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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2014, Vol. 34 Issue (12): 97-106    DOI: 10.13523/j.cb.20141214
技术与方法     
代谢工程改造Gluconobacter suboxydans生产2-酮基-D-葡萄糖酸
仪修南2, 李天明1, 王北辰3, 刘金雷1, 杜红燕1, 冯惠勇1
1. 河北科技大学 石家庄 050018;
2. 山东大学 济南 250100;
3. 西北农林科技大学 杨凌 712100
Production of 2-Keto-D-Gluconic Acid by Metabolically Engineered Gluconobacter suboxydans
YI Xiu-nan2, LI Tian-ming1, WANG Bei-chen3, LIU Jin-lei1, DU Hong-yan1, FENG Hui-yong1
1. Hebei University of Science and Technology, Shijiazhuang 050018, China;
2. ShanDong University, Jinan 250100, China;
3. Northwest University of Agriculture and Forest, Yangling 712100, China
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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42.2 g/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。
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    
Abstract:

2-keto-D-gluconic acid is an important precursor for D-isoascorbic acid and sodium D-isoascorbate, 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 by-product 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-D-gluconic acid without by-products 5-keto- gluconic acid after culturing in shake flask for 72 h and its 2-keto-D-gluconic 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.
Key words: 2-Keto-D-Gluconic Acid    Gluconobacter suboxydans    Glucose dehydrogenase    2-gluconate dehydrogenase
收稿日期: 2014-09-16 出版日期: 2014-12-25
ZTFLH:  Q789  
通讯作者: 冯惠勇     E-mail: fenghuiyong@163.com
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引用本文:

仪修南, 李天明, 王北辰, 刘金雷, 杜红燕, 冯惠勇. 代谢工程改造Gluconobacter suboxydans生产2-酮基-D-葡萄糖酸[J]. 中国生物工程杂志, 2014, 34(12): 97-106.

YI Xiu-nan, LI Tian-ming, WANG Bei-chen, LIU Jin-lei, DU Hong-yan, FENG Hui-yong. Production of 2-Keto-D-Gluconic Acid by Metabolically Engineered Gluconobacter suboxydans. China Biotechnology, 2014, 34(12): 97-106.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20141214    或    https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I12/97


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