Zhang Siqi, Zhou Jingwen, Zhang Guoqiang, Chen Jian
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, Jiangsu, China.
School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2020 Sep 25;36(9):1838-1848. doi: 10.13345/j.cjb.200003.
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 ARO4(K229L) and ARO7(G141S) 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.
对香豆酸是多种天然化合物的重要前体，如类黄酮和芪类化合物。它已广泛应用于生物医药、食品、营养和保健行业。与传统的植物提取物和化学合成相比，微生物合成对香豆酸等天然化合物因其生产周期短、转化效率高而备受关注。在此开发了一种产对香豆酸的酿酒酵母平台菌株。首先，敲除酪氨酸合成竞争途径基因ARO10和PDC5，并将ARO4(K229L)和ARO7(G141S)进行突变以解除酪氨酸的负反馈抑制。然后将来自约氏黄杆菌的酪氨酸氨裂解酶编码基因TAL整合到基因组中，获得对香豆酸产量为296.73 mg/L的C001。为进一步提高对香豆酸前体的积累，敲除了8个编码氨基酸和碳水化合物转运蛋白的基因，并增强了糖异生途径。结果表明，敲除GAL2并过表达EcppsA可使对香豆酸产量提高到475.11 mg/L。最后，分析了FjTAL锚定到酵母液泡对产物积累的影响，在FjTAL进行细胞内液泡定位后，获得了最高效价为593.04 mg/L的对香豆酸。通过增强前体供应、阻断竞争旁路途径以及采用亚细胞定位策略，为后续类黄酮和芪类化合物的合成提供了一种高效的产对香豆酸平台菌株。
