CN119410683A 一种生物发酵-全细胞催化级联生产己二酸的方法 （北京化工大学）

号一种生物发酵-全细胞催化级联生产己二酸2rhaB_M8(G27M/I374S)和pRSF_araB_M8(G27M/I374S)；所述烯还原酶基因M8(G27M/I374S)备成表达烯还原酶的菌株BL_M8(G27M/I374S)、BL_rhaB_M8(G27M/I374S)和BL_araB_M8(G27M/I374S)利用终浓度为1_10mM的鼠李糖诱导；菌株BL_araB_M8(G27M/I374S)利用终携带分子伴侣表达质粒的菌株额外添加终浓度0.5mg/以全细胞催化体系的体积计，粘糠酸的含量为403.权利要求1所述的全细胞催化剂或权利要求2所述的方法在催化生产己二酸中的应3[0001]本发明涉及生物技术领域，更具体的说是涉及一种生物发酵_全细胞催化级联生[0004]因此，提供一种生物发酵_全细胞催化级联生产己二酸的方法是本领域技术人员[0006]一种基于产粘糠酸基因工程菌的生物发酵_全细胞催化级联生产己二酸的方法，（1）构建携带烯还原酶基因M8(G27M/I374S)的重组质粒pRSF_M8(G27M/I374S)、pRSF_rhaB_M8(G27M/I374S)和pRSF_araB_M8(G27M/I374S)；所述烯还原酶基因M8(G27M/4诱导剂：菌株BL_M8(G27M/I374S)利用终浓度为0.5_1mM的IPTG诱导；菌株BL_携带分子伴侣表达质粒的菌株额外添加终浓度0.5mg/全细胞催化耦合级联的己二酸绿色合成方法对于合成尼龙66等高分子聚合物的可持续发5[0017]图1附图为本发明利用生物发酵_全细胞催化级联生物合成己二酸作用机理示意2OMnSO42O42O46[0032]利用本实验室前期构建的产粘糠酸的谷氨酸棒状杆菌MA2作为发酵菌株，首先将78板，利用引物M8(G27M/I374S)_F/R进行目的基因M8(G27M/I374S)的扩增；接着用NcoI和[0038]ATGAAATACAAAAAGCTATTTGAAACTGTGAAAATAAGGAATGTGGAACTCAAAAATCGTTATGCAATGGCACCAATGATGCCGCTGGGTCTTGCCGATGCAGAAGGCGGTTTCAACCAGCGCGGGATTGAGTATTATACAGCCCGTGCGCGCGGGGGAACCGCTCTGATTATTACCGGCGTCACTTTCGTTGATAATGAAGTGGAAGAGCACGGAATGCCAAACGTACCTTGCCCGACCCATAACCCTGTCCATTTTGTCCGGACTTCCAAAGAAATGACAGAGCGCATCCATGCATATGATTCGAAAATTTTTCTGCAAATGAGCGCCGGTTTTGGCCGGGTGACGATCCCGACAAACCTTGGCGAGTACCCGCCGGTTGCACCGTCGCCAATCCCGCATCGCTGGCTGGATAAAACATGTCGCGAACTGACAGTTGAAGAAATTCATTCCATTGTCCGCAAATTCGGGGATGGGGCGTTCAATGCGAAGCGCGCCGGATTTGACGGGGTGCAAATCCATGCTGTGCACGAAGGCTATTTGCTCGACCAGTTTGCGATTGCGTTTTTCAACAAACGTACCGATGCATACGGTGGCCCGCTTGAAAATCGCCTTCGTTTTGCCCGGGAAATTGTCGAGGAAATTAAACAGCGCTGTGGCGAAGATTTTCCTGTGACGCTCCGCTTCAGCCCGAAAAGTTTTATCAAGGATTGGCGGGAAGGGGCACTGCCTGGCGAGGAGTTTGAAGAAAAAGGCCGCGATTTGGATGAAGGCATCGAGGCAGCAAAGCTGCTCGTTTCCTACGGCTATGATGCTCTGGACGTCGATGTTGGTTCTTATGATTCATGGTGGTGGAGCCATCCTCCGATGTACCAGAAGAAGGGGCTTTACATTCCGTATGCCAGGCTGGTGAAGGAAGCTGTCGATGTGCCTGTCCTTTGCGCGGGCCGCATGGACAATCCGGATCTTGCACTTGCCGCACTGGAAGACGGAGCATGTGATATTATCAGCTTGGGCCGCCCGTTATTGGCTGACCCGGATTACGTCAATAAGCTCCGAATCGGGCAGGTTGCCGATATCCGCCCGTGTCTGTCATGCCATGAAGGCTGCATGGGTCGGAGCCAGGAGTATTCTTCCTTAGGCTGCGCAGTGAATCCGGCTGCCTGTCGAGAAAAAGAAGCAGCATTGACACCTGCTTTAAAAAAGAAACGCGTACTGATTGCAGGCGGCGGCGTGGCCGGATGCGAAGCTGCCCGTGTGCTTGCATTGCGCGGCCATGAACCGGTCATTTTTGAAAAATCGAACCGTTTAGGCGGCAACTTGATCCCTGGCGGCGCACCTGATTTTAAAGAAGATGACCTGGCGCTTGTTGCCTGGTATGAGCATACGTTGGAACGCCTTGGCGTAGAAATTCATTTGAATACTGCATTGACAAAAGAAGAAATTTTGGCTGCAAACGTGGATGCCGTGCTGATTGCAACGGGTTCGAATCCGAAAATTTTGCCGCTCGACGGAAAAAACAAAGTATTTACAGCAGAAGATGTTTTGCTCGATAAAGTGGATGCCGGGCAACATGTTGTCATTGTCGGCGGCGGTCTTGTCGGCTGCGAACTGGCTTTGAACCTTGCAGAAAAAGGAAAAGATGTCTCGCTTGTGGAAATGCAGGACAAACTGCTGGCAGTTAATGGTCCGCTTTGCCACGCTAACTCGGACATGCTGGAAAGACTCGTACCGTTTAAAGGTGTTCAAGTCTACACTTCTTCAAAAATAGTAGATACGACAGAAAAGACAGCCGTTGTGGATGTTGACGGCGAATTGCGTGAAATTGAAGCAGACAGCATTGTGCTCGCAGTCGGCTACTCGGCTGAAAAATCACTCTATGAAGATTTAAAGTTTGAGGTTGCCGATCTTCATGTGGTTGGCGATGCCCGCAAGGTCGCAAACATCATGTATGCCATCTGGGATGCT[0039]以质粒pRSF_M8(G27M/I374S)为模版，利用引物pRSF_F/pRSF_R进行质粒的线性[0040]以质粒pEcCas为模板，利用引物pRSF_rhaR_F/pRSF_rhaB_R扩增获得rhaB启动子的方法分别将rhaB启动子元件和araB启动子元件与线性化质粒pRSF_M8(G27M/I374S)ΔT79TTAATCTTTCTGCGAATTGAGATGACGCCACTGGCTGGGCGTCATCCCGGTTTCCCGGGTAAACACCACCGAAAAATAGTTACTATCTTCAAAGCCACATTCGGTCGAAATATCACTGATTAACAGGCGGCTATGCTGGAGAAGATATTGCGCATGACACACTCTGACCTGTCGCAGATATTGATTGATGGTCATTCCAGTCTGCTGGCGAAATTGCTGACGCAAAACGCGCTCACTGCACGATGCCTCATCACAAAATTTATCCAGCGCAAAGGGACTTTTCAGGCTAGCCGCCAGCCGGGTAATCAGCTTATCCAGCAACGTTTCGCTGGATGTTGGCGGCAACGAATCACTGGTGTAACGATGGCGATTCAGCAACATCACCAACTGCCCGAACAGCAACTCAGCCATTTCGTTAGCAAACGGCACATGCTGACTACTTTCATGCTCAAGCTGACCGATAACCTGCCGCGCCTGCGCCATCCCCATGCTACCTAAGCGCCAGTGTGGTTGCCCTGCGCTGGCGTTAAATCCCGGAATCGCCCCCTGCCAGTCAAGATTCAGCTTCAGACGCTCCGGGCAATAAATAATATTCTGCAAAACCAGATCGTTAACGGAAGCGTAGGAGTGTTTATCGTCAGCATGAATGTAAAAGAGATCGCCACGGGTAATGCGATAAGGGCGATCGTTGAGTACATGCAGGCCATTACCGCGCCAGACAATCACCAGCTCACAAAAATCATGTGTATGTTCAGCAAAGACATCTTGCGGATAACGGTCAGCCACAGCGACTGCCTGCTGGTCGCTGGCAAAAAAATCATCTTTGAGAAGTTTTAACTGATGCGCCACCGTGGCTACCTCGGCCAGAGAACGAAGTTGATTATTCGCAATATGGCGTACAAATACGTTGAGAAGATTCGCGTTATTGCAGAAAGCCATCCCGTCCCTGGCGAATATCACGCGGTGACCAGTTAAACTCTCGGCGAAAAAGCGTCGAAAAGTGGTTACTGTCGCTGAATCCACAGCGATAGGCGATGTCAGTAACGCTGGCCTCGCTGTGGCGTAGCAGATGTCGGGCTTTCATCAGTCGCAGGCGGTTCAGGTATCGCTGAGGCGTCAGTCCCGTTTGCTGCTTAAGCTGCCGATGTAGCGTACGCAGTGAAAGAGAAAATTGATCCGCCACGGCATCCCAATTCACCTCATCGGCAAAATGGTCCTCCAGCCAGGCCAGAAGCAAGTTGAGACGTGATGCGCTGTTTTCCAGGTTCTCCTGCAAACTGCTTTTACGCAGCAAGAGCAGTAATTGCATAAACAAGATCTCGCGACTGGCGGTCGAGGGTAAATCATTTTCCCCTTCCTGCTGTTCCATCTGTGCAACCAGCTGTCGCACCTGCTGCAATACGCTGTGGTTAACGCGCCAGTGAGACGGATACTGCCCATCCAGCTCTTGTGGCAGCAACTGATTCAGCCCGGCGAGAAACTGAAATCGATCCGGCGAGCGATACAGCACATTGGTCAGACACAGATTATCGGTATGTTCATACAGATGCCGATCATGATCGCGTACGAAACAGACCGTGCCACCGGTGATGGTATAGGGCTGCCCATTAAACACATGAATACCCGTGCCATGTTCGACAATCACAATTTCATGAAAATCATGATGATGTTCAGGAAAATCCGCCTGCGGGAGCCGGGGTTCTATCGCCACGGACGCGTTACCAGACGGAAAAAAATCCACACTATGTAATACGGTCATACTGGCCTCCTGATGTCGTCAACACGGCGAAATAGTAATCACGAGGTCAGGTTCTTACCTTAAATTTTCGACGGAAAACCACGTAAAAAACGTCGATTTTTCAAGATACAGCGTGAATTTTCAGGAAATGCGGTGAGCATCACATCACCACAATTCAGCAAATTGTGAACATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCATTTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACTCGCGAGAAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTAGCGATAGGCATCCGGGTGGTGCTCAAAAGCAGCTTCGCCTGACTAATGCGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAGGCAAACATGCTGTGCGACGCTGGCGATATCAAAATTGCTGTCAGCAAGATGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACAAGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAAACCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGGCCGTAGTGATGAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAGACAAATTCTCGTCCCTGATTTTTCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCGTTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCCAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCC进行化学转化，将构建好的pRSF_M8(G27M/I374S)、pRSF_rhaB_M8(G27M/I374S)和pRSF_表达烯还原酶的菌株BL_M8(G27M/I37