岩藻黄质关键前体新黄质的代谢工程合成

Metabolic engineering synthesis of neoxanthin,a key precursor of fucoxanthin

岩藻黄质是海洋藻类中常见的叶黄素之一,尤其在褐藻中含量较高,具有抑菌、抗炎、抗肥胖、抗糖尿病、抗肿瘤、调节肠道菌群和抗器官纤维化等多种生理活性。新黄质是岩藻黄质的可能前体物质,首次在大肠杆菌(Escherichia coli)中构建新黄质合成途径,首先构建了pTrc99a-crt EBIYZ质粒,该质粒携带5个来自于菠萝泛菌(Pantoea ananatis)的基因crt E、crt B、crt I、crt Y和crt Z,编码的蛋白分别是牻牛儿基牻牛儿基焦磷酸合成酶、八氢番茄红素合成酶、八氢番茄红素脱氢酶、番茄红素环化酶和β-胡萝卜素羟化酶。将该质粒转入大肠杆菌中,得到了产玉米黄质的工程菌E.coli BL21 pTrc99a-crt EBIYZ,玉米黄质产量(细胞干质量,下同)为0.70 mg·g^(-1)。以产玉米黄质工程菌为宿主,将来自三角褐指藻(Phaeodactylum tricornutum)的ZEP3基因去信号肽之后转入,得到产紫黄质的工程菌E.coli BL21 pTrc99a-crt EBIYZ p ACYCDuet-1-QZEP3。在紫黄质工程菌E.coli BL21 p Trc99a-crt EBIYZ p ACYCDuet-1-QZEP3的基础上转入新黄质合酶基因NSY,成功获得了产新黄质的工程菌E.coli BL21 NEO,新黄质产量为99.27μg·g^(-1),紫黄质产量为150.30μg·g^(-1),玉米黄质产量为119.77μg·g^(-1)。

Fucoxanthin is one of the common luteins in marine algae,especially in brown algae,which has antibacterial,antiinflammatory,anti-obesity,anti-diabetes,anti-cancer,regulating intestinal flora,anti-organ fibrosis and other physiological activities.Neoxanthin is a possible precursor of fucoxanthin.In this paper,we constructed a neoxanthin synthesis pathway in Escherichia coli for the first time and the plasmid pTrc99a-crtEBIYZ,which carries five genes(crtE,crtB,crtI,crtY and crtZ)from Pantoea ananatis,and codes for proteins(geranylgeranyl pyrophosphate synthase,phytoene synthase,lycopene cyclase andβ-carotene hydroxylase).We transferred the plasmid into E.coli,and obtained the zeaxanthin-producing E.coli BL21 pTrc99acrtEBIYZ.The yield of zeaxanthin(dry cell mass)was 0.70 mg·g^(-1).We obtained E.coli pTrc99a-crtEBIYZ pACYCDuet-1-QZEP3strain by transferring ZEP3 gene from Phaeodactylum tricornutum without signal peptide,and obtained E.coli BL21 NEO successfully by transferring the neoxanthin synthase gene NSY into E.coli BL21 pTrc99a-crtEBIYZ pACYCDuet-1-QZEP3.The concentrations of neoxanthin,violaxanthin and zeaxanthin were 99.27,150.30 and 119.77μg·g^(-1)dry cell mass,respectively.