产对香豆酸酿酒酵母工程菌株的构建与优化

Construction and optimization of p-coumaric acid-producing Saccharomyces cerevisiae

摘要:

对香豆酸是黄酮类、芪类等天然活性化合物的重要前体，在生物医药、食品等行业应用广泛。与传统植物提取和化学合成相比，微生物合成对香豆酸因其具有生产周期短、转化效率高等优势而得到广泛关注。为构建高产对香豆酸酵母工程菌株，以酿酒酵母为出发菌，通过敲除酪氨酸合成竞争路径基因ARO10和PDC5，突变芳香族氨基酸合成调控基因ARO4K229L与ARO7G141S、解除酪氨酸负反馈抑制、并整合酪氨酸解氨酶FjTAL，获得的工程菌C001对香豆酸产量为296.73 mg/L。为进一步提高对香豆酸合成前体积累，分别敲除8个与氨基酸、糖类等转运相关基因并强化糖异生途径，分析其对对香豆酸积累的影响。结果表明，敲除GAL2及过表达EcppsA，对香豆酸产量提高至475.11 mg/L。最后，分析了FjTAL蛋白锚定至酵母液泡对产物积累的影响，结果表明其定位液泡后对香豆酸产量明显提升，达到593.04 mg/L。通过强化前体物供应，阻断竞争旁路途径，利用亚细胞定位等策略有效提高对香豆酸产量，为后续黄酮类及芪类化合物的合成提供高效平台菌株，具有重要的应用前景。

Abstract:

p-Coumaric acid is an important precursor of various natural compounds, such as flavonoids and stilbenes. It has been widely used in biomedicine, food, nutrition and health care industries. Compared with traditional plant extracts and chemical synthesis, microbial synthesis of natural compounds such as p-coumaric acid has attracted wide attention due to its short production cycle and high conversion efficiency. Here a p-coumaric acid-producing Saccharomyces cerevisiae platform strain was developed. First, the tyrosine synthesis competition pathway genes ARO10 and PDC5 were knocked out, and ARO4K229L and ARO7G141S were mutated to release negative feedback inhibition from tyrosine. The tyrosine ammonia-lyase coding gene TAL from Flavobacterium johnsoniaeu was then integrated into genome and obtained C001 with yield of p-coumaric acid 296.73 mg/L. To further increase the accumulation of p-coumaric acid precursors, 8 genes encoding amino acids and carbohydrate transporters were knocked out and the gluconeogenesis pathway was enhanced. The results showed that GAL2 knockout and overexpression of EcppsA increased the yield of p-coumaric acid to 475.11 mg/L. Finally, the effect of FjTAL anchoring to yeast vacuoles on product accumulation was analyzed, and the highest titer of p-coumaric acid of 593.04 mg/L was obtained after intracellular vacuolar localization of FjTAL. It provided an efficient p-coumaric acid-producing platform strain for the subsequent synthesis of flavonoids and stilbene compounds by enhancing the supply of precursors, blocking the competitive bypass pathway, and using the strategy of subcellular localization.