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食品与发酵工业  2020, Vol. 46 Issue (13): 18-23    DOI: 10.13995/j.cnki.11-1802/ts.022717
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
大肠杆菌中三酶耦联合成α-酮异戊酸
李雅婷, 周丽, 周哲敏*
(江南大学 生物工程学院,江苏 无锡,214122)
Three enzyme coupling to synthesize α-ketoisovalerate in E. coli
LI Yating, ZHOU Li, ZHOU Zhemin*
(School of Biotechnology,Jiangnan University, Wuxi 214122, China)
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摘要 α-酮异戊酸是生物合成和化学合成领域中重要的中间体,在医药和化学合成领域中具有广泛的研究和应用价值。将α-酮异戊酸合成途径中的乙酰乳酸合成酶、乙酰乳酸异构还原酶、二羟基酸脱水酶3种关键酶编码基因BsalsS、EcilvC、EcilvD克隆于pETDuet-1载体上,在E. coli BL21(DE3)中加强α-酮异戊酸的合成途径,获得出发菌株SDC。进一步优化3个关键酶基因在质粒上的排列顺序,并分别在基因后添加终止子,通过比较菌株粗酶液催化丙酮酸钠底物合成α-酮异戊酸产物的能力,获得了菌株CTSDT,其α-酮异戊酸合成量比菌株SDC提高了2.28倍。菌株CTSDT以葡萄糖为碳源发酵16 h后,α-酮异戊酸产量达到1.70 g/L,是改造前菌株SDC的3.36倍。研究结果为大肠杆菌中α-酮异戊酸的高效发酵合成奠定了基础。
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李雅婷
周丽
周哲敏
关键词:  三酶耦联  α-酮异戊酸  基因排列顺序  生物合成  发酵    
Abstract: As an important intermediate product in the fields of biosynthesis and chemical synthesis, α-ketoisovalerate has been widely studied and applied in pharmaceutical and chemical synthesis field. Three key genes, BsalsS, EcilvC and EcilvD (encoding acetolactate synthase, acetolactate isomerase and dihydroxy acid dehydratase respectively), were cloned in this study into plasmid pETDuet-1 in order to enhance the synthesis of α-ketoisovalerate in E. coli BL21(DE3). And the original strain SDC were obtained. Furthermore, the orders of the three genes in plasmid were optimized to balance their expression. After comparing the co-expression result and the ability to catalyze the substrate sodium pyruvate to α-ketoisovalerate using the crude enzyme solution, the optimal strain CSD was obtained. Moreover, terminators of each gene in strain CSD to fine-tuning gene expression were added so as to get the final strain CTSDT. The α-ketoisovalerate conversion level of strain CTSDT was 2.28-fold high than that of strain SDC. Using glucose as the carbon source, strain CTSDT yielded α-ketoisovalerate of 1.70 g/L after 16 h cultivation, which was 3.36-fold higher than the original strain SDC. These results established foundation for efficient production of α-ketoisovalerate in E. coli BL21(DE3).
Key words:  three enzyme coupling    α-ketoisovalerate    gene order    biosynthesis    fermentation
收稿日期:  2019-11-05                出版日期:  2020-07-15      发布日期:  2020-08-04      期的出版日期:  2020-07-15
基金资助: 国家自然科学基金(31300087);江南大学自主科研课题重点项目(JUSRP51713B)
作者简介:  硕士研究生(周哲敏教授为通讯作者,E-mail:zhmzhou@jiangnan.edu.cn)
引用本文:    
李雅婷,周丽,周哲敏. 大肠杆菌中三酶耦联合成α-酮异戊酸[J]. 食品与发酵工业, 2020, 46(13): 18-23.
LI Yating,ZHOU Li,ZHOU Zhemin. Three enzyme coupling to synthesize α-ketoisovalerate in E. coli[J]. Food and Fermentation Industries, 2020, 46(13): 18-23.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.022717  或          http://sf1970.cnif.cn/CN/Y2020/V46/I13/18
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