广藿香醇是一种天然化学品,在化妆品行业有着较广泛的应用。相对于植物提取法,微生物发酵法合成广藿香醇对节约成本和保护环境有重要意义。在广藿香醇合酶PTS2上添加促溶标签,提高可溶表达,使重组菌株Escherichia coli BL21/pMev+pET28a-PTS2/T7A广藿香醇产量提高了39.74%。通过对PTS2进行同源比对、三维结构建模和分子表面氨基酸分析,确定了氨基酸突变位点,构建了PTS2突变体,表明E457、K28、K136和K343是影响酶活性的关键氨基酸位点。进一步,将合成法尼基焦磷酸的8个基因模块化分割,用T7启动子加强法尼基焦磷酸合成代谢途径,获得重组菌株BL21/pMev-T7M1M2+pET28a-PTS2,使广藿香醇产量提高了55.95%。最后,将最优的改造策略组合,得到重组菌株BL21/pMev-T7M1M2+pET28a-PTS2/T7A,广藿香醇产量累计提高了72.22%。该研究结果为大肠杆菌发酵合成广藿香醇等倍半萜化合物提供了借鉴。
As a kind of natural chemical, patchoulol is widely used in cosmetics industry. Compared with plant extraction, patchoulol production with microbial fermentation has great significance for saving cost and protecting environment. The solubility-enhancing tags were fused to patchoulol synthase PTS2 to improve soluble expression, and patchoulol production of strain Escherichia coli BL21/pMev-pET28a-PTS2T7A was increased by 39.74 %. Through the methods of homologous alignment, 3D structural modeling and analysis of surface amino acid, the amino-acid mutation sites of PTS2 were determined. Fermentation experiments using PTS2 mutants indicated that E457, K28, K136 and K343 were the key amino acid sites which can affect enzyme activity significantly. Furthermore, the eight genes of farnesyl pyrophosphate synthetic pathway were modularized, and strengthened by T7 promoter. Patchoulol production in the resulting strain BL21/pMev-T7M1M2+pET28a-PTS2 was increased by 55.95%. Finally, the optimal modification strategies were combined, and patchoulol production in the resulting strain BL21/pMev-T7M1M2+pET28a-PTS2T7A was cumulatively increased by 72.22%. The results provide reference for the production of patchoulol and other sesquiterpenes in E. coli.
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