食品与发酵工业 >

2020 , Vol. 46 >Issue 4: 28 - 33

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.021965

研究报告

常压室温等离子体诱变选育高产核黄素枯草芽孢杆菌

  • 郭佳欣 ,
  • 张培基 ,
  • 刘丁玉 ,
  • 洪坤强 ,
  • 陈涛 ,
  • 王智文
展开
  • 1(天津大学 化工学院,天津, 300350);
    2(系统生物工程教育部重点实验室,天津, 300350);
    3(合成生物学前沿科学中心,天津, 300350);
    4(天津化学化工协同创新中心合成生物学平台,天津, 300350)
硕士研究生(王智文副教授为通讯作者,E-mail: zww@tju.edu.cn)

收稿日期: 2019-08-12

  网络出版日期: 2020-04-07

基金资助

国家自然科学基金(21576200;21776209;21621004)

收起

Screening of high-yield riboflavin Bacillus subtilis strain by atmospheric and room temperature plasma

  • GUO Jiaxin ,
  • ZHANG Peiji ,
  • LIU Dingyu ,
  • HONG Kunqiang ,
  • CHEN Tao ,
  • WANG Zhiwen
Expand
  • 1(School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China);
    2(Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300350, China);
    3(Frontier Science Center for Synthetic Biology, Tianjin University, Tianjin 300350, China);
    4(SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350,China)

Received date: 2019-08-12

  Online published: 2020-04-07

Fold

摘要

该研究以BS120作为出发菌株,通过常压室温等离子体诱变(atmospheric and room temperature plasma,ARTP)技术进行诱变处理,第一轮以40 mg/L 8-氮鸟嘌呤为筛选拮抗物进行筛选,得到核黄素产量和得率分别提升61.60%和58.12%的菌株BSG1。第二轮诱变以300 mg/L寡霉素为筛选拮抗物进行筛选,筛选获得菌株BSG3,核黄素产量和得率较BS120分别提升83.59%和78.76%。将核黄素操纵子表达质粒pMX45转入BSG3中,得到菌株BSG5,核黄素产量达到(4 467.08±99.47) mg/L,得率为(42.56±1.25) mg/g葡萄糖,较BS120分别提高140.94%和120.52%,展现了良好的核黄素发酵性能和遗传稳定性。

关键词： 枯草芽孢杆菌; 核黄素; 常压室温等离子体诱变; 诱变选育; 8-氮鸟嘌呤; 寡霉素

本文引用格式

郭佳欣 , 张培基 , 刘丁玉 , 洪坤强 , 陈涛 , 王智文 . 常压室温等离子体诱变选育高产核黄素枯草芽孢杆菌[J]. 食品与发酵工业, 2020 , 46(4) : 28 -33 . DOI: 10.13995/j.cnki.11-1802/ts.021965

Abstract

Bacillus subtilis is an important riboflavin producing strain in microbial fermentation. It is significant importance to breed high-yield-riboflavin Bacillus subtilis strains in industrial production. In this work, the parent riboflavin-producing strain BS120 was treated with atmospheric and room temperature plasma (ARTP) for the following screening. In the first round, the production and yield of riboflavin for mutant strain BSG1 screened with 40 mg/L 8-azaguanine increased by 61.60% and 58.12%, respectively. In the second round, the production and yield of riboflavin for mutant strain BSG3 screened with 300 mg/L oligomycin increased by 83.59% and 78.76% compared with BS120, respectively. Riboflavin operon expression plasmid pMX45 was transformed into BSG3 to generate BSG5 in order to further increase the riboflavin production. The results showed that riboflavin production of BSG5 reached (4 467.08±99.47) mg/L, and the yield was (42.56±1.25) mg/g glucose, which was 140.94% and 120.52% higher than that of BS120 respectively. These mutant strains showed excellent riboflavin fermentation performance and genetic stability during the test.

Key words： Bacillus subtilis; riboflavin; ARTP; mutation breeding; 8-azaguanine; oligomycin

参考文献

[1] BANERJEE R, BATSCHAUER A. Plant blue-light receptors[J]. Planta, 2005, 220(3):498-502.
[2] WHITESELL J K. The Merck Index, 12th edition, CD-ROM(Macintosh): An encyclopedia of chemicals, drugs, and biologicals[J]. Journal of The American Chemical Society, 1998, 120(9):2 209-2 209.
[3] HASSAN I, CHIBBERI S. NASEEM I. Vitamin B2: A promising adjuvant in cisplatin based chemoradiotherapy by cellular redox management[J]. Food and Chemical Toxicology, 2013, 59:715-723.
[4] SCHETZEK S, HEINEN F, KRUSE S, et al. Headache in children: Update on complementary treatments[J]. Neuropediatrics, 2013, 44(1):25-33.
[5] SHERWOOD M, GOLDMAN R D. Effectiveness of riboflavin in pediatric migraine prevention[J]. Canadian Family Physician, 2014, 60(3):244-246;157.
[6] SCHALLMEY M, SINGH A, WARD OP. Developments in the use of Bacillus species for industrial production[J]. Canadian Journal of Microbiology, 2004, 50(1):1-17.
[7] BARBAU-PIEDNOIR E, DE KEERSMAECKER S C J, WUYTS V, et al. Genome sequence of EU-unauthorized genetically modified Bacillus subtilis strain 2014-3557 overproducing riboflavin, isolated from a vitamin B2 80% feed additive [J]. Microbiology Resource Announcements, 2015, 3(2):1-2.
[8] 吴亦楠, 邢新会, 张翀, 等. ARTP生物育种技术与装备研发及其产业化发展[J]. 生物产业技术, 2017(1):37-45.
[9] OTTENHEIM C, NAWRATH M, WU J. Microbial mutagenesis by atmospheric and room-temperature plasma (ARTP): The latest development[J]. Bioresources and Bioprocessing, 2018, 5(1):12.
[10] REVUELTA J L, LEDESMA-AMARO R, LOZANO-MARTINEZ P, et al. Bioproduction of riboflavin: A bright yellow history[J]. Journal of Industrial Microbiology & Biotechnology, 2017, 44 (4-5): 659-665.
[11] SHI T, WANG Y C, WANG Z W, et al. Deregulation of purine pathway in Bacillus subtilis and its use in riboflavin biosynthesis[J]. Microbial Cell Factories, 2014, 13(1):1-16.
[12] BUEY RM, LEDESMA-AMARO R, Balsera M, et al. Increased riboflavin production by manipulation of inosine 5'-monophosphate dehydrogenase in Ashbya gossypii[J]. Applied Microbiology and Biotechnology, 2015, 99(22): 9 577-9 589.
[13] WANG G,SHI T,CHEN T, et al. Integrated whole-genome and transcriptome sequence analysis reveals the genetic characteristics of a riboflavin-overproducing Bacillus subtilis[J]. Metabolic Engineering, 2018, 48:138-149.
[14] 张雪, 张晓菲, 王立言, 等. 常压室温等离子体生物诱变育种及其应用研究进展[J]. 化工学报, 2014, 65(7):2 676-2 684.
[15] 薛莹莹,林福兴,别小姝,等. ARTP诱变联合抗生素抗性选育纳豆激酶高产菌株[J]. 食品工业科技, 2019,40(33):93-97.
[16] 王智文. 产核黄素Bacillus subtilis中心代谢途径代谢工程和比较基因组学[D]. 天津: 天津大学, 2011.
[17] 李荣杰. 微生物诱变育种方法研究进展[J]. 河北农业科学, 2009, 13(10):73-76;78.
[18] 高琳, 邢天来, 王非, 等. 抗癌药物8-氮鸟嘌呤与DNA相互作用的电化学研究[J]. 河南大学学报(自然科学版), 2006(3):37-41.
[19] GOGIA S, PURANIK M. Solution structures of purine base analogues 6-chloroguanine, 8-azaguanine and allopurinol[J]. Journal of Biomolecular Structure & Dynamics, 2014, 32(1):27-35.
[20] 李志勇. 富含核黄素营养酵母的研究[D]. 天津: 天津科技大学,2004.
[21] DMYTRUK K V, YATSYSHYN V Y, SYBIRNA N O, et al. Metabolic engineering and classic selection of the yeast Candida famata (Candida flareri) for construction of strains with enhanced riboflavin production[J]. Metabolic Engineering, 2010, 13(1):82-88.
[22] 林秀萍, 刘永宏, 李季伦. 寡霉素的研究进展[J]. 中国抗生素杂志, 2012, 37(9):662-665;670.
[23] 范海鸣, 刘艳霞. 线粒体ATP合酶中寡霉素敏感相关蛋白的研究进展[J]. 中国药学杂志, 2008,43(9):641-643.
[24] KOEBMANN B J, WESTERHOFF H V, SNOEP J L, et al. The glycolytic flux in Escherichia coli is controlled by the demand for ATP[J]. Journal of Bacteriology, 2002, 184(14):3 909-3 916.
[25] 陈涛. 基于基因组重排的产核黄素枯草芽孢杆菌的代谢工程[D]. 天津: 天津大学, 2004.
[26] WAGNER J M, LIU L Q, YUAN S F, et al. A comparative analysis of single cell and droplet-based FACS for improving production phenotypes: Riboflavin overproduction in Yarrowia lipolytica[J]. Metabolic Engineering, 2018, 47: 346-356.
Options
文章导航

/

〈 〉

技术支持：北京玛格泰克科技发展有限公司