产3-羟脯氨酸重组菌的构建及发酵优化

Construction of Recombinant Escherichia coli Strains Producing cis-3-Hydroxyproline and Preliminary Optimization of the Fermentation Conditions

DOI：10.3969/j.issn.1673-1689.2017.03.003

中文关键词: 脯氨酸-3-羟化酶 顺式-3-羟脯氨酸 密码子优化 重组菌构建 发酵优化

英文关键词: proline-3-hydroxylase,cis-3-hydroxyproline,codon optimization,construction of recombinant Escherichia coli,fermentation optimize

基金项目:

摘要点击次数: 179

全文下载次数: 521

中文摘要:

顺式-3-羟基-L-脯氨酸（顺式-3-羟脯氨酸）可用于合成多种抗癌药物，具有重要的商业价值，目前大多通过添加IPTG来诱导表达脯氨酸-3-羟化酶，采用两步法生物合成顺式-3-羟脯氨酸。作者通过目的基因优化设计，引入强启动子色氨酸串联启动子（Ptrp2）来避免异源表达时的诱导剂使用，构建重组质粒pES-Ptrp2-P3H，成功构建了重组大肠杆菌BL21（DE3） / pET21a-Ptrp2-P3H，优化后的脯氨酸-3-羟化酶基因（P3H）改变了168个碱基，GC含量由原来的64.83%降低到49.31%。该菌在初步优化培养基（葡萄糖1 g/dL，甘油0.125 g/dL，胰蛋白胨1.6 g/dL，（NH4）2SO4 0.5 g/dL，K2HPO4 0.1 g/dL，NaCl 0.2 g/dL，FeSO4 1 mmol/L，MgSO4 0.5 g/dL，CaCl2 0.015 g/dL，脯氨酸10 g/L，pH 7.5）上能一步法原位合成顺式-3-羟脯氨酸，摇瓶发酵24 h，产量0.8 g/L，比优化前提高一倍以上，为进一步开展顺-3-羟脯氨酸产业化提供了依据。

英文摘要:

The cis-3-hydroxy-L-proline can be used to synthesize many anticancer drugs with important commercial value. At present，cis-3-hydroxyproline is produced with IPTG-inducible recombinants in two steps. The subject of this study is to construct a recombinant with high proline-3-hydroxylase activity and generate cis-3-hydroxyproline without adding IPTG which is expensive and toxic .We constructed the recombinant plasmid pES-Ptrp2-P3H and transformed E. coli BL21（DE3） successfully. Compared with the original gene sequence of proline 3-hydroxylase，168 nucleotides were changed and the GC percentage was reduced from 64.83 % to 49.31%. The preliminarily optimized medium was 1 g/dL glucose，0.125 g/dL glycerinum，1.6 g/dL tryptone，0.5 g/dL（NH4）2SO4，0.1 g/dL K2HPO4，0.2 g/dL NaCl，1 mmol/L FeSO4，0.5 g/dL MgSO4，0.015 g/dL CaCl2，10 g/L L-proline，and pH valve at 7.5. At shaking flask level，cis-3-hydroxyproline was accumulated to about 0.8 g/L in 24 h，which was one times higher than before. It provides the basis for the industrialization of cis-3-hydroxyproline bioproduction .

查看全文 查看/发表评论 下载PDF阅读器