DataSheet2_Efficient production of anthocyanins in Saccharomyces cerevisiae by introducing anthocyanin transporter and knocking out endogenous degrading enzymes.xlsx

Anthocyanins are natural pigments found in various plants. As multifunctional natural compounds, anthocyanins are widely used in food, pharmaceuticals, health products, and cosmetics. At present, the anthocyanins are heterologously biosynthesized in prokaryotes from flavan-3-ols, which is rather expensive. This study aimed to metabolically engineer Saccharomyces cerevisiae for anthocyanin production. Anthocyanin production has been extensively studied to understand the metabolic pathway enzymes in their natural hosts, including CHS (chalcone synthase); FLS (flavonol synthase); CHI (chalcone isomerase); F3H (flavanone 3-hydroxylase); F3′H (flavonoid 3′-hydroxylase); F3′5′H (flavonoid 3′,5′-hydroxylase); DFR (dihydroflavonol 4-reductase); ANS (anthocyanidin synthase); LAR (leucoanthocyanidin reductase); and UFGT (flavonoid 3-O-glucosyltransferase). The anthocyanin transporter MdGSTF6 was first introduced and proven to be indispensable for the biosynthesis of anthocyanins. By expressing MdGSTF6, FaDFR, PhANS0, and Dc3GT and disrupting EXG1 (the main anthocyanin-degrading enzyme), the BA-22 strain produced 261.6 mg/L (254.5 mg/L cyanidin-3-O-glucoside and 7.1 mg/L delphinidin-3-O-glucoside) anthocyanins from 2.0 g/L dihydroflavonols, which was known to be the highest titer in eukaryotes. Finally, 15.1 mg/L anthocyanins was obtained from glucose by expressing the de novo biosynthesis pathway in S. cerevisiae, which is known to be the highest de novo production. It is the first study to show that through the introduction of a plant anthocyanin transporter and knockout of a yeast endogenous anthocyanin degrading enzyme, the anthocyanin titer has been increased by more than 100 times.

花青素是广泛存在于多种植物中的天然色素。作为一类多功能天然化合物，花青素被广泛应用于食品、医药、保健品及化妆品领域。目前，花青素多在原核生物中以黄烷-3-醇为底物进行异源生物合成，但该方法成本高昂。本研究旨在通过代谢工程改造酿酒酵母（Saccharomyces cerevisiae）以实现花青素的生物合成。目前已有大量研究针对花青素天然宿主中的代谢通路酶展开解析，相关酶类包括查尔酮合酶（CHS, chalcone synthase）、黄酮醇合酶（FLS, flavonol synthase）、查尔酮异构酶（CHI, chalcone isomerase）、黄烷酮3-羟化酶（F3H, flavanone 3-hydroxylase）、黄酮类3'-羟化酶（F3'H, flavonoid 3'-hydroxylase）、黄酮类3',5'-羟化酶（F3'5'H, flavonoid 3',5'-hydroxylase）、二氢黄酮醇4-还原酶（DFR, dihydroflavonol 4-reductase）、花青素合酶（ANS, anthocyanidin synthase）、无色花青素还原酶（LAR, leucoanthocyanidin reductase）以及黄酮类3-O-葡萄糖基转移酶（UFGT, flavonoid 3-O-glucosyltransferase）。本研究首次引入花青素转运蛋白MdGSTF6，并证实其对花青素生物合成不可或缺。通过共表达MdGSTF6、FaDFR、PhANS0与Dc3GT，并敲除主要的花青素降解酶EXG1，工程菌株BA-22以2.0 g/L二氢黄酮醇为底物，可合成261.6 mg/L花青素（其中含254.5 mg/L矢车菊素-3-O-葡萄糖苷、7.1 mg/L飞燕草素-3-O-葡萄糖苷），该产量为目前已知真核生物体系中的最高滴度。最终，本研究通过在酿酒酵母中构建从头生物合成通路，以葡萄糖为底物实现了15.1 mg/L的花青素合成，该结果为目前已知的最高从头合成产量。本研究首次证实，通过引入植物来源的花青素转运蛋白并敲除酵母内源花青素降解酶，可将花青素的合成滴度提升100倍以上。