产麦角硫因大肠杆菌工程菌株的构建与优化

Construction and optimization of ergothioneine-producing Escherichia coli

麦角硫因(ergothioneine,ERG)是一种天然的抗氧化剂,广泛应用于化妆品、食品以及医药领域。相比于传统植物提取和化学合成方法,微生物发酵合成麦角硫因具有周期短、成本低等优点,因而受到广泛关注。为构建高产麦角硫因的大肠杆菌工程菌株,本研究以大肠杆菌(Escherichia coli)BL21(DE3)为出发菌株,通过引入耻垢分枝杆菌(Mycobacterium smegmatis)来源的麦角硫因合成基因簇egtABCDE以及粟酒裂殖酵母(Schizosaccharomyces pombe)来源的Egt1,构建了从头发酵合成麦角硫因的工程菌株E1-A1,麦角硫因发酵浓度为(95.58±3.20)mg/L。为进一步提高麦角硫因的产量,对前体氨基酸组氨酸、甲硫氨酸以及半胱氨酸合成路径中关键酶的表达进行了强化,结果表明,单一表达基因serA^(T410STOP)、thrA以及两者共表达时,麦角硫因发酵浓度分别提高至(134.83±4.22)mg/L、(130.26±3.34)mg/L以及(144.97±5.40)mg/L。最后,采用分批补料发酵策略,菌株E1-A1-thrA-serA^(*)在无机盐培养基和丰富培养基培养中的产量分别为548.75 mg/L和710.53 mg/L。

Ergothioneine(ERG)is a natural antioxidant that has been widely used in the fields of food,medicine and cosmetics.Compared with traditional plant extraction and chemical synthesis approaches,microbial synthesis of ergothioneine has many advantages,such as the short production cycle and low cost,and thus has attracted intensive attention.In order to engineer an ergothioneine high-yielding Escherichia coli strain,the ergothioneine synthesis gene cluster egtABCDE from Mycobacterium smegmatis and egt1 from Schizosaccharomyces pombe were introduced into E.coli BL21(DE3)to generate a strain E1-A1 harboring the ergothioneine biosynthesis pathway.As a result,(95.58±3.2)mg/L ergothioneine was produced in flask cultures.To further increase ergothioneine yield,the relevant enzymes for biosynthesis of histidine,methionine,and cysteine,the three precursor amino acids of ergothioneine,were overexpressed.Individual overexpression of serA^(T410STOP) and thrA resulted in an ergothioneine titer of(134.83±4.22)mg/L and(130.26±3.34)mg/L,respectively,while co-overexpression of serA^(T410STOP) and thrA increased the production of ergothioneine to(144.97±5.40)mg/L.Eventually,by adopting a fed-batch fermentation strategy in 3 L fermenter,the optimized strain E1-A1-thrA-serA^(*) produced 548.75 mg/L and 710.53 mg/L ergothioneine in glucose inorganic salt medium and rich medium,respectively.