麦角硫因是一种普遍存在于高等动植物体内的天然抗氧化剂,具有减少细胞氧化损伤、抗炎和治疗相关疾病的能力,被广泛应用于食品、化妆品和医药行业。与传统的植物提取法及化学合成法相比,通过微生物合成法制备麦角硫因具有周期短、成本低和产量高等优势。该研究以酿酒酵母Saccharomyces cerevisiae CEN.PK113-5D为出发菌株,利用CRISPR/Cas9系统向其基因组中引入异源酶基因,构建具有完整麦角硫因生物合成途径的酿酒酵母工程菌。为进一步提高其产量,过表达组氨酸特异性转运蛋白Hip1p后,在仅有0.5 mmol/L组氨酸供给条件下,菌株胞内游离组氨酸水平显著提高,且麦角硫因产量提高10%。最终经摇瓶发酵72 h产量达(6.88±0.11) mg/L,而在5 L罐补料分批发酵策略下,培养72 h后麦角硫因产量达237.34 mg/L。该研究为后续代谢工程改造产麦角硫因酿酒酵母工程菌株提供靶基因参考。
Ergothioneine is a natural antioxidant that exists in higher animals and plants.It has the ability in reducing cell oxidative damage, anti-inflammation and treating related diseases, and it has been widely used in food, cosmetics and pharmaceutical industries.Compared with traditional plant extraction methods and chemical synthesis methods, the preparation of ergothioneine by microbial synthesis method has the advantages of short cycle, low cost and high yield.Here, Saccharomyces cerevisiae CEN.PK113-5D was used as the original strain, and CRISPR/Cas9 system was used to introduce heterologous genes into its genome to construct engineered strain with complete ergothioneine biosynthesis pathway.In order to further increase the yield of ergothioneine, the overexpression of the histidine specific transporter Hip1p showed that the yield of ergothioneine was increased by 10% under the condition of only 0.5 mmol/L histidine supply, and the final yield reached (6.88±0.11) mg/L after 72 h shake-flask fermentation.Besides, the intracellular free histidine level of the strain was significantly increased.Under the fed-batch fermentation strategy in a 5 L bioreactor, the yield of ergothioneine reached 237.34 mg/L through fermentation for 72 h.This study provided target gene reference for subsequent metabolic engineering modification of engineered strain for production of ergothioneine.
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