利用基因工程改造的大肠杆菌合成蒎烯

Efficient Synthesis of Pinene by Using Genetically Engineered Escherichia coli

目的:以大肠杆菌为底盘细胞,利用合成生物学手段导入外源杂合蒎烯合成代谢途径,构建高效合成蒎烯的微生物工厂。方法:将来源于酵母和粪肠球菌的异源杂合甲羟戊酸(MVA)代谢途径和来源于北美巨冷杉的牻牛儿焦磷酸合酶(GPPS)和蒎烯合成酶(PS)基因序列共同导入大肠杆菌,构建催化蒎烯合成的大肠杆菌工程菌。首先优化合成GPPS、PS基因,再分别以pETDuet-1和pET-24a(+)载体为基础构建GPPS、PS共表达和融合表达载体,并分别转化大肠杆菌获得工程菌E.hzh01和E.hzh02,摇瓶培养后利用GC-MS技术检测E.hzh01和E.hzh02的蒎烯产量。进一步获取MVA代谢途径相关酶基因mvaE、mvaS、ERG12、ERG8、ERG19、IDI序列,分别利用多顺反子模型和Bio?Brick方法构建2种不同方案的异源MVA代谢途径,再将异源MVA代谢途径和GPPS、PS融合表达基因共同转化大肠杆菌,构建完整蒎烯合成代谢工程菌株E.hzh03和E.hzh05。结果:摇瓶培养E.hzh01和E.hzh02的蒎烯产量分别为0.85和1.86 mg/L,结果表明融合表达更有利于蒎烯的合成。E.hzh03和E.hzh05摇瓶培养得到的蒎烯产量分别为6.32和19.26 mg/L。结论:利用融合表达载体和多顺反子模型导入异源蒎烯代谢途径构建大肠杆菌工程菌,可以显著提高蒎烯的产量,为工业生物合成蒎烯奠定了一定的基础。

Objective:The microbial-host for synthetic pinene was constructed,and the heterozygous biosynthesis pathway of pinene was transformed into the chassis cell E.coli.Methods:The gene of external hybrid meva?lonic acid(MVA)pathway derived from yeast and Enterococcus faecalis and the geranyldiphosphate synthase(GPPS)and pinene synthase(PS)coexpression genes derived from the Abies grandis were transformed into the E.coli using the idea of synthetic biology.Induced their expression to construct engineering E.coli containing complete pinene metabolic pathway.Firstly,the codons of GPPS and PS were optimized and the two genes were synthesized artificially.GPPS and PS co-expression vector and fusion expression vector were constructed based on pETDuet-1 and pET-24a(+)vector respectively,and transformed them into E.coli to obtain engineering bacteria E.hzh01 and E.hzh02,then use the GC-MS technology to further detect the pinene titer.Secondly,the primers were designed according to the gene sequences of MVA metabolic pathway related enzymes(mvaE,mvaS,ERG12,ERG8,ERG19,IDI)and then cloned them respectively by PCR.Then the MVA metabolic pathways were constructed by two different protocols using the polycistronic model and the BioBrick principle method.The MVA pathway and the GPPS-PS fusion expression genes were co-transformed into E.coli was named E.hzh03 and E.hzh05.Results:The pinene yields of E.hzh01 and E.hzh02 were 0.85 and 1.86 mg/L,which showed that fusion-expression was more favorable for pinene synthesis.The pinene yields of E.hzh03 and E.hzh05 were 6.32 and 19.26 mg/L respectively.Conclusion:The yield of pinene can be significantly improved by fusion expression vector and polycistronic model to introduce MVA metabolic pathway into E.coli.The experiment results provide the fundamental data for industrial production.