| 大肠杆菌合成1,2,4-丁三醇的途径优化 |
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| 引用本文： | 孙雷,杨帆,朱泰承,李兴华,孙红兵,李寅,许正宏,张延平. 大肠杆菌合成1,2,4-丁三醇的途径优化[J]. 生物工程学报, 2016, 32(1): 51-63 |
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| 作者姓名： | 孙雷 杨帆 朱泰承 李兴华 孙红兵 李寅 许正宏 张延平 |
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| 作者单位： | 1 江南大学药学院，江苏 无锡 214122,2 中国科学院微生物研究所，北京 100101；3 中国科技大学生命科学院，安徽 合肥 230022,2 中国科学院微生物研究所，北京 100101,2 中国科学院微生物研究所，北京 100101,4 中国科学院天津工业生物技术研究所，天津 300308,2 中国科学院微生物研究所，北京 100101,1 江南大学药学院，江苏 无锡 214122,2 中国科学院微生物研究所，北京 100101 |
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| 基金项目： | 中国科学院知识创新工程重大项目 (No. KSCX2-EW-G-5)，北京市自然科学基金 (No. 31170039) 资助。 |
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| 摘 要： | 1,2,4-丁三醇(BT)是一种在工业中有多种用途的重要的非天然化合物。文中通过将外源基因xdh和mdlC导入大肠杆菌BW25113表达,并敲除了xylA、xylB、yagE、yjhH、yiaE和ycdW等木糖和中间产物代谢旁路基因,构建了能够将D-木糖转化为BT的重组菌株。为优化BT合成途径,针对BT合成途径中的限速步骤——3-脱氧-D-甘油-戊酮糖酸的脱羧反应,进行了新酶的筛选和评价,获得了可显著提高反应效率的新的2-酮酸脱羧酶——KivD,并构建了表达该酶的重组菌株BW-025。在此基础上,通过初步条件优化,将BT产量提高至2.38g/L;进一步调节途径中各个酶的表达量,探究了它们对BW-025合成BT的影响,最终获得了BT产量较BW-025提高了48.62%的重组菌株BW-074。
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| 关 键 词： | D-木糖，1 2 4-丁三醇，大肠杆菌，2-酮酸脱羧酶，途径优化 |
| 收稿时间： | 2015-03-25 |
Optimization of 1,2,4-butanetriol synthetic pathway in Escherichia coli |
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| Lei Sun,Fan Yang,Taicheng Zhu,Xinghua Li,Hongbing Sun,Yin Li,Zhenghong Xu and Yanping Zhang. Optimization of 1,2,4-butanetriol synthetic pathway in Escherichia coli[J]. Chinese journal of biotechnology, 2016, 32(1): 51-63 |
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| Authors: | Lei Sun Fan Yang Taicheng Zhu Xinghua Li Hongbing Sun Yin Li Zhenghong Xu Yanping Zhang |
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| Affiliation: | 1 Laboratory of Pharmaceutical Engineering, School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, Jiangsu, China,2 CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; 3 School of Life Sciences, University of Science and Technology of China, Hefei 230022, Anhui, China,2 CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China,2 CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China,4 National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China,2 CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China,1 Laboratory of Pharmaceutical Engineering, School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, Jiangsu, China and 2 CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China |
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| Abstract: | 1,2,4-Butanetriol (BT) is an important non-natural chemical with a variety of industrial applications. A recombinant Escherichia coli biosynthesizing BT from D-xylose was constructed by heterologously expressing xdh and mdlC, and knocking out competing pathway genes including xylA, xylB, yjhE, yagH and ycdW. To optimize BT synthesis pathway, the third catalytic step that catalyzes the decarboxylation reaction of 3-deoxy-D-glycero-pentulosonic acid was identified as a potential bottleneck. Consequently, 2-keto acid decarboxylases from three different microorganisms were screened, and the kivD gene from Lactococcus lactis was found to increase BT titer by 191%. The improved strain BW-025 reached a final BT titer of 2.38 g/L under optimized transformation conditions. Attempts on synthetic pathway optimization were also made by fine-tuning the expression levels of each enzyme involved in the whole pathway based on BW-025. As a result, an xdh overexpressed recombinant strain, BW-074 was finally generated, with 48.62% higher BT production than that of BW-025. |
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| Keywords: | D-xylose 1 2 4-butanetriol Escherichia coli 2-keto acid decarboxylase pathway optimization |
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