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食品与发酵工业  2021, Vol. 47 Issue (1): 1-7    DOI: 10.13995/j.cnki.11-1802/ts.024789
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
微液滴适应性进化强化大肠杆菌耐受高浓度L-山梨糖
曾伟主1,2, 单小玉1,2, 房峻1,2, 周景文1,2*
1(工业生物技术教育部重点实验室(江南大学),江苏 无锡,214122)
2(江南大学 未来食品科学中心,江苏 无锡,214122)
Enhanced tolerance of Escherichia coli to L-sorbose by microdroplet aided adaptive laboratory evolution
ZENG Weizhu1,2, SHAN Xiaoyu1,2, FANG Jun1,2, ZHOU Jingwen1,2*
1(Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi 214122, China)
2(Science Center for Future Foods, Jiangnan University, Wuxi 214122, China)
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摘要 前期研究构建了1株组成型表达山梨糖脱氢酶(sorbose dehydrogenase, SDH)的大肠杆菌工程菌,该菌株能利用L-山梨糖生产维生素C前体2-酮基-L-古龙酸(2-keto-L-gulonic acid, 2-KLG),但对底物L-山梨糖的耐受性较差。为解决这一问题,对该菌株进行适应性进化,并强化了进化菌株发酵生产2-KLG的能力。首先,应用基于微流控技术的全自动高通量微生物液滴培养系统,将出发菌株在不同浓度梯度的L-山梨糖培养基中生长、传代,获得能够耐受高浓度L-山梨糖的进化菌株。在摇瓶上进一步验证,最终获得了1株能耐受高浓度L-山梨糖的进化菌株2-F6。然后在2-F6中共表达了能促进2-酮基-L-古龙酸积累的山梨酮脱氢酶(sorbosone dehydrogenase, SNDH),并在摇瓶水平上对接种量、发酵温度、SNDH诱导时间、IPTG诱导浓度以及L-山梨糖添加量进化了优化,在最优条件下,2-KLG的产量达6.05 g/L。最终,将摇瓶发酵条件放大至5 L发酵罐后,2-KLG的产量为5.70 g/L。研究结果为一菌一步发酵法生产维生素C前体2-KLG提供了参考。
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曾伟主
单小玉
房峻
周景文
关键词:  2-酮基-L-古龙酸  大肠杆菌  适应性进化  L-山梨糖  高浓度耐受性    
Abstract: An engineered Escherichia coli constitutively expressing sorbose dehydrogenase (SDH) was previously constructed for producing the vitamin C precursor 2-keto-L-gulonic acid (2-KLG) with L-sorbose as substrate. However, this strain has a poor tolerance to L-sorbose. In order to solve this problem, adaptive laboratory evolution was carried out. Firstly, to enhance the tolerance of L-sorbose, the starting strain was grown and subcultured in the medium with different gradient concentration of L-sorbose by applying Microbial Microdroplet Culture System designed based on microfluidic technology. An evolutionary strain 2-F6 that could tolerate high concentration of L-sorbose was obtained after further verifying on the shaking flasks. Then, the sorbosone dehydrogenase (SNDH) was expressed in 2-F6 to promote the strength of 2-KLG production. The inoculation amount, fermentation temperature, induction time for SNDH, IPTG concentration and L-sorbose addition were optimized in the shaking flasks, and the titer of 2-KLG reached 6.05 g/L under the optimized optimal conditions. Finally, after scaling up in a 5 L fermenter, 2-KLG of 5.70 g/L was obtained. The results obtained could provide references for the production of vitamin C precursor 2-KLG with one-step fermentation process.
Key words:  2-keto-L-gulonic acid    Escherichia coli    adaptive laboratory evolution    L-sorbose    high concentration tolerance
收稿日期:  2020-06-18      修回日期:  2020-07-16                发布日期:  2021-02-03      期的出版日期:  2021-01-15
基金资助: 国家重点研发计划项目(2019YFA09004900);国家自然科学基金重点项目(31830068)
作者简介:  硕士,助理研究员(周景文教授为通讯作者,E-mail:zhoujw1982@jiangnan.edu.cn)
引用本文:    
曾伟主,单小玉,房峻,等. 微液滴适应性进化强化大肠杆菌耐受高浓度L-山梨糖[J]. 食品与发酵工业, 2021, 47(1): 1-7.
ZENG Weizhu,SHAN Xiaoyu,FANG Jun,et al. Enhanced tolerance of Escherichia coli to L-sorbose by microdroplet aided adaptive laboratory evolution[J]. Food and Fermentation Industries, 2021, 47(1): 1-7.
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http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.024789  或          http://sf1970.cnif.cn/CN/Y2021/V47/I1/1
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