本文選題：反式-4-羥脯氨酸　切入點(diǎn)：起始效率　出處：《湖南農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：反式-4-羥脯氨酸為手性亞氨基酸,是一種被廣泛應(yīng)用于醫(yī)療美容,化工,畜牧領(lǐng)域的生產(chǎn)原料。本文構(gòu)建了一株能夠高效表達(dá)反式-4-脯氨酸羥化酶基因的重組大腸桿菌,并對得到的重組菌株DH5α/pUC-19-pH進(jìn)行了搖瓶發(fā)酵工藝優(yōu)化,主要結(jié)果如下：1,優(yōu)化反式-4-脯氨酸羥化酶編碼區(qū)序列：使用JCAT軟件優(yōu)化了反式-4-脯氨酸羥化酶的編碼區(qū)基因序列,改變了141個(gè)堿基(替換了138個(gè)同義密碼子),使其CAI指數(shù)由0.3965優(yōu)化至0.9277,C/G比由73.6263%優(yōu)化至59.7069%,優(yōu)化后的mRNA翻譯延伸效率顯著提高。2,優(yōu)化反式-4-脯氨酸羥化酶非編碼區(qū)序列：在RBS區(qū)前端添加一段來源于T7噬菌體的g10-L翻譯增強(qiáng)子序列,以提高外源基因的表達(dá)量；設(shè)計(jì)RBS區(qū),使其內(nèi)部包含一段以AAGGA為核心的強(qiáng)SD序列,使得mRNA能夠高效的與原核生物核糖體結(jié)合,同時(shí),將T7 g10-L序列置于SD序列上游能有力地提高下游基因編碼蛋白的表達(dá)水平；使用RNAstructure軟件優(yōu)化TIR區(qū)的二級結(jié)構(gòu),使其自由能∧G由-12.8升高到了-7.6,從而優(yōu)化mRNA的翻譯起始效率。3,選擇色氨酸啟動子并進(jìn)行簡化,在構(gòu)建的反式-4-脯氨酸羥化酶基因末端添加來源于PET載體的TrpLABCDE轉(zhuǎn)錄終止序列,以保護(hù)序列的穩(wěn)定性。為了方便后續(xù)實(shí)驗(yàn),在表達(dá)盒的5’端加入限制性內(nèi)切酶Sal Ⅰ位點(diǎn),在3’端加入Nde Ⅰ酶切位點(diǎn)；使用T載體質(zhì)粒pUC-19成功構(gòu)建重組菌DH5α/pUC-19-pH,并對比大腸桿菌JM109,C43,BL21以及XL-1優(yōu)化宿主菌,對比pCDFDuet-1, pACYCDuet-1, pETDuet-1, pRSFDuet-1, pCOLAduet-1優(yōu)化質(zhì)粒載體,最終得到比酶活最高的菌株為DH5a/pUC-19-pH,達(dá)到0.0108 U/mg。4,測定DH5α/pUC-19-pH的生長曲線及發(fā)酵曲線,進(jìn)行搖瓶發(fā)酵的單因素條件優(yōu)化,最佳結(jié)論值如下：溫度37℃,L-脯氨酸濃度200 mM,亞鐵離子濃度2 mM,鎂離子濃度0.02%,胰蛋白胨8 g/L,碳氮比為1,得到的培養(yǎng)基配方如下：葡萄糖20g/L,胰蛋白胨8 g/L,L-脯氨酸200 mM,硫酸亞鐵2 mM,硫酸鎂0.2g/L,硫酸銨10 g/L,磷酸氫二鉀1g/L,氯化鈉2 g/L,檸檬酸2 g/L,氯化鈣0.015g/L。優(yōu)化后反式-4-脯氨酸羥化酶的比酶活在發(fā)酵16 h時(shí)達(dá)到了0.301 U/mg,較最近的報(bào)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 偽 / 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，

本文編號：1577627