代谢工程改造热带假丝酵母生产鼠尾草酸  

Metabolic engineering of Candida tropicalis for carnosic acid production

作　　者：霍达 陈献忠[1] 杨海泉[1] 曹钰[1] HUO Da;CHEN Xianzhong;YANG Haiquan;CAO Yu(School of Biotechnology,Key Laboratory of Industrial Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《食品与发酵工业》2024年第3期45-51,I0002,I0003,共9页Food and Fermentation Industries

摘　　要：鼠尾草酸(carnosic acid,CA)是一种酚类二萜化合物,广泛存在于鼠尾草等唇科类植物中。CA具有抗肿瘤等多种生理以及药理活性,在日化、食品、医疗领域应用价值巨大。热带假丝酵母具有鲁棒性强、耐高温、产油脂等特性,因而在萜类化合物高效生产方面极具潜力。该研究根据热带假丝酵母密码子偏好性对来源于鼠尾草的铁锈醇合酶基因CYP76AH24和鼠尾草酸合酶基因CYP76AK6、来源于拟南芥的细胞色素还原酶基因ATR1进行全序列密码子优化以及基因合成。以生产鼠尾草酸前体次丹参酮二烯热带假丝酵母1C2PM04作为出发菌株,将上述基因整合到其基因组中成功合成鼠尾草酸。利用模块化工程与酵母不同亚细胞区室的空间组合,将上述3条基因以模块化的方式导入胞质和过氧化物酶体中进行表达,鼠尾草酸产量分别为10.25 mg/L和4.64 mg/L。通过下调角鲨烯合酶基因ERG9,降低法尼基焦磷酸池的消耗进而改变代谢通量进一步提高鼠尾草酸产量,效价达到26.3 mg/L。将细胞色素P450酶和细胞色素还原酶进行融合表达,测试以刚性linker(E3AK,E3AK×2,E3AK×3)和柔性linker(G4S,G4S×2,G4S×3)为连接肽,融合表达CYP76AH24和ATR1,鼠尾草酸产量提高1.54倍。该文通过增加CPR拷贝数,增加电子供体,进一步提高鼠尾草酸表达水平,产量达到78.24 mg/L。Carnosic acid(CA)is a phenolic diterpene compound,which is widely present in rosemary,sage and other labiaceae plants.Since CA has various physiological and pharmacological activities,it can be applied in the area of daily chemical,food,and medicine.In the biosynthesis of terpenoids,Candida tropicalis is a highly efficient production platform,because of its strong robustness,high temperature resistance,oil production and other properties.In this study,according to the codon preference and specificity of C.tropicalis,the whole sequence codon optimization and gene synthesis of the ferruginol synthase genes CYP76AH24 and the CA synthase CYP76AK6 from sage and the cytochrome reductase ATR1(CPR)from Arabidopsis were carried out.These genes were integrated into the genome of C.tropicalis 1C2PM04,which produces miltiradiene,the precursor of CA,and CA was successfully synthesized.Using modular engineering and the spatial combination of different subcellular compartments of yeast,the above three genes were introduced into the cytoplasm and peroxisome,and the CA production was 10.25 and 4.64 mg/L,respectively.By down-regulating squalene synthase ERG9,the consumption of farnesyl pyrophosphate pool was reduced and the metabolic flux was changed to further improve the CA production,and the titer reached 26.3 mg/L.Through the strategy of fusion expression of cytochrome P450 enzyme and cytochrome reductase,the rigid linker(E3AK,E3AK×2,E3AK×3)and flexible linker(G4S,G4S×2,G4S×3)were used as linker peptides to express CYP76AH24 and ATR1.The yield of CA increased by 1.54 times.By increasing the copy number of CPR and increasing the electron donor,the CA expression level was further improved,and the yield reached 78.24 mg/L.

关 键 词：热带假丝酵母 鼠尾草酸 细胞色素P450 融合蛋白 代谢工程 

分 类 号：TQ920.6[轻工技术与工程—发酵工程]