代谢工程改造Gluconobacter suboxydans生产2-酮基-D-葡萄糖酸  

Production of 2-Keto-D-Gluconic Acid by Metabolically Engineered Gluconobacter suboxydans

作　　者：仪修南 李天明[2] 王北辰[3] 刘金雷[2] 杜红燕[2] 冯惠勇[2] 

机构地区：[1]山东大学,济南250100 [2]河北科技大学,石家庄050018 [3]西北农林科技大学,杨凌712100

出　　处：《中国生物工程杂志》2014年第12期97-106,共10页China Biotechnology

摘　　要：2-酮基-D-葡萄糖酸是重要的抗氧化剂和食品添加剂——D-异抗坏血酸的重要前体。弱氧化葡糖酸杆菌(Gluconobacter suboxydans)具有丰富的周质空间氧化还原酶类,可将葡萄糖氧化为葡萄糖酸再氧化为2-酮基-D-葡萄糖酸。以提高2-酮基-D-葡萄糖酸的产量和减少副产物为目标,采用同源重组染色体修饰策略,将编码甘油脱氢酶的基因gldh置换为编码葡萄糖脱氢酶的基因gdh,将编码山梨醇脱氢酶的基因sdh置换为编码2-酮-D-葡萄糖酸脱氢酶的基因ga-2-dh。经PCR、酶活性显色及发酵产物HPLC检测验证表明:构建的工程菌株gdh和ga-2-dh基因被强化而gldh和sdh被敲除;使用10%的葡萄糖复合培养基,摇瓶发酵72h,工程菌2KGA3发酵液中没有副产物5-酮基-葡萄糖酸,2-酮基-D-葡萄糖酸的含量终浓度达到72.3 g/L,比野生菌株提高42.2g/L,工程菌和野生菌的2-D-KGA质量转化率分别为72.3%和30.1%,工程菌比野生菌提高1.4倍。构建获得的工程菌,不需要外加抗生素,可以保持稳定遗传,对于工业化规模生产具有一定优势,为获得可产业化显示的优势遗传资源打下了基础。2-keto-D-gluconic acid is an important precursor for D-isoascorbic acid and sodium Disoascorbate,a significant antioxidant and food additive. Gluconobacter suboxydans has lots of oxidases in the periplasmic space and can be used to produce 2-keto-D-gluconic acid through two-step oxidation-reduction reactions： the oxidation from glucose to gluconic acid,catalyzed by the glucose dehydrogenase and the oxidation from gluconic acid to 2-keto-D-gluconic acid,catalyzed by the 2-gluconate dehydrogenas. To inactivate other byproduct forming pathways and improving the production of 2-keto-D-gluconic acid,a genetically stable strain by integrating glucose dehydrogenase（ gdh） and 2-gluconate dehydrogenase（ ga-2-dh） genes into the chromosome of Gluconobacter suboxydans to replace glycerol dehydrogenase（ gldh） and sorbitol dehydrogenase（ sdh） gene respectively was constructed. By the verifying PCR,color reaction and HPLC,the characteristics of engineered strain indicated that gdh and ga-2-dh gene were strengthened and gldh and sdh gene were knocked out. Using compound medium with 10% glucose as carbon sources,the engineered strain could produce 72. 3 g / L 2-keto-Dgluconic acid without by-products 5-keto- gluconic acid after culturing in shake flask for 72 h and its 2-keto-Dgluconic acid production increased 1. 4 fold comparing to J12 wild type. Genetically stable strains constructed can metabolize glucose to produce2-keto-D-gluconic acid without the addition of antibiotics which is favorable for the industrial production.

关 键 词：2-酮基-D-葡萄糖酸 弱氧化葡萄糖酸杆菌 葡萄糖氧化酶 2-酮-D-葡萄糖酸脱氢酶 

分 类 号：Q789[生物学—分子生物学]