH水溶液调pH至9.0；
含抗性的60LB液体培养基包括：胰蛋白胨10g/L，酵母提取物5g/L，氯化钠60g/L，蒸馏水定容至1L，5M NaOH水溶液调pH至9.0，接种前加25mg/L氯霉素或/和100mg/L 壮观霉素，(固体培养基中添加2％琼脂)。
OD600nm测定：采用紫外分光光度计TU-1801(北京普析)测600nm波长下的吸光度。
高效液相色谱检测柠檬酸和衣康酸含量，采用安捷伦1100高效液相色谱仪，色谱柱为Aminex HPX-87H(300mm×7.8mm,9μm,Bio-Rad)，5mM H2SO4为流动相，流速0.4mL/min，进样量10μL，紫外检测器(210nm波长下)，柱温60℃。
Escherichia coli S17-1λpir，购自Biovector质粒载体菌种细胞基因保藏中心，20191011，中国，http://www.biovector.net/。
实施例1嗜盐单胞菌底盘细胞的构建
在野生型嗜盐单胞菌Halomonas sp.TD01(已于2010年11月19日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏登记号为CGMCC No.4353，保藏地址：北京市朝阳区北辰西路1号院3号)基因组上整合Mmp1 RNA聚合酶表达单元，得到Halomonassp.TD1.0；
再将Halomonas sp.TD1.0基因组上异柠檬酸脱氢酶编码基因icd的起始密码子ATG替换为TTG，得到Halomonas sp.TD2.0；
所述异柠檬酸脱氢酶编码基因icd的核苷酸序列如SEQ ID NO.1所示；
具体包括以下步骤：
(1)在野生型嗜盐单胞菌Halomonas sp.TD01基因组上整合Mmp1RNA聚合酶表达单元，得到Halomonas sp.TD1.0，以实现当IPTG存在时类T7启动子的激活转录，具体操作方法记载在如下文献中：Zhao H,Zhang H M,Chen X,Li T,Wu Q,et al.(2016).Novel t7-likeexpression systems used for halomonas.Metabolic Engineering,128，公众可根据文献内容改造Halomonas sp.TD01获得Halomonas sp.TD1.0；
(2)进一步利用CRISPR/Cas9技术(方法记录在如下文献中：Qin Q,Ling C,ZhaoY, Yang T,Yin J,Guo Y,et al.(2018).Crispr/cas9editing genome of extremophilehalomonas spp. Metabolic Engineering,S1096717618300053.)，将Halomonas sp.TD1.0基因组上异柠檬酸脱氢酶编码基因icd的起始密码子ATG替换为TTG，以弱化其表达强度，得到Halomonas sp.TD2.0 作为本发明的底盘细胞。
实施例2：表达载体的构建
(1)构建高拷贝表达载体pN59-PMmp1-RBS-cadA-acn
以SEQ ID No.9所示PMmp1-F1和以SEQ ID No.10所示RBS-R1为引物，以SEQ IDNo.3 所示PMmp1片段为模板，PCR扩增获得PMmp1-RBS片段(其中所述RBS为大肠杆菌Escherichia coli MG1655强核糖体结合位点，其核苷酸序列如SEQ ID NO.4所示)；以SEQID No.11所示 cadA-F和以SEQ ID No.12所示cadA-R为引物，以SEQ ID No.5所示人工合成的cadA基因为模板，PCR扩增获得cadA片段；以SEQ ID No.13所示acn-F1和以SEQ ID No.14所示acn-R1 为引物，以Corynebacterium glutamicum ATCC13032基因组为模板，PCR扩增获得acn片段(所述acn核苷酸序列如SEQ ID NO.6所示)；进一步利用融合PCR技术，将PMmp1-RBS、 cadA和acn融合得到PMmp1-RBS-cadA-acn融合片段；再以SEQ ID No.15所示pN59-F和以 SEQ ID No.16所示pN59-R为引物，以SEQ ID No.2所示的pN59质粒(pSEVA341,TheStandard European Vector Architecture(SEVA):a coherent platform for theanalysis and development of complex prokaryotic phenotypes(2013).Nucleicacids research,41,75)为模板，PCR扩增获得线性pN59，通过CPEC组装技术，将PMmp1-RBS-cadA-acn片段组装至高拷贝表达载体pN59 多克隆位点区域，构建获得高拷贝表达载体pN59-PMmp1-RBS-cadA-acn。重组产物先化转至大肠杆菌Trans 5α(北京全式金生物，货号：CD201-01，https://www.transgen.com.cn/)，PCR验证(验证引物为SEQ ID No.29所示F24和SEQ ID No.30所示R24为引物，正确的转化子扩增片段为4.5kb)和测序验证，将正确的转化子培养提取质粒，质粒电转至大肠杆菌S17-1λpir，挑取单菌落PCR验证(验证引物为SEQID No.29所示F24和SEQ ID No.30所示R24为引物，正确的转化子扩增片段为4.5kb)和测序验证，得到含pN59-PMmp1-RBS-cadA-acn质粒的大肠杆菌S17-1λpir。
所述高拷贝表达载体pN59的核苷酸序列如SEQ ID NO.2所示；
所述PMmp1为IPTG诱导型启动子，其核苷酸序列如SEQ ID NO.3所示；
所述RBS为大肠杆菌Escherichia coli MG1655强核糖体结合位点，其核苷酸序列如SEQ ID NO.4所示；
所述cadA为土曲霉Aspergillus terreus来源的经密码子优化得到的顺乌头酸脱羧酶基因，其核苷酸序列如SEQ ID NO.5所示；
所述acn为谷氨酸棒状杆菌Corynebacterium glutamicum ATCC13032来源的顺乌头酸酶基因，其核苷酸序列如SEQ ID NO.6所示；
(2)构建低拷贝表达载体pN85-PMmp1-RBS-GroESL-PMmp1-RBS-acn
以SEQ ID No.17所示PMmp1-F2和以SEQ ID No.18所示RBS-R2为引物，以SEQ IDNo.3 所示PMmp1片段为模板，PCR扩增获得PMmp1-RBS片段(其中所述RBS为大肠杆菌Escherichia coli MG1655强核糖体结合位点，其核苷酸序列如SEQ ID NO.4所示)；以SEQID No.19所示GroESL-F和以SEQ ID No.20所示GroESL-R为引物，以Halomonas sp.TD01基因组为模板，PCR扩增获得GroESL片段，将PMmp1-RBS片段和GroESL片段融合获得 PMmp1-RBS-GroESL融合片段；以SEQ ID No.21所示pN85-F1和以SEQ ID No.22所示pN85-R1 为引物，以SEQ ID No.7所示pN85质粒(pSEVA321,The Standard European Vector Architecture(SEVA):a coherent platform for the analysis and development of complexprokaryotic phenotypes (2013).Nucleic acids research,41,75)为模板，PCR扩增获得全长线性pN85，通过CPEC组装技术，将PMmp1-RBS-GroESL片段组装至低拷贝表达载体pN85多克隆位点区域，构建获得 pN85-PMmp1-RBS-GroESL，转至大肠杆菌Trans 5α，PCR验证(验证引物为SEQ ID No.29所示F24和SEQ ID No.30所示R24为引物，正确的转化子扩增片段为2.2kb)和测序验证，将正确的转化子培养提取得到质粒pN85-PMmp1-RBS-GroESL。
进一步以SEQ ID No.23所示PMmp1-F3和以SEQ ID No.24所示RBS-R3为引物，以SEQID No.3所示PMmp1片段为模板，PCR扩增获得PMmp1-RBS片段(其中所述RBS为大肠杆菌Escherichia coli MG1655强核糖体结合位点，其核苷酸序列如SEQ ID NO.4所示)；以SEQID No.25所示acn-F2和以SEQ ID No.26所示acn-R2为引物，以Corynebacteriumglutamicum ATCC13032基因组为模板，PCR扩增获得acn片段(所述acn核苷酸序列如SEQ IDNO.6所示)，将PMmp1-RBS片段和acn片段融合获得PMmp1-RBS-acn融合片段；
最后以SEQ ID No.27所示pN85-F2和以SEQ ID No.28所示pN85-R2为引物，以得到的质粒pN85-PMmp1-RBS-GroESL为模板，PCR扩增获得全长线性pN85-PMmp1-RBS-GroESL，通过CPEC组装技术，将PMmp1-RBS-acn片段组装至线性质粒pN85-PMmp1-RBS-GroESL的 GroESL基因后，构建获得低拷贝表达载体pN85-PMmp1-RBS-GroESL-PMmp1-RBS-acn。重组产物先化转至大肠杆菌Trans 5α，PCR验证(验证引物为SEQ ID No.29所示F24和SEQ ID No.30 所示R24为引物，正确的转化子扩增片段为5.2kb)和测序验证，将正确的转化子培养提取得到质粒pN85-PMmp1-RBS-GroESL-PMmp1-RBS-acn，再将该质粒电转至大肠杆菌S17-1λpir，挑取PCR验证(验证引物为SEQ ID No.29所示F24和SEQ ID No.30所示R24为引物，正确的转化子扩增片段为5.2kb)和测序验证，得到含pN85-PMmp1-RBS-GroESL-PMmp1-RBS-acn 质粒的大肠杆菌S17-1λpir。
所述低拷贝表达载体pN85的核苷酸序列如SEQ ID NO.7所示；
所述GroESL是嗜盐单胞菌Halomonas sp.TD01来源的分子伴侣基因，其核苷酸序列如 SEQ ID NO.8所示；
实施例3：重组嗜盐单胞菌的构建
将实施例2中含pN59-PMmp1-RBS-cadA-acn质粒的大肠杆菌S17-1λpri与实施例1所述的嗜盐单胞菌Halomonas sp.TD2.0接合。
具体为将含pN59-PMmp1-RBS-cadA-acn质粒的大肠杆菌S17-1λpri接种于5mL含25mg/L氯霉素抗性的LB液体培养基中，将Halomonas sp.TD2.0接种于5mL 60LB液体培养基中，分别于37℃，200rpm培养12h。收集1mL含pN59-PMmp1-RBS-cadA-acn质粒的大肠杆菌S17-1λpri菌液和1mL Halomonas sp.TD2.0菌液，分别用LB液体培养基和60LB液体培养基洗涤两次，以1:1比例混匀，滴种于20LB固体培养基上，37℃接合12h。最后涂布于含25mg/L氯霉素抗性60LB固体平板上，挑取单菌落PCR验证(验证引物为SEQ ID No.29 所示F24和SEQ IDNo.30所示R24为引物，正确的转化子扩增片段为4.5kb)和测序验证，得到重组嗜盐单胞菌Halomonas sp.IA01。
将实施例2中含pN85-PMmp1-RBS-GroESL-PMmp1-RBS-acn质粒的大肠杆菌S17-1λpir与重组嗜盐单胞菌Halomonas sp.IA01接合。最后接合产物涂布于含25mg/L氯霉素和100mg/L 壮观霉素抗性的60LB固体平板上，挑取单菌落PCR验证(验证引物为SEQ ID No.29所示 F24和SEQ ID No.20所示GroESL-R为引物，正确的扩增片段为2.2kb)和测序验证，得到重组嗜盐单胞菌Halomonas sp.IA02。
实施例4重组嗜盐单胞菌IA02催化柠檬酸制备衣康酸的应用，包括如下步骤：
(1)将重组嗜盐单胞菌Halomonas sp.IA02接种于5mL含25mg/L氯霉素和100mg/L壮观霉素抗性的60LB液体培养基，37℃，200rpm培养12h，再以1％接种量转接于100mL 含25mg/L氯霉素和100mg/L壮观霉素抗性的60LB液体培养基中，37℃，200rpm培养 OD600nm为1，添加1mM IPTG诱导剂，转至30℃，200rpm条件下诱导培养16h；
(2)将步骤(1)获得的培养液，于4500rpm，4℃离心10min，PB高渗缓冲液洗涤2 次，用PB高渗缓冲液重悬，测定OD600nm，收集获得重组嗜盐单胞菌Halomonas sp.IA02：
所述PB高渗缓冲液配置为0.2M Na2HPO4水溶液和0.2MNaH2PO4水溶液以1:2.17体积比混合，添加NaCl使终浓度60g/L；
(3)按比例，向容器中加入底物柠檬酸，使终浓度为550mM，步骤(2)获得的重组嗜盐单胞菌，使终浓度为30OD600nm/mL，Trixon X-100使体积分数为0.1％，余量为pH是 6.0的0.01M PB缓冲液，混合均匀后在35℃反应，得到衣康酸。反应52h，生成436mM IA，柠檬酸转化率为81.83％。
实施例5重组嗜盐单胞菌IA02催化柠檬酸制备衣康酸的应用，包括如下步骤：
(1)将重组嗜盐单胞菌Halomonas sp.IA02接种于5mL含25mg/L氯霉素和100mg/L壮观霉素抗性的60LB液体培养基，37℃，200rpm培养12h，再以1％接种量转接于100mL 含25mg/L氯霉素和100mg/L壮观霉素抗性的60LB液体培养基中，37℃，200rpm培养OD600nm为2，添加1mM IPTG诱导剂，转至30℃，200rpm条件下诱导培养12h；
(2)将步骤(1)获得的培养液，于4000rpm，10℃离心20min，PB高渗缓冲液洗涤3次，用PB高渗缓冲液重悬，测定OD600nm，收集获得重组嗜盐单胞菌Halomonas sp.IA02：
所述PB高渗缓冲液配置为0.2M Na2HPO4水溶液和0.2MNaH2PO4水溶液以1:2.17体积比混合，添加NaCl使终浓度60g/L；
(3)按比例，向容器中加入底物柠檬酸使终浓度为600mM，步骤(2)获得的重组嗜盐单胞菌Halomonas sp.IA02，使终浓度为30OD600nm/mL，Trixon X-100使体积分数为0.6％，余量为pH是6.5的0.01M PB缓冲液，混合均匀后在30℃反应，得到衣康酸。反应52h，生成425mMIA，柠檬酸转化率为81.8％。
实施例6重组嗜盐单胞菌IA02催化柠檬酸制备衣康酸的应用，包括如下步骤：
(1)将重组嗜盐单胞菌Halomonas sp.IA02接种于5mL含25mg/L氯霉素和100mg/L壮观霉素抗性的60LB液体培养基，37℃，200rpm培养12h，再以1％接种量转接于100mL 含25mg/L氯霉素和100mg/L壮观霉素抗性的60LB液体培养基中，37℃，200rpm培养 OD600nm为3，添加1mM IPTG诱导剂，转至30℃，200rpm条件下诱导培养8h；
(2)将步骤(1)获得的培养液，于8000rpm，6℃离心10min，PB高渗缓冲液洗涤2 次，用PB高渗缓冲液重悬，测定OD600nm，收集获得重组嗜盐单胞菌Halomonas sp.IA02：
所述PB高渗缓冲液配置为0.2M Na2HPO4水溶液和0.2MNaH2PO4水溶液以1:2.17体积比混合，添加NaCl使终浓度60g/L；
(3)按比例，向容器中加入底物柠檬酸使终浓度为500mM，步骤(2)获得的重组嗜盐单胞菌Halomonas sp.IA02，使终浓度为30OD600nm/mL，Trixon X-100使体积分数为0.6％，余量为pH是6.3的0.01M PB缓冲液，混合均匀后在30℃反应，得到衣康酸。反应36h柠檬酸转化率为95.20％，生成451.45mM(58.73g/L)衣康酸。结果如图2所示。
实施例7补料法生产衣康酸应用
为进一步提高产量，通过补料法进行衣康酸的生产。
(1)将重组嗜盐单胞菌Halomonas sp.IA02接种于5mL含25mg/L氯霉素和100mg/L壮观霉素抗性的60LB液体培养基，37℃，200rpm培养12h，再以1％接种量转接于100mL 含25mg/L氯霉素和100mg/L壮观霉素抗性的60LB液体培养基中，37℃，200rpm培养 OD600nm为3，添加1mM IPTG诱导剂，转至30℃，200rpm条件下诱导培养10h；
(2)将步骤(1)获得的培养液，于4500rpm，4℃离心10min，PB高渗缓冲液洗涤2 次，用PB高渗缓冲液重悬，测定OD600nm，收集获得重组嗜盐单胞菌Halomonas sp.IA02：
所述PB高渗缓冲液配置为0.2M Na2HPO4水溶液和0.2MNaH2PO4水溶液以1:2.17体积比混合，添加NaCl使终浓度60g/L；
(3)按比例，向容器中加入底物柠檬酸使终浓度为500mM，步骤(2)获得的重组嗜盐单胞菌Halomonas sp.IA02，使终浓度为30OD600nm/mL，Trixon X-100使体积分数为0.5％，余量为pH是6.3的0.01M PB缓冲液，混合均匀后在30℃反应。反应过程中监测衣康酸和柠檬酸剩余量，前10h分2次补加柠檬酸三钠(固体形式)维持450-500mM，衣康酸不再增加终止反应。整个反应周期共54h，总底物添加为674mM，共消耗529mM柠檬酸，转化率为78.49％，生成489mM(63.6g/L)衣康酸。
序列表
<110> 天津大学
清华大学
<120> 重组嗜盐单胞菌及构建方法及催化柠檬酸制备衣康酸的应用
<160> 30
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2238
<212> DNA
<213> 盐单胞菌(Halomonas sp. )
<400> 1
atgtctaaaa cgccgaagat catttacacg ctcaccgacg aagcgcctgc gctcgcgaca 60
tactctttgc taccgattat tgacgctttc accgatgctg ctggtatcga ggtggaaacc 120
cgggatatct ccctggcggc tcgcgttctc tctttatttc cagactactt gactgaagag 180
cagcgtattg aagaccactt ggccgaattg ggtgcacttg ccaaggttcc tgaagccaat 240
atcatcaaat taccgaatat cagcgcctct atgccgcagc tgcgtgctgt gatcaaagag 300
ctgcaagaac aagggtacaa gctgcctgag tatccggacg acccaagcag cgatgaagag 360
aaagatatca gagcgcgcta cgacaaagtc aaaggcagcg cagttaaccc tgtattgcgt 420
gaaggtaatt ccgaccgtcg cgcaccgaaa gccgtgaaag agtacgcccg caagtatccg 480
cactctatgg gggagtggag ccaggcgtca cgtacccatg tttcgcacat gcatagtggc 540
gacttctacc atggtgagaa gtcgatgacc ctggatcgcg cccgcaacgt caagatggag 600
ctgatcaccg ccagcggtga aaccaaggtg ctcaagcctc gggtcgagct gctggaaggc 660
gaaatcatcg acagcatgtt tatgagcaag aaagcgctgc tggattttta cgagcgtgaa 720
atcgaggatg cgcgtgagac cggtgtgatg ttctcattgc acgtaaaagc gacgatgatg 780
aaggtctcgc accccatcgt gtttgggcac tgcgtcaaaa ttttctacaa ggacgccttt 840
gagaagcacg gtgagctctt caaagagttg ggtgtcgatg ttaacaacgg catcgctaat 900
ctctacgata aaattgcgac cttgccagaa tctcaacgtg atgaagtcat tagtgacttg 960
catgcgtgcc atgacaagcg cccagagcta gcaatggttg attcggctcg cggcatcacc 1020
aacttccact cacccagcga tgtaattgtt gacgcttcaa tgccggcgat gattcgtgct 1080
ggcggtaaga tgtacggtgc tgatggtcgt ctcaaagacg tcaaagccgt catgcctgag 1140
tcgacgttcg cgcgtatcta tcaggagatg atcaacttct gtaagtggca cggcgcgttt 1200
gatccggcca ctatgggtac cgttcctaat gttggtctga tggcgcagaa agccgaagag 1260
tatggctctc acgataagac cttcgaggtg ccggaagatg gtgttgctaa catcactgac 1320
ctggacacgg gcgaagtact gctgtcgcag actgtggagc agggcgatat ctggcgtatg 1380
tgtcaggtca aagatgcacc gattcgcgat tgggtgaagt tggcggttga gcgctgccgt 1440
gattctggta tgccgacggt attctggctc gacccttacc gcccgcacga gaacgagttg 1500
atcaagaagg tcaaaacgta cctcaaggat cacgacactg atgggcttga aatccacgtc 1560
atgtctcaag ttcgggcgat gcgttacacc cttgagcgtg tgattcgtgg tctcgacacg 1620
atctctgtga caggtaacat tctccgcgat tacctgaccg acctatttcc gattctcgag 1680
ctgggcacta gtgccaaaat gctttccatc gtgccgttga tggatggcgg tggtctgttt 1740
gaaacgggcg ctggtggttc cgcacccaag cacgttcagc agttgcttga agaaaaccac 1800
ctacgttggg acagccttgg agagttcctg gctctagttg cttctttgga acacctcgcc 1860
aaacgttttg gtaatgaccg tgctaagctg ctggcgaaag cgttggatga agctaacggc 1920
aagttccttg aaagcaataa atcaccttca cgcaaagtgg gtgagcttga taaccgtgga 1980
agccacttct atctggcgct gtattgggca gaagcattgg cagcgcagga tcaagacgct 2040
gaactgaaaa cacgctttgc tcgtttggta gaaacgctga aagccaatga agctacgatc 2100
attgaagagc taaacagcgt gcaaggacag cctgttgatc tcaagggtta ctaccacccg 2160
aacggagagt tggctagcca gactatgcgc ccgagtaaga cgctcaacga agcgcttgcg 2220
atggtcgcga acggttaa 2238
<210> 2
<211> 3376
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ttaattaaag cggataacaa tttcacacag gaggccgcct aggccgcggc cgcgcgaatt 60
cgagctcaag cttgcggccg cgtcgtgact gggaaaaccc tggcgactag tcttggactc 120
ctgttgatag atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt 180
tgccgccggg cgttttttat tggtgagaat ccaggggtcc ccaataatta cgatttaaat 240
tggcgaaaat gagacgttga tcggcacgta agaggttcca actttcacca taatgaaata 300
agatcactac cgggcgtatt ttttgagtta tcgagatttt caggagctaa ggaagctaaa 360
atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa 420
cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat 480
attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt 540
cacattcttg cccgcctgat gaatgctcat ccggaatttc gtatggcaat gaaagacggt 600
gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa 660
acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat 720
tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag 780
aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg 840
gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc 900
gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat 960
gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa 1020
tttgactttt gtccttttcc gctgcataac cctgcttcgg ggtcattata gcgatttttt 1080
cggtatatcc atcctttttc gcacgatata caggattttg ccaaagggtt cgtgtagact 1140
ttccttggtg tatccaacgg cgtcagccgg gcaggatagg tgaagtaggc ccacccgcga 1200
gcgggtgttc cttcttcact gtcccttatt cgcacctggc ggtgctcaac gggaatcctg 1260
ctctgcgagg ctggccgtag gccggccgat aatctcatga ccaaaatccc ttaacgtgag 1320
ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 1380
ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 1440
tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 1500
cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct 1560
gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 1620
gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 1680
tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 1740
ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 1800
gacaggcatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 1860
ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 1920
tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccgtg 1980
aaaggcaggc cggtccgtgg tggccacggc ctctaggcca gatccagcgg catctgggtt 2040
agtcgagcgc gggccgcttc ccatgtctca ccagggcgag cctgtttcgc gatctcagca 2100
tctgaaatct tcccggcctt gcgcttcgct ggggccttac ccaccgcctt ggcgggcttc 2160
ttcggtccaa aactgaacaa cagatgtgtg accttgcgcc cggtctttcg ctgcgcccac 2220
tccacctgta gcgggctgtg ctcgttgatc tgcgtcacgg ctggatcaag cactcgcaac 2280
ttgaagtcct tgatcgaggg ataccggcct tccagttgaa accactttcg cagctggtca 2340
atttctattt cgcgctggcc gatgctgtcc cattgcatga gcagctcgta aagcctgatc 2400
gcgtgggtgc tgtccatctt ggccacgtca gccaaggcgt atttggtgaa ctgtttggtg 2460
agttccgtca ggtacggcag catgtctttg gtgaacctga gttctacacg gccctcaccc 2520
tcccggtaga tgattgtttg cacccagccg gtaatcatca cactcggtct tttccccttg 2580
ccattgggct cttgggttaa ccggacttcc cgccgtttca ggcgcagggc cgcttctttg 2640
agctggttgt aggaagattc gatagggaca cccgccatcg tcgctatgtc ctccgccgtc 2700
actgaataca tcacttcatc ggtgacaggc tcgctcctct tcacctggct aatacaggcc 2760
agaacgatcc gctgttcctg aacactgagg cgatacgcgg cctcgaccag ggcattgctt 2820
ttgtaaacca ttgggggtga ggccacgttc gacattcctt gtgtataagg ggacactgta 2880
tctgcgtccc acaatacaac aaatccgtcc ctttacaaca acaaatccgt cccttcttaa 2940
caacaaatcc gtcccttaat ggcaacaaat ccgtcccttt ttaaactcta caggccacgg 3000
attacgtggc ctgtagacgt cctaaaaggt ttaaaaggga aaaggaagaa aagggtggaa 3060
acgcaaaaaa cgcaccacta cgtggccccg ttggggccgc atttgtgccc ctgaaggggc 3120
gggggaggcg tctgggcaat ccccgtttta ccagtcccct atcgccgcct gagagggcgc 3180
aggaagcgag taatcagggt atcgaggcgg attcaccctt ggcgtccaac cagcggcacc 3240
agcggcgcct gagaggggcg cgcccagctg tctagggcgg cggatttgtc ctactcagga 3300
gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc 3360
gttttatttg atgcct 3376
<210> 3
<211> 129
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cccatgagtt aattatattt gtggcattat agggaattgt gagcgctcac aattagctgt 60
caccggatgt gctttccggt ctgatgagtc cgtgaggacg aaacagcctc tacaaataat 120
tttgtttaa 129
<210> 4
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tactagagaa agaggagaaa tactag 26
<210> 5
<211> 1473
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgaccaaac agtctgctga ttctaatgcc aaatctggtg tgacctctga aatttgccac 60
tgggcctcta acctggccac tgatgatatt ccctctgatg tgttggaaag ggccaaatac 120
ctgattctgg atgggattgc atgtgcctgg gtgggtgcta gggtgccctg gtctgaaaaa 180
tatgtgcaag ccaccatgtc ttttgaaccc cctggtgcct gtagggtgat tggctatggt 240
cagaaactgg gccctgtggc tgctgccatg accaactctg cctttattca agccactgaa 300
ctggatgatt accactctga agcccccctg cactctgcct ctattgtgct gcctgctgtg 360
tttgctgcct ctgaagtgct ggctgaacaa ggcaaaacca tttctggcat tgatgtgatt 420
ctggctgcca ttgtgggctt tgaatctggc cctaggattg gcaaagccat ttatggctct 480
gatctactga acaatggctg gcattgtggt gctgtatatg gtgcccctgc tggtgccctg 540
gccactggca aactgctggg cctgacccct gattctatgg aagatgccct gggcattgcc 600
tgcacccaag cctgtggcct gatgtctgct cagtatggtg gcatggtgaa gagggtgcag 660
catggttttg cagctaggaa tggcttatta ggtggcctgc tggcccatgg tggctatgaa 720
gccatgaaag gtgtgctgga aaggtcttat ggtggctttc tgaaaatgtt taccaaaggc 780
aatggtaggg aaccccccta caaagaagaa gaagtggtgg ctggcctggg ctctttttgg 840
cacaccttta ccattaggat taaactgtat gcctgctgtg gcctggtgca tggccctgtg 900
gaagccattg aaaacctgca aggtaggtac cctgagctgc taaatagggc caacctgtct 960
aacattaggc atgtgcatgt gcagctgtct actgcctcta actctcactg tggctggatt 1020
cctgaagaaa ggcccatttc ttctattgct ggtcagatgt ctgtggccta cattctggct 1080
gtgcagctgg tggatcagca gtgcctgctg tctcagtttt ctgaatttga tgataacctg 1140
gaaaggcctg aagtgtggga tctggctagg aaagtgacct cttctcagtc tgaagaattt 1200
gatcaagatg gcaactgcct gtctgctggt agggtgagga ttgaatttaa tgatggctct 1260
tctattactg aatctgtgga aaaacccctg ggtgtgaaag aacccatgcc caatgaaagg 1320
attctgcaca aatataggac cctggctggc tctgtgactg atgaatctag ggtgaaagaa 1380
attgaagatc tggtgctggg cctggatagg ctgactgata tttctcccct gctggaactg 1440
ctgaactgcc ctgtgaaatc tcccctggtg taa 1473
<210> 6
<211> 2832
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum)
<400> 6
atggagctca ctgtgactga aagcaagaac tccttcaatg ctaagagcac ccttgaagtt 60
ggcgacaagt cctatgacta cttcgccctc tctgcagtgc ctggcatgga gaagctgccg 120
tactccctca aggttctcgg agagaacctt cttcgtaccg aagacggcgc aaacatcacc 180
aacgagcaca ttgaggctat cgccaactgg gatgcatctt ccgatccaag catcgaaatc 240
cagttcaccc cagcccgtgt tctcatgcag gacttcaccg gtgtcccttg tgtagttgac 300
ctcgcaacca tgcgtgaggc agttgctgca ctcggtggcg accctaacga cgtcaaccca 360
ctgaacccag ccgagatggt cattgaccac tccgtcatcg tggaggcttt cggccgccca 420
gatgcactgg ctaagaacgt tgagatcgag tacgagcgca acgaggagcg ttaccagttc 480
ctgcgttggg gttccgagtc cttctccaac ttccgcgttg ttcctccagg aaccggtatc 540
gtccaccagg tcaacattga gtacttggct cgcgtcgtct tcgacaacga gggccttgca 600
tacccagata cctgcatcgg taccgactcc cacaccacca tggaaaacgg cctgggcatc 660
ctgggctggg gcgttggtgg cattgaggct gaagcagcaa tgctcggcca gccagtgtcc 720
atgctgatcc ctcgcgttgt tggcttcaag ttgaccggcg agatcccagt aggcgttacc 780
gcaactgacg ttgtgctgac catcaccgaa atgctgcgcg accacggcgt cgtccagaag 840
ttcgttgagt tctacggctc cggtgttaag gctgttccac tggctaaccg tgcaaccatc 900
ggcaacatgt ccccagagtt cggctccacc tgtgcgatgt tcccaatcga cgaggagacc 960
accaagtacc tgcgcctcac cggccgccca gaagagcagg ttgcactggt cgaggcttac 1020
gccaaggcgc agggcatgtg gctcgacgag gacaccgttg aagctgagta ctccgagtac 1080
ctcgagctgg acctgtccac cgttgttcct tccatcgctg gccctaagcg cccacaggac 1140
cgcatccttc tctccgaggc aaaggagcag ttccgtaagg atctgccaac ctacaccgac 1200
gacgctgttt ccgtagacac ctccatccct gcaacccgca tggttaacga aggtggcgga 1260
cagcctgaag gcggcgtcga agctgacaac tacaacgctt cctgggctgg ctccggcgag 1320
tccttggcta ctggcgcaga aggacgtcct tccaagccag tcaccgttgc atccccacag 1380
ggtggcgagt acaccatcga ccacggcatg gttgcaattg catccatcac ctcttgcacc 1440
aacacctcta acccatccgt gatgatcggc gctggcctga tcgcacgtaa ggcagcagaa 1500
aagggcctca agtccaagcc ttgggttaag accatctgtg caccaggttc ccaggttgtc 1560
gacggctact accagcgcgc agacctctgg aaggaccttg aggccatggg cttctacctc 1620
tccggcttcg gctgcaccac ctgtattggt aactccggcc cactgccaga ggaaatctcc 1680
gctgcgatca acgagcacga cctgaccgca accgcagttt tgtccggtaa ccgtaacttc 1740
gagggacgta tctcccctga cgttaagatg aactacctgg catccccaat catggtcatt 1800
gcttacgcaa tcgctggcac catggacttc gacttcgaga acgaagctct tggacaggac 1860
caggacggca acgacgtctt cctgaaggac atctggcctt ccaccgagga aatcgaagac 1920
accatccagc aggcaatctc ccgtgagctt tacgaagctg actacgcaga tgtcttcaag 1980
ggtgacaagc agtggcagga actcgatgtt cctaccggtg acaccttcga gtgggacgag 2040
aactccacct acatccgcaa ggcaccttac ttcgacggca tgcctgtcga gccagtggca 2100
gtcaccgaca tccagggcgc acgcgttctg gctaagctcg gcgactctgt caccaccgac 2160
cacatctccc ctgcttcctc cattaagcca ggtacccctg cagctcagta cttggatgag 2220
cacggtgtgg aacgccacga ctacaactcc ctgggttcca ggcgtggtaa ccacgaggtc 2280
atgatgcgcg gcaccttcgc caacatccgc ctccagaacc agctggttga catcgcaggt 2340
ggctacaccc gcgacttcac ccaggagggt gctccacagg cgttcatcta cgacgcttcc 2400
gtcaactaca aggctgctgg cattccgctg gtcgtcttgg gcggcaagga gtacggcacc 2460
ggttcttccc gtgactgggc agctaagggc actaacctgc tcggaattcg cgcagttatc 2520
accgagtcct tcgagcgtat tcaccgctcc aacctcatcg gtatgggcgt tgtcccactg 2580
cagttccctg caggcgaatc ccacgagtcc ctgggccttg acggcaccga gaccttcgac 2640
atcaccggac tgaccgcact caacgagggc gagactccta agactgtcaa ggtcaccgca 2700
accaaggaga acggcgacgt cgtcgagttc gacgcagttg tccgcatcga caccccaggt 2760
gaggctgact actaccgcca cggcggcatc ctgcagtacg tgctgcgtca gatggctgct 2820
tcttctaagt aa 2832
<210> 7
<211> 3478
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cccttgcaat gctggatttt ctgcctgtgg acagcccctc aaatgtcaat aggtgcgccc 60
ctcatctgtc agcactctgc ccctcaagtg tcaaggatcg cgcccctcat ctgtcagtag 120
tcgcgcccct caagtgtcaa taccgcaggg cacttatccc caggcttgtc cacatcatct 180
gtgggaaact cgcgtaaaat caggcgtttt cgccgatttg cgaggctggc cagctccacg 240
tcgccggccg aaatcgagcc tgcccctcat ctgtcaacgc cgcgccgggt gagtcggccc 300
ctcaagtgtc aacgtccgcc cctcatctgt cagtgagggc caagttttcc gcgaggtatc 360
cacaacgccg gcggccctac atggctctgc tgtagtgagt gggttgcgct ccggcagcgg 420
tcctgatccc ccgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggcgc 480
gcccagctgt ctagggcggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac 540
agataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga tgcctttaat 600
taaagcggat aacaatttca cacaggaatg ctgagaccct agctagggag ctctctagaa 660
agcttctcac ggccgcgtcg tgactgggaa aaccctggcg actagtcttg gactcctgtt 720
gatagatcca gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg 780
ccgggcgttt tttattggtg agaatccagg ggtccccaat aattacgatt taaattggcg 840
aaaatgagac gttgatcggc acgtaagagg ttccaacttt caccataatg aaataagatc 900
actaccgggc gtattttttg agttatcgag attttcagga gctaaggaag ctaaaatgga 960
gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt 1020
tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac 1080
ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat 1140
tcttgcccgc ctgatgaatg ctcatccgga atttcgtatg gcaatgaaag acggtgagct 1200
ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt 1260
ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca 1320
agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat 1380
gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa 1440
tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa 1500
ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg 1560
cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaatttga 1620
cttttgtcct tttccgctgc ataaccctgc ttcggggtca ttatagcgat tttttcggta 1680
tatccatcct ttttcgcacg atatacagga ttttgccaaa gggttcgtgt agactttcct 1740
tggtgtatcc aacggcgtca gccgggcagg ataggtgaag taggcccacc cgcgagcggg 1800
tgttccttct tcactgtccc ttattcgcac ctggcggtgc tcaacgggaa tcctgctctg 1860
cgaggctggc cgtaggccgg ccgcgatgca ggtggctgct gaacccccag ccggaactga 1920
ccccacaagg ccctagcgtt tgcaatgcac caggtcatca ttgacccagg cgtgttccac 1980
caggccgctg cctcgcaact cttcgcaggc ttcgccgacc tgctcgcgcc acttcttcac 2040
gcgggtggaa tccgatccgc acatgaggcg gaaggtttcc agcttgagcg ggtacggctc 2100
ccggtgcgag ctgaaatagt cgaacatccg tcgggccgtc ggcgacagct tgcggtactt 2160
ctcccatatg aatttcgtgt agtggtcgcc agcaaacagc acgacgattt cctcgtcgat 2220
caggacctgg caacgggacg ttttcttgcc acggtccagg acgcggaagc ggtgcagcag 2280
cgacaccgat tccaggtgcc caacgcggtc ggacgtgaag cccatcgccg tcgcctgtag 2340
gcgcgacagg cattcctcgg ccttcgtgta ataccggcca ttgatcgacc agcccaggtc 2400
ctggcaaagc tcgtagaacg tgaaggtgat cggctcgccg ataggggtgc gcttcgcgta 2460
ctccaacacc tgctgccaca ccagttcgtc atcgtcggcc cgcagctcga cgccggtgta 2520
ggtgatcttc acgtccttgt tgacgtggaa aatgaccttg ttttgcagcg cctcgcgcgg 2580
gattttcttg ttgcgcgtgg tgaacagggc agagcgggcc gtgtcgtttg gcatcgctcg 2640
catcgtgtcc ggccacggcg caatatcgaa caaggaaagc tgcatttcct tgatctgctg 2700
cttcgtgtgt ttcagcaacg cggcctgctt ggcttcgctg acctgttttg ccaggtcctc 2760
gccggcggtt tttcgcttct tggtcgtcat agttcctcgc gtgtcgatgg tcatcgactt 2820
cgccaaacct gccgcctcct gttcgagacg acgcgaacgc tccacggcgg ccgatggcgc 2880
gggcagggca gggggagcca gttgcacgct gtcgcgctcg atcttggccg tagcttgctg 2940
gactatcgag ccgacggact ggaaggtttc gcggggcgca cgcatgacgg tgcggcttgc 3000
gatggtttcg gcatcctcgg cggaaaaccc cgcgtcgatc agttcttgcc tgtatgcctt 3060
ccggtcaaac gtccgattca ttcaccctcc ttgcgggatt gccccggaat taattccccg 3120
gatcgatccg tcgatcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc 3180
cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccggt 3240
tcgcttgctg tccataaaac cgcccagtct agctatcgcc atgtaagccc actgcaagct 3300
acctgctttc tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc 3360
atccggggtc agcaccgttt ctgcggactg gctttctacg tggctgccat ttttggggtg 3420
aggccgttcg cggccgaggg gcgcagcccc tggggggatg ggaggcccgc gttagcgg 3478
<210> 8
<211> 1996
<212> DNA
<213> 盐单胞菌(Halomonas sp.)
<400> 8
atgaatatcc gtcctttgca cgatcgcgtc gttgttcgtc gcgtggaaga agagcagaaa 60
accgctggcg gcatcgtgct accgggcaac gctcaggaaa aacccactcg cggtgaagtc 120
ctggcagttg gtaatggccg cattctcgac aacggtgacg ttcgcccgtt ggacgttaaa 180
gttggcgact cggtgatttt caaagacggt ttcggcgttg aaaaacagaa aatcaacggc 240
gaagaagttc tgatcatgag cgaagccgat atcttggcag tcgtcgaagg ctaaataccg 300
cctcgatcat ttacttttcc ttttaaattt caatgtttag gaaataacga acatggcagc 360
taagcaagtt aagttttccg atgatgcccg caaacgtatg gcgcgtggcg tagacgttct 420
agctaacgca gtaaaagtta ccctcggccc gaaaggtcgt aacgtggtac tggacaagtc 480
ttttggctcc cccaccgtca ccaaagacgg cgtatcggtt gccaaagaga ttgaactgaa 540
agacaagttc gagaacatgg gcgctcagat ggttaaggaa gttgcttcca aaacttctga 600
tgctgcaggc gacggcacca cgacagcaac ggttctggcc caggcaatta tcgctgaagg 660
cctgaaaggc gtgactgctg gcatgaaccc gatggacctg aagcgcggca tcgaccaagc 720
agttagcgcg gcagtaaaag aagttcaggc gatgtctgtc ccctgcaccg acactaagtc 780
tatcgcccag gtaggtacta tttctgccaa cggtgacaag cgcatcggtg agatcatcgc 840
tgaagcgatg gaaaaagttg gtaaagaagg cgtcatcact gttgatgaag gtcgtggctt 900
cgaagacgag ctggaagtcg ttgaagggat gcagtttgat cgcggctacc tctcgcccta 960
ctttgtcact aaccaagaca ccatgtctgt cgagctagaa gacccttaca ttctgctcgt 1020
tgacaagaaa atctccaaca tccgcgaact actgccggtg ctggaagccg ttgctaagca 1080
aggcaaaccg ctcgcgatta tcgctgaaga tatcgaaggc gaagcgctag caacgctggt 1140
tgtaaataac atgcgcggta tcgttaaagt tgctgctgct aaggcacccg gcttcggtga 1200
ccgtcgtaaa gcgatgctgc aagatatcgc tatcctgacc aatggcaccg tcatttctga 1260
agaagttggc ctgacgcttg agcaagcgaa cctggatcac ctgggtaccg ctaagcgcat 1320
gacgatgtct aaagagaaca ccaccatcat cgatggcgct ggtgtagaaa acgacatcga 1380
agcacgcgtt aaccaaatcc gtgcgcaaat cgaagaaacg tcttcggatt acgacaaaga 1440
gaagttgcaa gagcgcgttg ccaaactggc cggcggtgtt gctgttattc gtgtcggtgc 1500
ggctaccgaa gtagaaatga aagagaagaa agcacgtgtt gaagatgctc tgcactcaac 1560
ccgcgctgca gtagaagaag gcgttgtacc tggcggcggt actgcactgg ttcgcgcaat 1620
ggctaaagta caaggcctga ctggcgacaa cgaagaccaa aaccacggta tcaacatggc 1680
gctgcgcgct atgcagtctc cgctgcgcca gattgttact aacgccggtg aagaagccgc 1740
tgtggttatc aatcgcgtga aagacggtga aggcaacttc ggctacaacg cacaaactgg 1800
tgagtacggc gacctgttcg aaatgggtgt tctggacccg gctaaagtaa cccgtactgc 1860
actgcagtcc gctggctctg ttgctggtct gatgatcacc actgagtgca tgatcgctga 1920
cgatccagaa gagcaagacg ctgccccaga catgggtggc atgggcggaa tgggtggcat 1980
gggcggcatg atgtag 1996
<210> 9
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ggccgcgcga attcgagctc cccatgagtt aattatattt 40
<210> 10
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tcagcagact gtttggtcat ctagtatttc tcctctttct ctagtattaa acaaaattat 60
ttgtag 66
<210> 11
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
agaaagagga gaaatactag atgaccaaac agtctgctga 40
<210> 12
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ctagtatttc tcctctttct ctagtatcta gaggatcctt acaccagggg agatttca 58
<210> 13
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ctctagatac tagagaaaga ggagaaatac tagatggagc tcactgtgac tga 53
<210> 14
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ttttcccagt cacgacgcgg ccgcaagctt ttacttagaa gaagcagcca 50
<210> 15
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
aagcttgcgg ccgcgtcgtg actgggaaaa ccctggcg 38
<210> 16
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gagctcgaat tcgcgcggcc gcggcctagg cggcctcctg 40
<210> 17
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
agaccctagc tagggagctc cccatgagtt aattatattt 40
<210> 18
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ctagtatttc tcctctttct ctagtattaa acaaaattat ttgtag 46
<210> 19
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
agaaagagga gaaatactag atgaatatcc gtcctttgca 40
<210> 20
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gacgcggccg tgaggaattc ctacatcatg ccgcccatgc 40
<210> 21
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gaattcctca cggccgcgtc gtgactggga aaaccctggc 40
<210> 22
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
gagctcccta gctagggtct cagc 24
<210> 23
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
gcatgggcgg catgatgtag gaattcccca tgagttaatt atatttgtgg 50
<210> 24
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
catatgctag tatttctcct ctttctctag ta 32
<210> 25
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
agaaagagga gaaatactag catatgatgg agctcactgt gactga 46
<210> 26
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ttcccagtca cgacgcggcc gtgagttact tagaagaagc agcca 45
<210> 27
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
ctcacggccg cgtcgtgact gggaa 25
<210> 28
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
aattaactca tggggaattc ctacatcatg ccgcccatgc 40
<210> 29
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
agcggataac aatttcacac agga 24
<210> 30
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
cgccagggtt ttcccagtca cgac 24
Claims (4)
1.一种重组嗜盐单胞菌的构建方法，其特征是包括如下步骤：
(1)在野生型嗜盐单胞菌Halomonas sp.TD01保藏登记号为CGMCCNo.4353基因组上整合Mmp1 RNA聚合酶表达单元，得到Halomonas sp.TD1.0；
再将Halomonas sp.TD1.0基因组上异柠檬酸脱氢酶编码基因icd的起始密码子ATG替换为TTG，得到Halomonas sp.TD2.0；
所述异柠檬酸脱氢酶编码基因icd的核苷酸序列如SEQ ID NO.1所示；
(2)在高拷贝表达载体pN59多克隆位点区域插入PMmp1-RBS-cadA-acn，得到高拷贝表达载体pN59-PMmp1-RBS-cadA-acn；
所述高拷贝表达载体pN59的核苷酸序列如SEQ ID NO.2所示；
所述PMmp1为IPTG诱导型启动子，其核苷酸序列如SEQ ID NO.3所示；
所述RBS为大肠杆菌Escherichia coli MG1655强核糖体结合位点，其核苷酸序列如SEQ ID NO.4所示；
所述cadA为土曲霉Aspergillus terreus来源的经密码子优化得到的顺乌头酸脱羧酶基因，其核苷酸序列如SEQ ID NO.5所示；
所述acn为谷氨酸棒状杆菌Corynebacterium glutamicum ATCC13032来源的顺乌头酸酶基因，其核苷酸序列如SEQ ID NO.6所示；
(3)在低拷贝表达载体pN85多克隆位点区域插入PMmp1-RBS-GroESL，得到表达载体pN85-PMmp1-RBS-GroESL；
在表达载体pN85-PMmp1-RBS-GroESL的GroESL基因后再插入PMmp1-RBS-acn，得到低拷贝表达载体pN85-PMmp1-RBS-GroESL-PMmp1-RBS-acn；
所述低拷贝表达载体pN85的核苷酸序列如SEQ ID NO.7所示；
所述GroESL是嗜盐单胞菌Halomonassp.TD01来源的分子伴侣基因，其核苷酸序列如SEQ ID NO.8所示；
(4)向步骤(1)获得的Halomonas sp.TD2.0中导入高拷贝表达载体pN59-PMmp1-RBS-cadA-acn，得到Halomonas sp.IA01；
(5)向步骤(4)获得的Halomonas sp.IA01中导入低拷贝表达载体pN85-PMmp1-RBS-GroESL-PMmp1-RBS-acn，得到重组嗜盐单胞菌Halomonas sp.IA02。
2.权利要求1的构建方法构建的重组嗜盐单胞菌。
3.权利要求2的重组嗜盐单胞菌催化柠檬酸制备衣康酸的应用。
4.根据权利要求3所述的应用，其特征是包括如下步骤：
(1)将权利要求2的重组嗜盐单胞菌进行诱导培养；
(2)将步骤(1)获得的培养液，于4000-8000rpm，4-10℃离心10-20min，PB高渗缓冲液洗涤2-3次，用PB高渗缓冲液重悬，测定OD600nm，收集获得重组嗜盐单胞菌；
(3)按比例，向容器中加入底物柠檬酸，使终浓度为500-600mM，步骤(2)获得的重组嗜盐单胞菌，使终浓度为30OD600nm/mL，Trixon X-100使体积分数为0.1％-0.6％，余量为pH是6.0-6.5的0.01M PB缓冲液，混合均匀后在30-35℃反应，得到衣康酸。
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| Publication number | Priority date | Publication date | Assignee | Title |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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Non-Patent Citations (3)
| Title |
|---|
| Substrate profiling and tolerance testing of Halomonas TD01 suggest its potential application in sustainable manufacturing of chemicals;Zhang, Jing等;《JOURNAL OF BIOTECHNOLOGY》;20200527;全文 * |
| 嗜盐单胞菌ST-1的分离及其石油烃类降解特性的研究;秦春艳;《生物技术世界》;20160315(第03期);全文 * |
| 水体中盐单胞菌属(Halomonas)细菌实时荧光定量PCR检测方法的建立及应用;张艳等;《应用与环境生物学报》;20160225(第01期);全文 * |
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