基于重组氧化葡萄糖酸杆菌生物合成吡咯喹啉醌  

Synthesis of pyrroloquinoline quinone by recombinant Gluconobacter oxydans

作　　者：叶润乐 李沣 丁凡 赵振辉 陈晟 袁建锋 Runle Ye;Feng Li;Fan Ding;Zhenhui Zhao;Sheng Chen;Jianfeng Yuan(Xingzhi College,Zhejiang Normal University,Lanxi 321100,Zhejiang,China;Key Laboratory of Wildlife Biotechnology and Conservation and Utilization of Zhejiang Province,Zhejiang Normal University,Jinhua 321004,Zhejiang,China)

机构地区：[1]浙江师范大学行知学院,浙江兰溪321100 [2]浙江师范大学浙江省野生动物生物技术与保护利用重点实验室,浙江金华321004

出　　处：《生物工程学报》2020年第6期1138-1149,共12页Chinese Journal of Biotechnology

基　　金：浙江省教育厅一般项目(No.Y201840796);国家级大学生创新创业训练计划项目(No.201913276014)资助。

摘　　要：吡咯喹啉醌(Pyrroloquinoline quinone,PQQ)是一种重要的氧化还原酶辅基,具有多种生理生化功能,在食品、医药卫生及农业等领域具有广泛的应用。文中采用重组氧化葡萄糖酸杆菌生物合成吡咯喹啉醌。首先构建丙酮酸脱羧酶基因GOX1081敲除的重组菌G.oxydans T1,减少副产物乙酸的形成。然后利用筛选的内源性组成型启动子P0169融合表达pqqABCDE基因簇及tldD基因,构建重组菌G.oxydans T2。最后对发酵培养基添加物和发酵条件进行优化。结果显示重组菌G.oxydans T1、G.oxydans T2生物量较野生菌分别提高43.02%和38.76%,而PQQ的产量分别是野生菌的4.82倍和20.5倍。进一步优化G.oxydans T2碳源及培养条件,最终PQQ产量达(51.3241±0.8997)mg/L,是野生菌的345.62倍。通过基因工程手段,可以有效提高氧化葡萄糖酸杆菌的生物量和合成PQQ的产量,为改善PQQ生物合成效率奠定基础。Pyrroloquinoline quinone(PQQ),an important redox enzyme cofactor,has many physiological and biochemical functions,and is widely used in food,medicine,health and agriculture industry.In this study,PQQ production by recombinant Gluconobacter oxydans was investigated.First,to reduce the by-product of acetic acid,the recombinant strain G.oxydans T1 was constructed,in which the pyruvate decarboxylase(GOX1081)was knocked out.Then the pqqABCDE gene cluster and tldD gene were fused under the control of endogenous constitutive promoter P0169,to generate the recombinant strain G.oxydans T2.Finally,the medium composition and fermentation conditions were optimized.The biomass of G.oxydans T1 and G.oxydans T2 were increased by 43.02%and 38.76%respectively,and the PQQ production was 4.82 and 20.5 times higher than that of the wild strain,respectively.Furthermore,the carbon sources and culture conditions of G.oxydans T2 were optimized,resulting in a final PQQ yield of(51.32±0.8997 mg/L),345.6 times higher than that of the wild strain.In all,the biomass of G.oxydans and the yield of PQQ can be effectively increased by genetic engineering.

关 键 词：氧化葡萄糖酸杆菌 吡咯喹啉醌 生物合成 代谢工程 

分 类 号：Q936[生物学—微生物学]