聚羟基丁酸酯合成引发的高密度生长大肠杆菌的多位点突变分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0004

生物技术通报 ›› 2020, Vol. 36 ›› Issue (7): 112-118.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0004

聚羟基丁酸酯合成引发的高密度生长大肠杆菌的多位点突变分析

陈桥^1,2, 吴海英^1,3, 王宗寿⁴, 谢雨康^1,2, 李宜青^1,3, 孙俊松^1,2,3

1.中国科学院上海高等研究院,上海 201210;
2.中国科学院大学,北京 100049;
3.上海科技大学,上海 201210;
4.福建省南平市畜牧站,南平 353000

收稿日期:2020-01-03 出版日期:2020-07-26 发布日期:2020-07-28
作者简介:陈桥,男,硕士研究生,研究方向：微生物代谢工程;E-mail：chenqiao@sari.ac.cn
基金资助:
福建省STS合作项目（2018T3021）

Multiple-site Mutations in Escherichia coli Capable of High-density Growing Induced from the Biosynthesis of Polyhydroxybutyrate

CHEN Qiao^1,2, WU Hai-ying^1,3, WANG Zong-shou⁴, XIE Yu-kang^1,2, LI Yi-qing^1,3, SUN Jun-song^1,2,3

1. Shanghai Advanced Research Institute,Chinese Academy of Sciences,Shanghai 201210;
2. University of Chinese Academy of Sciences,Beijing 100049;
3. Shanghai Tech University,Shanghai 201210;
4. Animal Husbandry Station of Nanping City,Nanping 353000

Received:2020-01-03 Published:2020-07-26 Online:2020-07-28

摘要/Abstract

摘要： 聚-3-羟基丁酸酯（PHB）的合成可以通过在重组大肠杆菌中过表达phaCAB操纵子实现。提升菌株的性能被认为是降低PHB生产成本的关键因素之一。在大肠杆菌S17-3中表达PHB的合成质粒pBhya-CAB,却可以自发实现菌株的高密度生长,摇瓶生长时的最高OD₆₀₀可达40左右。全基因组扫描测序和转录组测序分析比对表明,一些编码碳代谢流相关蛋白如磷酸葡萄糖异构酶（pgi）和6-磷酸葡萄糖脱氢酶（zwf）发生了阅读框序列或转录水平的改变。在S17-3中敲除zwf会导致转化菌株生长密度的降低,而在模式大肠杆菌BW25113中敲除pgi则会引起转化子的生长密度变高,揭示了S17-3高密度生长与自身碳代谢流的优化密切相关。

关键词: 大肠杆菌, 聚-3-羟基丁酸酯, 高密度生长, 磷酸葡萄糖异构酶, 6-磷酸葡萄糖脱氢酶

Abstract: Biosynthesis of polyhydroxybutyrate（PHB）in Escherichia coli can be achieved by the overexpression of the operon phaCAB. Improving strain performance is regarded as the one of the key factors to lower the cost of PHB bio-production. An E. coli mutant S17-3,when having synthetized plasmid pBhya-CAB of expressing PHB,is able to grow at high density,and the maximal OD₆₀₀ reached about 40 when growing in shake flasks. Comparative analysis of whole-genome scan sequencing and transcriptome sequencing revealed that mutations in reading frame sequence or at transcription level occurred in the carbon metabolic flux-related proteins,such as pgi of encoding phosphoglucose isomerase and zwf of encoding glucose-6-phosphate 1-dehydrogenase. Null deletion of zwf in S17-3 diminished the growth density of the strain,while pgi knockout in BW25113 promoted the transformant cells’ growth density. Therefore,it is revealed that the high-density growth of S17-3 is closely related to the optimization of itself carbon metabolic flux.

Key words: Escherichia coli, polyhydroxybutyrate, high-density growth, phosphoglucose isomerase, glucose-6-phosphate 1-dehydrogenase

陈桥, 吴海英, 王宗寿, 谢雨康, 李宜青, 孙俊松. 聚羟基丁酸酯合成引发的高密度生长大肠杆菌的多位点突变分析[J]. 生物技术通报, 2020, 36(7): 112-118.

CHEN Qiao, WU Hai-ying, WANG Zong-shou, XIE Yu-kang, LI Yi-qing, SUN Jun-song. Multiple-site Mutations in Escherichia coli Capable of High-density Growing Induced from the Biosynthesis of Polyhydroxybutyrate[J]. Biotechnology Bulletin, 2020, 36(7): 112-118.

参考文献

[1] Chen GQ, Patel MK.Plastics derived from biological sources:Present and future:A technical and environmental review[J]. Chemical Reviews, 2012, 112(4):2082-2099.
[2] Shah AA, Hasan F, Hameed A, et al.Biological degradation of plastics:A comprehensive review[J]. Biotechnology Advances, 2008, 26(3):246-265.
[3] Koller M, Braunegg G.Potential and prospects of continuous polyhy-droxyalkanoate(PHA)production[J]. Bioengineering(Basel), 2015, 2(2):94-121.
[4] Li ZB, Loh XJ.Recent advances of using polyhydroxyalkanoate-based nanovehicles as therapeutic delivery carriers[J]. Wiley Interdisciplinary Reviews-Nanomedicine and Nanobiotechnology, 2017, 9(3):e1429.
[5] Chen GQ, Jiang XR, Guo Y.Synthetic biology of microbes synthesizing polyhydroxyalkanoates(PHA)[J]. Synth Syst Biotechnol, 2016, 1(4):236-242.
[6] Pantani R, Turng LS.Manufacturing of advanced biodegradable polymeric components[J]. Journal of Applied Polymer Science, 2015, 132(48):42889.
[7] Jendrossek D, Handrick R.Microbial degradation of polyhydroxyal-kanoates[J]. Annu Rev Microbiol, 2002, 56:403-432.
[8] Shiraki M, Endo T, Saito T.Fermentative production of(R)-(-)-3-hydroxybutyrate using 3-hydroxybutyrate dehydrogenase null mutant of Ralstonia eutropha and recombinant Escherichia coli[J]. Journal of Bioscience and Bioengineering, 2006, 102(6):529-534.
[9] Tseng HC, Harwell CL, Martin CH.et al Biosynthesis of chiral 3-hydroxyvalerate from single propionate-unrelated carbon sources in metabolically engineered E. coli[J]. Microb Cell Fact, 2010, 9:96.
[10] Arifin Y, Sabri S, Sugiarto H, et al.Deletion of cscR in Escherichia coli W improves growth and poly-3-hydroxybutyrate(PHB)production from sucrose in fed batch culture[J]. J Biotechnol, 2011, 156(4):275-278.
[11] Kleman GL, Strohl WR.Acetate metabolism by Escherichia coli in high-cell-density fermentation[J]. Applied and Environmental Microbiology, 1994, 60(11):3952-3958.
[12] Phue JN, Noronha SB, Bhattacharyya R.et al Glucose metabolism at high density growth of E. coli B and E. coli K:Differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and northern blot analyses[J]. Biotechnology and Bioengineering, 2005, 91(5):649-649.
[13] Li ZP, Nimtz M, Rinas U.The metabolic potential of Escherichia coli BL21 in defined and rich medium[J]. Microbial Cell Factories, 2014, 13:45.
[14] Wu H, Chen S, Ji M, et al.Activation of colanic acid biosynthesis linked to heterologous expression of the polyhydroxybutyrate pathway in Escherichia coli[J]. Int J Biol Macromol, 2019, 128:752-760.
[15] 凌翓, 纪明华, 段海燕, 等. 木糖酸氧化途径在枯草芽孢杆菌的构建及转化乙醇酸的研究[J]. 生物技术通报, 2019, 35(6):76-82.
[16] Kabir MM, Shimizu K.Gene expression patterns for metabolic pathway in pgi knockout Escherichia coli with and without phb genes based on RT-PCR[J]. Journal of Biotechnology, 2003, 105(1-2):11-31.
[17] Conway T, Yi KC, Egan SE, et al.Locations of the zwf, edd, and eda genes on the Escherichia coli physical map[J]. J Bacteriol, 1991, 173(17):5247-5248.

聚羟基丁酸酯合成引发的高密度生长大肠杆菌的多位点突变分析

Multiple-site Mutations in Escherichia coli Capable of High-density Growing Induced from the Biosynthesis of Polyhydroxybutyrate

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	陈彩萍, 任昊, 龙腾飞, 何冰, 鲁兆祥, 孙坚. 大肠杆菌Nissle 1917对炎症性肠病治疗作用的研究进展[J]. 生物技术通报, 2023, 39(6): 109-118.
[2]	唐瑞琪, 赵心清, 朱笃, 汪涯. 大肠杆菌对木质纤维素水解液抑制物的胁迫耐受性[J]. 生物技术通报, 2023, 39(11): 205-216.
[3]	李仁瀚, 张乐乐, 刘春立, 刘秀霞, 白仲虎, 杨艳坤, 李业. 基于紫色杆菌素生物合成途径的L-色氨酸生物传感器的构建[J]. 生物技术通报, 2023, 39(10): 80-92.
[4]	高伟欣, 黄火清, 赵晶, 张鑫, 杨宁, 杨浩萌. 应用于基因编辑的核糖核蛋白复合体的构建与活性验证[J]. 生物技术通报, 2022, 38(8): 60-68.
[5]	孙曼銮, 葛赛, 卜佳, 朱壮彦. 大肠杆菌核糖核酸酶调控机制研究[J]. 生物技术通报, 2022, 38(3): 234-245.
[6]	李晓芳, 刘慧燕, 潘琳, 艾治宇, 李一鸣, 张恒, 方海田. 常温常压等离子体诱变选育高产L-异亮氨酸大肠杆菌[J]. 生物技术通报, 2022, 38(1): 150-156.
[7]	吴蓉, 曹佳睿, 曹君, 刘飞翔, 杨猛, 苏二正. 南极假丝酵母脂肪酶B基因在大肠杆菌中的表达和发酵优化[J]. 生物技术通报, 2021, 37(2): 138-148.
[8]	王凯凯, 王晓璐, 苏小运, 张杰. 大肠杆菌双质粒CRISPR-Cas9系统的优化及应用[J]. 生物技术通报, 2021, 37(12): 252-264.
[9]	张春晨, 胡双艳, 阮海华. 人源溶菌酶在大肠杆菌中的表达与复性研究[J]. 生物技术通报, 2020, 36(3): 153-161.
[10]	王琦, 颜春蕾, 高洪伟, 吴薇, 杨庆利. 基于核酸适配体传感器检测食品致病菌的研究进展[J]. 生物技术通报, 2020, 36(11): 245-258.
[11]	刘新平, 谭玉萌, 张雪, 冯雁, 杨广宇. 神经节苷脂氟化寡糖在大肠杆菌中的生物合成[J]. 生物技术通报, 2019, 35(8): 162-169.
[12]	李冉, 黄玉清, 贾振华. 大肠杆菌代谢途径改造策略与应用研究进展[J]. 生物技术通报, 2019, 35(8): 232-237.
[13]	陈相好, 刘芳, 王彩霞, 陈峥宏, 洪伟, 蔡梦迪, 张峥嵘, 綦廷娜, 廖永慧, 谷俊莹, 崔古贞. 高效严谨型大肠杆菌Targetron基因打靶系统的构建[J]. 生物技术通报, 2019, 35(6): 213-220.
[14]	张良, 陈小青, 宋佳宇, 毛然然, 姜倩雯, 林向民. 巴洛沙星胁迫下大肠杆菌的比较蛋白质组学研究[J]. 生物技术通报, 2019, 35(3): 103-109.
[15]	张永丽, 袁伟, 杨清香. 纳米TiO₂和四环素暴露对耐药大肠杆菌的影响[J]. 生物技术通报, 2019, 35(11): 124-131.