产对香豆酸酿酒酵母菌株的构建及优化

Construction and Optimization of p-coumaric Acid Producing Saccharomyces cerevisiae

对香豆酸(p-coumaric acid)作为苯丙素类物质、芪类物质及黄酮类物质的重要前体化合物,在生物医药、化妆品及食品工业中均有广泛的应用价值。以酿酒酵母作为底盘菌株,利用合成生物学原理构建一株高产对香豆酸的人工酵母细胞。通过对比不同拷贝数的酪氨酸解氨酶(tyrosine ammonia lyase)合成的对香豆酸产量,发现随着基因拷贝数的增加对香豆酸的产量也相应提高;同时对酪氨酸的负反馈调控相关的蛋白质进行氨基酸定点突变得到Aro4pK229L和Aro7pG141S,利用delta位点将突变后的基因整合至酵母基因组,并挑取24株构建成功的酵母细胞进行发酵验证,发现菌株最高产量与最低产量相差28.87mg/L;为了进一步增加对香豆酸的代谢通量,对生成芳香醇类物质的旁路基因ARO10和PDC5进行敲除,发现同时敲除两个基因的菌株对香豆酸的产量最高,是敲除前产量的2.05倍(从42.71mg/L到87.56mg/L)。此外,通过设计前体酪氨酸的梯度添加实验,发现当添加1mmol/L的酪氨酸时,对香豆酸产量达到峰值(174.57±0.30)mg/L,相较于未添加时提高了将近1倍。通过运用合成生物学原理在酿酒酵母中实现了对香豆酸的高产,为后续的芪类化合物和黄酮类化合物生物合成奠定了基础。

p-coumaric acid is widely used in bio-pharmaceutical,cosmetics and food industry as a very important precursor compound of phenylpropanoids,stilbenes and flavonoids. Saccharomyces cerevisiae was used as the host cell to synthesize p-coumaric acid with the methods of synthetic biology. It was demonstrated that the multi-copies plasmid harbored TAL showed a larger accumulation of p-coumaric acid; meanwhile,to eliminate the feedback inhibition of L-tyrosine,specific amino acid mutation Aro4p^K229 Land Aro7p^G141S was obtained. The relevant mutated gene was integrated into yeast genome using delta site integration. 24 strains were picked out to verify the production of p-coumaric acid,the different yield between the highest strain and the lowest strain was 28. 87mg/L. To strength the metabolic flux to p-coumaric,gene ARO10 and PDC5 which involved in the biosynthesis of byproduct aromatic alcohols were knocked out. Production of p-coumaric acid in strain with two gene knock out was improved to 87. 56mg/L,2. 05-fold to the control one. Furthermore,when 1mmol/L Ltyrosine was added,the production of p-coumaric acid arrived the peak,about（ 174. 57 ± 0. 30） mg/L. The pcoumaric acid over-producing S. cerevisiae using the synthetic biology method as well as lay a foundation of the biosynthesis of subsequent stilbenes and flavonoids has been successfuly constructed.