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

作　　者：刘春筱 夏媛媛 齐丽娜 杨海泉[1,2] 陈磊 沈微[1,2] 陈献忠[1,2] Chunxiao Liu;Yuanyuan Xia;Lina Qi;Haiquan Yang;Lei Chen;Wei Shen;Xianzhong Chen(Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,Jiangsu,China;School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《生物工程学报》2021年第12期4243-4253,共11页Chinese Journal of Biotechnology

基　　金：江苏省自然科学基金(No.BK20171138)资助。

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EscherichiacoliBL21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1.81g/L,同时发现几乎没有副产物积累。5L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。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 YMG5 A*R using CRISPR-Cas9 technology. Meanwhile, the pathway for production of acetic acid was deleted, resulting in a recombinant strain YMGRD1 H1. Shake flask fermentation showed that strain YMGRD1 H1 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.

关 键 词：大肠杆菌 羟化酶 酪醇 羟基酪醇 CRISPR-Cas9 

分 类 号：TQ923[轻工技术与工程—发酵工程]