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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  2018, Vol. 38 Issue (9): 81-87    DOI: 10.13523/j.cb.20180912
综述     
大肠杆菌丁醇耐受机制及耐受菌选育研究进展 *
贺雪婷1,2,张敏华2,3,4,洪解放2,3,马媛媛2,3,**()
1 天津大学化工学院 天津 300072
2 天津大学石油化工技术开发中心 天津 300072
3 天津大学绿色合成与转化教育部重点实验室 天津 300072
4 天津大学内燃机燃烧学国家重点实验室 天津 300072
Research Progress on Butanol-Tolerant Strain and Tolerance Mechanism of Escherichia coli
Xue-ting HE1,2,Min-hua ZHANG2,3,4,Jie-fang HONG2,3,Yuan-yuan MA2,3,**()
1 School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
2 Tianjin R&D Center for Petrochemical Technology, Tianjin University, Tianjin 300072, China;
3 Key Laboratory for Green Chemical Technology of Ministry of Education, Tianjin University, Tianjin 300072, China
4 State Key Laboratory of Engines, Tianjin University, Tianjin 300072, China
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摘要：

随着全球变暖和能源危机日益加剧,生物丁醇因能用作清洁能源和重要化学品而备受关注。大肠杆菌(Escherichia coli)由于具有优良的遗传操作性能成为丁醇生产的底盘菌,但丁醇对细胞的毒害作用已成为提高工程菌丁醇产量的瓶颈,因而增强E. coli丁醇耐受性是提高工程菌丁醇产量的必要前提。为此,需要详细了解E. coli丁醇耐受机制。丁醇可破坏细胞膜的屏障作用、扰乱物质转运和传递功能,细胞产生与热激、渗透等胁迫类似的生理应答反应,通过转录与翻译调节应答丁醇胁迫。从上述几个方面综述了E. coli丁醇耐受机制,并总结了运用基因工程理性设计获得丁醇耐受菌株的研究进展。然而目前丁醇耐受机制尚未完全揭示,限制了理性设计策略的应用,因此概括了运用定向进化获得耐受丁醇菌株并解析丁醇耐受功能基因的反向代谢工程策略在此方面的研究进展。同时也关注和评述了最新的组合策略、化学修饰方法提高E. coli丁醇耐受性的研究。最后总结和展望了提高底盘菌株E. coli丁醇耐受性的关键策略。
关键词： 大肠杆菌;  丁醇耐受;  基因工程;  定向进化    
Abstract:

Biobutanol has been attracting much attention as a clean fuel and chemical due to that the use of fossil fuels lead to aggravation of global warming and energy crisis. Escherichia coli is an ideal candidate for butanol production because it is easy to manipulate genetically. Butanol toxicity has been a bottleneck for industrial-scale biobutanol production, so the improvement in butanol tolerance is essential for high titer butanol production. Butanol destroyed the barrier and transport functions of cell membrane, and cell produces physiological response, which is similar to that of heat shock, osmotic stress, etc. Cell regulates transcription and translation to resist butanol stress. In the light of the above points, the butanol tolerance mechanism of E. coli and recent advances in development of butanol-tolerant strains by rational design strategy are summarized in this review. Nevertheless, the mechanism has not been yet fully elucidated, which limits the use of rational design strategy. There is also concern about the application of inverse metabolic engineering in this area, which means that the butanol-tolerant strains are obtained through directed evolution and the functional genes are further revealed. In addition, the progress on application of the latest strategies for improving butanol tolerance, such as combined strategy, chemical modification, and propose the potential key points for enhancing butanol tolerance of E. coli were reviewed.
Key words: Escherichia coli    Butanol tolerance    Genetic engineering    Directed evolution
收稿日期: 2018-03-30 出版日期: 2018-10-12
基金资助: * 国家自然科学基金(NSFC 30900033);天津市自然科学基金(18JCYBJC24200)
通讯作者: 马媛媛     E-mail: myy@tju.edu.cn
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引用本文:

贺雪婷,张敏华,洪解放,马媛媛. 大肠杆菌丁醇耐受机制及耐受菌选育研究进展 *[J]. 中国生物工程杂志, 2018, 38(9): 81-87.

Xue-ting HE,Min-hua ZHANG,Jie-fang HONG,Yuan-yuan MA. Research Progress on Butanol-Tolerant Strain and Tolerance Mechanism of Escherichia coli. China Biotechnology, 2018, 38(9): 81-87.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180912    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I9/81

机制 方法 工程菌株丁醇耐受性 文献
改变膜的生理
特性或转运
系统的特性		 过表达顺反异构酶基因cti 0.6%(V/V)丁醇条件下的生长速率比对照菌高16% [14]
外排泵基因acrB的定向进化 0.7%(V/V)丁醇条件下的生长速率比对照菌高25% [15]
构建Pgntk-acrBv2负反馈调节系统 0.7%(V/V)丁醇条件下的最大细胞密度是对照菌的1.4倍 [16]
同时过表达脂肪酸合成基因、铁转运相关蛋白基因feoA及流泵基因srpABC 1%、1.5%、2%(V/V)丁醇条件下的生长比对照菌分别提高了4倍、5倍、9倍 [17]
启动胁迫类似
生理应答		 过表达E. coli菌株的热激蛋白基因groESL 0.75%(V/V)丁醇、1.25% (V/V) 2-丁醇、20%(V/V)丁三醇条件下的细胞活力比对照菌株分别增加2.8倍、3倍、4倍 [18]
过表达丙酮丁醇梭菌的groESL基因 0.8%、1%(V/V)丁醇条件下生长比对照菌分别高27%、9%;相对耐受性(RT%)1)比对照菌分别提高了58%、56% [19]
同时过表达grpE、groESL、clpB基因 1%(V/V)丁醇条件下的CFU数为野生型菌株的3.9倍 [20]
过表达罗非鱼的金属硫蛋白TMT清除活性氧簇 丁醇耐受性提高至1.5%(V/V) [21]
调节胞内转录 下调转录调节因子Fur 丁醇耐受性提高 [22]
表1  基因工程策略提高E. coli丁醇耐受性
策略 方法 丁醇耐受性 文献
基因组工程 适应性进化和质子照射 0.9%(V/V)丁醇条件下细胞的最大OD600从1.5提高到4 [7]
构建基因组DNA文库 最大生长速率比对照菌株增加超过100%;0.9%和1.1%(V/V)丁醇条件下最大OD600分别增加62%和13% [26]
适应性进化 获得的菌株可耐受13g/L的丁醇 [32]
适应性进化 全基因组测序和功能鉴定发现acrA,、gatY、 tnaA、yhbJ和marCRAB的突变对耐受性的提高具有显著的作用,同时缺失这5个基因获得的突变株在6g/L丁醇下的生长高于对照菌株 [33]
适应性培养 除可耐受辛酸外,还可耐受丁醇和异丁醇,在0.6%(V/V)丁醇条件下的比生长速率比对照菌高15% [34]
实时可视性进化和基因组改组技术 2%(V/V)丁醇存在下存活率增加10~100倍 [18]
转录工程 构建RNA聚合酶σ70亚基突变文库 突变株B8可耐受2%(V/V)的丁醇 [27]
构建RNA聚合酶的α亚基突变文库 0.75%和0.9%(V/V)丁醇条件下的耐受性约为野生型菌株2倍 [28]
构建人工转录因子(ATFs)文库 筛选到的菌株BT能够耐受1.5%(V/V)的丁醇 [29]
构建cAMP-CRP复合物,用ePCR和DNA改组技术获得突变文库 筛选到的菌株能耐受 1.5% (V/V) 的丁醇,且具有热抵抗力的增加 [35]
构建外源转录因子irrE基因的随机突变文库 对丁醇耐受性提高了10~100倍 [30]
组合策略 构建人工转录因子文库(ATFs)并过表达脂肪酸合成基因fabDx3和铁转运相关蛋白基因feoA 构建ATFs筛选到的菌株BT能耐受1.5%(V/V)丁醇;过表达上述三类基因后在1%、1.5%、2%(V/V)丁醇条件下的生长提高了4倍、5倍、9倍 [14,29]
化学修饰 固定插膜分子COE1-5C 3.5%(V/V)丁醇条件下比生长率从0.032/h提高到0.094/h [36]
表2  定向进化、组合策略或化学修饰提高丁醇耐受性
[1] 王庆龙, 刘莉, 史吉平 , 等. 丁醇基因在大肠杆菌中表达的现状与展望. 中国生物工程杂志, 2014,34(6):90-97.
doi: 10.13523/j.cb.20140613
Wang Q L, Liu L, Shi J P , et al. Current status and prospects of the expression of butanol pathway in Escherichia coli. China Biotechnology, 2014,34(6):90-97.
doi: 10.13523/j.cb.20140613
[2] 刘娅, 刘宏娟, 张建安 , 等. 新型生物燃料——丁醇的研究进展. 现代化工, 2008,28(6):28-31,33.
Liu Y, Liu H J, Zhang J A , et al. Research progress in new biofuel butanol. Modern Chemical Industry, 2008,28(6):28-31,33.
[3] Jiang Y, Liu J, Jiang W , et al. Current status and prospects of industrial bio-production of n-butanol in China. Biotechnol Adv, 2015,33(7):1495-1501.
[4] Dong H, Zhao C, Zhang T , et al. Engineering Escherichia coli cell factories for n-butanol production. Adv Biochem Eng Biotechnol, 2016,155:141-163.
[5] Liu S, Qureshi N, Hughes S R . Progress and perspectives on improving butanol tolerance. World J Microbiol Biotechnol, 2017,33(3):51.
doi: 10.1007/s11274-017-2220-y
[6] 戴宗杰, 董红军, 朱岩 , 等. 生物丁醇代谢工程的研究进展. 生物加工过程, 2013,11(2):58-64.
Dai Z J, Dong H J, Zhu Y , et al. Metabolic engineering for biobutanol production: a review. Chinese Journal of Bioprocess Engineering, 2013,11(2):58-64.
[7] Jeong H, Lee S W, Kim S H , et al. Global functional analysis of butanol-sensitive Escherichia coli and its evolved butanol-tolerant strain. J Microbiol Biotechnol, 2017,27(6):1171-1179.
[8] Shen C R, Lan E I, Dekishima Y , et al. Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli. Appl Environ Microbiol, 2011,77(9):2905-2915.
doi: 10.1128/AEM.03034-10
[9] Ohtake T, Pontrelli S, Laviña W A , et al. Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli. Meta Eng, 2017,41:135-143.
doi: 10.1016/j.ymben.2017.04.003
[10] Dong H, Zhao C, Zhang T , et al. A systematically chromosomally engineered Escherichia coli efficiently produces butanol. Meta Eng, 2017,44:284-292.
doi: 10.1016/j.ymben.2017.10.014
[11] Jang Y S, Lee S Y . Recent advances in biobutanol production. Industrial Biotechnol, 2015,11(6):316-321.
doi: 10.1089/ind.2015.0023
[12] Knoshau E P, Zhang M , Butanol tolerance in a selection of microorganisms. Appl Biochem Bioyechnol, 2009,153(1-3):13-20.
doi: 10.1007/s12010-008-8460-4
[13] Rau M H, Calero P, Lennen R M , et al. Genome-wide Escherichia coli stress response and improved tolerance towards industrially relevant chemicals. Microb Cell Fact, 2016,15(1):176.
doi: 10.1186/s12934-016-0577-5
[14] Tan Z, Yoon J M, Nielsen D R , et al. Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables. Meta Eng, 2016,35:105-113.
doi: 10.1016/j.ymben.2016.02.004
[15] Fisher M A, Boyarskiy S, Yamada M R , et al. Enhancing tolerance to short-chain alcohols by engineering the Escherichia coli AcrB efflux pump to secrete the non-native substrate n-butanol. ACS Synth Biol, 2014,3(1):30-40.
doi: 10.1021/sb400065q
[16] Boyarskiy S, López S D, Kong N , et al. Transcriptional feedback regulation of efflux protein expression for increased tolerance to and production of n-butanol. Meta Eng, 2016,33(1):130-137.
doi: 10.1016/j.ymben.2015.11.005
[17] Bui L M, Lee J Y, Geraldi A , et al. Improved n-butanol tolerance in Escherichia coli by controlling membrane related functions. J Biotechnol, 2015,204:33-44.
doi: 10.1016/j.jbiotec.2015.03.025
[18] Zingaro K A, Papoutsakis E T . GroESL overexpression imparts Escherichia coli tolerance to i-, n-, and 2-butanol, 1,2,4-butanetriol and ethanol with complex and unpredictable patterns. Meta Eng, 2013,15(1):196-205.
doi: 10.1016/j.ymben.2012.07.009
[19] Abdelaal A S, Ageez A M , Abd EI-Hadi A E , et al. Genetic improvement of n-butanol tolerance in Escherichia coli by heterologous overexpression of groESL operon from Clostridium acetobutylicum. 3 Biotech, 2015,5(4):401-410.
[20] Zingaro K A, Papoutsakis E T . Toward a semisynthetic stress response system to engineer microbial solvent tolerance. mBio, 2012,3(5):e00308-312.
[21] Chin W C, Lin K H, Chang J J , et al. Improvement of n-butanol tolerance in Escherichia coli by membrane-targeted tilapia metallothionein. Biotechnol Biofuels, 2013,6(1):130.
doi: 10.1186/1754-6834-6-130
[22] Sandoval N R, Papoutsakis E T . Engineering membrane and cell-wall programs for tolerance to toxic chemicals: Beyond solo genes. Curr Opin Microbial, 2016,33:56-66.
doi: 10.1016/j.mib.2016.06.005
[23] Reyes L H, Almario M P, Winkler J , et al. Visualizing evolution in real time to determine the molecular mechanisms of n-butanol tolerance in Escherichia coli. Meta Eng, 2012,14(5):579-590.
doi: 10.1016/j.ymben.2012.05.002
[24] 冯言, 刘马峰, 程安春 . 革兰氏阴性菌亚铁离子转运系统的组成及作用机制. 微生物学报, 2016,56(7):1061-1069.
doi: 10.13343/j.cnki.wsxb.20150403
Feng Y, Liu M F, Cheng A C . Component and functional mechanism of the ferrous iron acquisition system in gram-negative bacteria-a review. Acta Microbiologica Sinica, 2016,56(7):1061-1069.
doi: 10.13343/j.cnki.wsxb.20150403
[25] Jones D P . Radical-free biology of oxidative stress. Am J Physiol Cell Physiol, 2008,295(4):C849-C868.
doi: 10.1152/ajpcell.00283.2008
[26] Freedman B G, Zu T N, Wallace R S , et al. Raman spectroscopy detects phenotypic differences among Escherichia coli enriched for 1-butanol tolerance using a metagenomic DNA library. Biotechnol J, 2016,11(7):877-889.
doi: 10.1002/biot.v11.7
[27] Si H M, Zhang F, Wu A N , et al. DNA microarray of global transcription factor mutant reveals membrane-related proteins involved in n-butanol tolerance in Escherichia coli. Biotechnol Biofuels, 2016,9(1):114.
doi: 10.1186/s13068-016-0527-9
[28] Klein-Marcuschamer D, Santos C N, Yu H , et al. Mutagenesis of the bacterial RNA polymerase alpha subunit for improvement of complex phenotypes. Appl Environ Microbiol, 2009,75(9):2705-2711.
doi: 10.1128/AEM.01888-08
[29] Lee J Y, Yang K S, Jang S A , et al. Engineering butanol-tolerance in Escherichia coli with artificial transcription factor libraries. Biotechnol Bioeng, 2011,108(4):742-749.
doi: 10.1002/bit.22989
[30] Chen T, Wang J, Yang R , et al. Laboratory-evolved mutants of an exogenous global regulator, IrrE from Deinococcus radiodurans, enhance stress tolerances of Escherichia coli. PLoS One, 2011,6(1):e16228.
doi: 10.1371/journal.pone.0016228
[31] 周筱飞, 陈婷婷, 田野 , 等. 反向代谢工程研究进展. 台湾农业探索, 2015,1:73-79.
Zhou X F, Chen T T, Tian Y , et al. Advances on inverse metabolic engineering. Taiwan Agricultural Research, 2015,1:73-79.
[32] Zhu L, Cai Z, Zhang Y , et al. Engineering stress tolerance of Escherichia coli by stress-induced mutagenesis (SIM)-based adaptive evolution. Biotechnol J, 2014,9(1):120-127.
doi: 10.1002/biot.v9.1
[33] Atsumi S, Wu T Y, Machado I M , et al. Evolution, genomic analysis, and reconstruction of isobutanol tolerance in Escherichia coli. Mol Syst Biol, 2010,21(6):449.
[34] Royce L A, Yoon J M, Chen Y , et al. Evolution for exogenous octanoic acid tolerance improves carboxylic acid production and membrane integrity. Meta Eng, 2015,29:180-188.
doi: 10.1016/j.ymben.2015.03.014
[35] Zhang H, Chong H, Ching C B , et al. Engineering global transcription factor cyclic AMP receptor protein of Escherichia coli for improved 1-butanol tolerance. Appl Microbiol Biotechnol, 2012,94(4):1107-1117.
doi: 10.1007/s00253-012-4012-5
[36] Hinks J, Wang Y, Matysik A , et al. Increased microbial butanol tolerance by exogenous membrane insertion molecules. Chem Sus Chem, 2015,8(21):3718-3726.
doi: 10.1002/cssc.201500194
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