摘要
为了利用合成生物学技术高效制备黄芩素,该研究以合成白杨素的酿酒酵母(Saccharomyces cerevisiae)合成底盘细胞Ch-9为基础构建黄芩素合成途径,将来自拟南芥的辅酶A连接酶(4CL)突变体4AT及同工酶CLL-7基因,整合到产生肉桂酸脱羧酶的基因PAD1位点,通过同源重组实现基因置换敲除,菌株BA-C2-1摇瓶发酵白杨素产量为40.21 mg/L。将来自苹果的烯脂酰还原酶基因MdECR整合替换至酿酒酵母中DBR基因位点,强化表达乙酰辅酶A合成酶的基因ACS来增强乙酰辅酶A的供应。结果表明,重组酵母菌株BA-C3-4摇瓶发酵白杨素产量为70.25 mg/L,比改造前的底盘细胞Ch-9提高7.2倍;在通过强化表达ACS基因,增强乙酰辅酶A后,菌株BA-C4-22摇瓶发酵白杨素产量为125.04 mg/L,对比未强化表达ACS基因的实验组提高了1.78倍;将BA-C4-22菌株整合SbaiCYP82D4基因以及ATR2基因,使合成终点延伸到黄芩素,其中菌株BA-B1-5发酵液黄芩素的产量为70.94 mg/L。
In order to efficiently prepare baicalein using synthetic biology techniques,a synthesis pathway of baicalein was constructed based on the synthetic chassis cell Ch-9 from Saccharomyces cerevisiae,which synthesizes chrysin.The coenzyme A ligase(4CL)mutant 4AT and the homologous enzyme CLL-7 gene from the Arabidopsis thaliana were integrated into the PAD1 gene site of the cinnamic acid decarboxylase.Gene substitution knockout was achieved through homologous recombination,and the yield of chrysin in shake flask fermentation of strain BA-C2-1 was 40.21 mg/L.The alkenyl reductase gene MdECR from apples was integrated and replaced into DBR gene site in S.cerevisiae,and enhance the expression of the ACS gene for acetyl CoA synthase to increase the supply of acetyl CoA.The results showed that the yield of chrysin by shake flask fermentation of recombinant yeast strain BA-C3-4 was 70.25 mg/L,which was 7.2 times higher than that of the original chassis cell Ch-9;After enhancing the expres-sion of ACS gene and acetyl CoA,the yeast strain BA-C4-22 produced chrysin 125.04 mg/L in shake flask fermentation,which was 1.78 times higher than the experimental group without enhanced expression of ACS gene;Finally,the BA-C4-22 strain was integrated with the SbaiCYP82D4 gene and ART2 gene,to extend the synthesis endpoint to baicalein,the yield of baicalein in fermentation broth of strain BA-B1-5 was 70.94 mg/L.
作者
王舜
王鉴
汪方奕
于潇洁
江会峰
何如怡
田宇
刘志国
WANG Shun;WANG Jian;WANG Fangyi;YU Xiaojie;JIANG Huifeng;HE Ruyi;TIAN Yu;LIU Zhiguo(College of Biopharmaceuticals and Engineering,Wuhan Polytechnic University,Wuhan 430040,China;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China)
出处
《中国酿造》
北大核心
2025年第4期112-119,共8页
China Brewing
基金
天津市重大科技攻关项目(TSBICIP-KJGG-010)。
关键词
白杨素
黄芩素
酿酒酵母改造
异源合成
chrysin
baicalein
Saccharomyces cerevisiae modification
heterologous synthesis
