在大肠杆菌中拓展合成途径合成异胡椒醇

Expanding the pathway to synthesize isopiperitenol in Escherichia coli

摘要:

天然异胡椒醇作为一种植物来源香料，具有很高的经济价值。目前国内外还未见利用大肠杆菌工程菌株转化生产异胡椒醇的报道。【目的】构建工程菌，采用生物合成方法获得天然香料异胡椒醇。【方法】对来自胡椒薄荷的细胞色素P450家族的柠檬烯-3-羟化酶（LIM3H）基因PM2进行改造，截短LIM3H的N端疏水区，分别添加17α短肽或2B1短肽增加其亲水性，采用CO差示光谱法检测LIM3H活性；将N端疏水区截短后的来自拟南芥（Arabidopsis thaliana）的NADPH-细胞色素P450还原酶基因（trATR）和修饰后LIM3H基因（Modi-LIM3H）融合后表达，最终以柠檬烯为底物进行全细胞转化。【结果】3种N端修饰LIM3H基因均检测到LIM3H表达和异胡椒醇生成，其中17α短肽修饰后的蛋白表达量最高，异胡椒醇产量达到1.94 mg/L。【结论】本研究在大肠杆菌中增加外源LIM3H与ATR，成功催化柠檬烯生成异胡椒醇，为天然异胡椒醇的生产提供了新的方法。

关键词:柠檬烯-3-羟化酶;大肠杆菌;N端修饰;异胡椒醇

Abstract:

As a plant-derived aromatic compound, isopiperitenol has a high economic value. Little is known about the production of isopiperitenol in Escherichia coli engineering strains. [Objective] To construct the engineering strains of E. coli for the biosynthesis of natural isopiperitenol. [Methods] We modified the limonene-3-hydroxylase (LIM3H) gene PM2 of cytochrome P450 family from Mentha×piperita by truncating the N-terminal hydrophobic region or adding 17α short peptide or 2B1 short peptide of LIM3H to increase its hydrophilicity. The activity of LIM3H was detected by CO differential spectroscopy. The NADPH-cytochrome P450 reductase gene with truncated N-terminal hydrophobic region (trATR) from Arabidopsis thaliana was fused with the modified LIM3H (Modi-LIM3H) gene and the fusion gene was expressed in E. coli. The obtained fusion protein was used to catalyze the production of isopiperitenol from limonene. [Results] LIM3H expression and isopiperitenol production were detected in all the strains with the LIM3H N-terminus modified in three different ways. The LIM3H modified by addition of 17α short peptide showed the highest protein level, and the strain carrying this modified gene had the highest yield (1.94 mg/L) of isopiperitenol.[Conclusion] In this study, exogenous LIM3H and ATR were introduced into E. coli and the fusion protein catalyzed the production of isopiperitenol from limonene, which provided a new method for the production of natural isopiperitenol.

Key words:limonene-3-hydroxylase;Escherichia coli;N-terminal modification;isopiperitenol