齐墩果酸因其在保肝、抗炎和抗肿瘤等方面具有突出的生物活性,已被广泛应用于医药领域。目前,齐墩果酸植物提取法效率低,微生物合成法受途径中异源细胞色素P450酶表达水平低和电子传递不平衡等限制,导致齐墩果酸生产无法满足市场需求。该研究以实验室保存的一株敲除GAL80的酿酒酵母菌株出发,构建了齐墩果酸前体物质β-香树脂醇的合成路径,并通过对甲羟戊酸途径及角鲨烯合成途径关键基因进行过量表达,得到一株β-香树脂醇产量为125.8 mg/L的菌株。以该菌株为底盘,导入适配性较高的产齐墩果酸的CYP716A12和ATR1基因,成功构建了齐墩果酸合成途径。通过平衡途径中CYP716A12和ATR1的表达,发现高的CYP716A12∶ATR1表达比率有利于提高齐墩果酸产量。结合增强乙酰辅酶A供应和NADPH再生,齐墩果酸摇瓶水平和5 L-发酵罐水平产量分别达到304.0 mg/L和680.8 mg/L,为目前报道最高产量。该研究为利用酿酒酵母合成齐墩果酸和下游产物相关研究奠定了基础。
Oleanolic acid has been widely used in the pharmaceutical field, based on its outstanding biological activities in liver protection, anti-inflammatory, and anti-tumor aspects.At present, the production of oleanolic acid cannot meet the market demand, because of the low efficiency of the plant extraction method for oleanolic acid, and limitation of the microbial synthesis method by the low expression levels of heterologous P450 enzymes in the pathway and imbalanced electron transfer.In this study, based on a strain of Saccharomyces cerevisiae with GAL80 knocked out that preserved in the laboratory, the synthetic pathway of b-amyrin that a precursor of oleanolic acid was constructed.By further overexpressing key genes in the MVA pathway and squalene synthesis pathway, the accumulation of b-amyrin reached 125.75 mg/L.After introducing the CYP716A12 and ATR1 with high adaptability for producing oleanolic acid, the oleanolic acid synthesis pathway was successfully constructed.Then, the strategy of balancing the expression of CYP716A12 and ATR1 was implemented, results presented that a high expression ratio of CYP716A12 to ATR1 is beneficial for improving the production of oleanolic acid.By increasing the cytosolic acetyl-CoA level and strengthening the NADPH regeneration system, the oleanolic acid titer was increased to 303.98 mg/L in a shake flask and to 680.8 mg/L in a 5 L-fermenter, respectively, which is the highest oleanolic acid titer reported to date.This study laid a foundation for the synthesis of oleanolic acid and its downstream products by saccharomyces cerevisiae.
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