反式-4-羟基脯氨酸基因工程菌的构建及摇瓶发酵工艺优化

发布时间：2018-03-07 03:01

  本文选题：反式-4-羟脯氨酸　切入点：起始效率　出处：《湖南农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：反式-4-羟脯氨酸为手性亚氨基酸,是一种被广泛应用于医疗美容,化工,畜牧领域的生产原料。本文构建了一株能够高效表达反式-4-脯氨酸羟化酶基因的重组大肠杆菌,并对得到的重组菌株DH5α/pUC-19-pH进行了摇瓶发酵工艺优化,主要结果如下：1,优化反式-4-脯氨酸羟化酶编码区序列：使用JCAT软件优化了反式-4-脯氨酸羟化酶的编码区基因序列,改变了141个碱基(替换了138个同义密码子),使其CAI指数由0.3965优化至0.9277,C/G比由73.6263%优化至59.7069%,优化后的mRNA翻译延伸效率显著提高。2,优化反式-4-脯氨酸羟化酶非编码区序列：在RBS区前端添加一段来源于T7噬菌体的g10-L翻译增强子序列,以提高外源基因的表达量；设计RBS区,使其内部包含一段以AAGGA为核心的强SD序列,使得mRNA能够高效的与原核生物核糖体结合,同时,将T7 g10-L序列置于SD序列上游能有力地提高下游基因编码蛋白的表达水平；使用RNAstructure软件优化TIR区的二级结构,使其自由能∧G由-12.8升高到了-7.6,从而优化mRNA的翻译起始效率。3,选择色氨酸启动子并进行简化,在构建的反式-4-脯氨酸羟化酶基因末端添加来源于PET载体的TrpLABCDE转录终止序列,以保护序列的稳定性。为了方便后续实验,在表达盒的5’端加入限制性内切酶Sal Ⅰ位点,在3’端加入Nde Ⅰ酶切位点；使用T载体质粒pUC-19成功构建重组菌DH5α/pUC-19-pH,并对比大肠杆菌JM109,C43,BL21以及XL-1优化宿主菌,对比pCDFDuet-1, pACYCDuet-1, pETDuet-1, pRSFDuet-1, pCOLAduet-1优化质粒载体,最终得到比酶活最高的菌株为DH5a/pUC-19-pH,达到0.0108 U/mg。4,测定DH5α/pUC-19-pH的生长曲线及发酵曲线,进行摇瓶发酵的单因素条件优化,最佳结论值如下：温度37℃,L-脯氨酸浓度200 mM,亚铁离子浓度2 mM,镁离子浓度0.02%,胰蛋白胨8 g/L,碳氮比为1,得到的培养基配方如下：葡萄糖20g/L,胰蛋白胨8 g/L,L-脯氨酸200 mM,硫酸亚铁2 mM,硫酸镁0.2g/L,硫酸铵10 g/L,磷酸氢二钾1g/L,氯化钠2 g/L,柠檬酸2 g/L,氯化钙0.015g/L。优化后反式-4-脯氨酸羟化酶的比酶活在发酵16 h时达到了0.301 U/mg,较最近的报道提高了45.4%。
[Abstract]:Trans -4-hydroxyproline, a chiral amino acid, is widely used as a raw material in the fields of medical beauty, chemical industry and animal husbandry. A recombinant Escherichia coli expressing trans--4-proline hydroxylase gene was constructed in this paper. The recombinant strain DH5 伪 / pUC-19-pH was optimized for shaking flask fermentation. The main results were as follows: 1. The sequence of trans-4-proline hydroxylase coding region was optimized. The coding region gene sequence of trans--4-proline hydroxylase was optimized by JCAT software. By changing 141 bases (replacing 138 synonymous codon), the CAI index was optimized from 0.3965 to 0.9277C / G than from 73.6263% to 59.7069. The optimized translation extension efficiency of mRNA was significantly improved by .2and the sequence of non-coding region of trans-4-proline hydroxylase was optimized. : a G10-L translation enhancer sequence derived from the T7 phage was added to the front of the RBS region. In order to improve the expression of foreign genes, the RBS region was designed to contain a strong SD sequence with AAGGA as the core, which enabled mRNA to efficiently bind to prokaryote ribosomes, at the same time, Placing the T7g10-L sequence upstream of the SD sequence can improve the expression level of the downstream gene coding protein, and optimize the secondary structure of the TIR region by using RNAstructure software. In order to optimize the translation initiation efficiency of mRNA from -12.8 to -7.6, the tryptophan promoter was selected and simplified, and the TrpLABCDE transcriptional termination sequence derived from the PET vector was added to the end of the constructed trans--4-proline hydroxylase gene. In order to protect the stability of the sequence, the restriction endonuclease Sal 鈪，

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