代谢工程改造大肠杆菌合成羟基酪醇

Metabolic engineering of Escherichia coli for production of hydroxytyrosol

摘要:

羟基酪醇是重要精细化学品，作为天然抗氧化剂被广泛应用于食品、医药领域。利用合成生物学技术生产羟基酪醇具有重要意义。本文克隆并功能鉴定了来源于大肠杆菌Escherichia coli BL21的羟化酶编码基因HpaBC，结果表明该酶的两个亚基均能成功表达并能催化酪醇生成羟基酪醇。通过CRISPR-Cas9技术将由tac启动子调控的HpaBC基因表达盒整合到前期构建的酪醇高产菌株YMG5A*R基因组中，同时删除副产物乙酸的合成途径，获得大肠杆菌代谢工程菌株YMGRD1H1。摇瓶发酵实验结果表明，重组菌株能够直接利用葡萄糖生产羟基酪醇，产量达到1.81 g/L，同时发现几乎没有副产物积累。5 L发酵罐规模的流加补料发酵实验表明，羟基酪醇最高产量达到2.95 g/L，是目前文献报道以葡萄糖从头合成羟基酪醇的最高水平。通过系统改造大肠杆菌，实现了羟基酪醇的大量合成，为进一步构建具有工业应用潜力的羟基酪醇细胞工厂奠定了基础，也为拓展芳香族化合物的微生物制造路线提供了有益的参考。

Abstract:

Hydroxytyrosol is an important fine chemical and is widely used in food and medicine as a natural antioxidant. Production of hydroxytyrosol through synthetic biology is of important significance. Here we cloned and functionally characterized a hydroxylase encoding gene HpaBC from Escherichia coli BL21, and both subunits of this enzyme can be successfully expressed to convert the tyrosol into hydroxytyrosol. A HpaBC gene integration expression cassette under the tac promoter was constructed, and integrated into the genome of a tyrosol hyper-producing E. coli YMG5A*R using CRISPR-Cas9 technology. Meanwhile, the pathway for production of acetic acid was deleted, resulting in a recombinant strain YMGRD1H1. Shake flask fermentation showed that strain YMGRD1H1 can directly use glucose to produce hydroxytyrosol, reaching a titer of 1.81 g/L, and nearly no by-products were detected. A titer of 2.95 g/L was achieved in a fed-batch fermentation conducted in a 5 L fermenter, which is the highest titer for the de novo synthesis of hydroxytyrosol from glucose reported to date. Production of hydroxytyrosol by engineered E. coli lays a foundation for further construction of hydroxytyrosol cell factories with industrial application potential, adding another example for microbial manufacturing of aromatic compounds.