研究报告

质粒复制原点在地衣芽孢杆菌中的功能鉴定及理性设计

  • 张高阳 ,
  • 李由然 ,
  • 肖丰旭 ,
  • 石贵阳
展开
  • 1(江南大学 生物工程学院,江苏 无锡,214122)
    2(江南大学,粮食发酵与食品生物制造国家工程研究中心,江苏 无锡,214122)
第一作者:硕士研究生(李由然副教授为通信作者,E-mail:liyouran@jiangnan.edu.cn)

收稿日期: 2024-01-08

  修回日期: 2024-03-28

  网络出版日期: 2024-10-14

基金资助

国家重点研发计划(2020YFA0907704);国家自然科学基金(32172174)

Development and application of strong origin of replication of Bacillus licheniformis

  • ZHANG Gaoyang ,
  • LI Youran ,
  • XIAO Fengxu ,
  • SHI Guiyang
Expand
  • 1(School of Biotechnology, Jiangnan University, Wuxi 214122, China)
    2(National Engineering Research center of Cereal Fermentation and Food Biomanufacturing Technology, Jiangnan University, Wuxi 214122, China)

Received date: 2024-01-08

  Revised date: 2024-03-28

  Online published: 2024-10-14

摘要

食品安全级工业微生物地衣芽胞杆菌(Bacillus licheniformis)与模式微生物相比,严重缺乏质粒载体等合成生物学标准化遗传工具,导致其研究与应用受到限制。该研究首先建立了质粒特性在地衣芽孢杆菌中的评价方法,由此鉴定获得了高效复制原点。在此基础上进一步解析了复制原点中茎环结构影响质粒拷贝数和稳定性的分子机制,并理性设计了系列人工复制原点。结果表明,枯草芽孢杆菌来源的复制原点ORIT5拷贝数是pHY300内源复制原点的2.15倍(达到40.2),同时具有更高的稳定性。3′端增加1个motif为TCCGCC茎环的人工复制原点pT1,拷贝数进一步提升至62.2,稳定性提高36%。以麦芽寡糖基海藻糖合成酶为报告蛋白,携带pT1复制原点的重组菌发酵最高酶活力达到8 726.7 U/mL,比出发菌株提高了43.9%。人工复制原点的开发与应用为地衣芽孢杆菌表达系统的改进和完善奠定了基础。

本文引用格式

张高阳 , 李由然 , 肖丰旭 , 石贵阳 . 质粒复制原点在地衣芽孢杆菌中的功能鉴定及理性设计[J]. 食品与发酵工业, 2024 , 50(18) : 1 -8 . DOI: 10.13995/j.cnki.11-1802/ts.038515

Abstract

Compared to model microorganisms, the food-safety grade industrial bacteria Bacillus licheniformis has a serious lack of standardized genetic tools for synthetic biology, such as plasmid vectors, which limits its use in research and applications.Using a technique we developed for assessing plasmid characteristics in Bacillus licheniformis, we were able to identify and obtain extremely effective replication primordia in this work.A variety of artificial replication origins were logically created based on the additional analysis of the molecular process by which the stem-loop structure in the replication origin influences plasmid copy number and stability.According to the findings, the replication origin ORIT5 from B.subtilis had a copy number that was 2.15 times larger and more stable than the endogenous replication origin pHY300 (up to 40.2).With the addition of a TCCGCC stem-loop at the 3′ end, pT1, an artificial replication origin, enhanced stability by 36% and raised copy number to 62.2.The maximal enzyme activity of the recombinant bacterial fermentation bearing the pT1 replication origin was 8 726.7 U/mL, which was 43.9% greater than that of the starting strain, using malt oligosaccharide-based alginate synthase as the reporter protein.The creation and use of the artificial replication origin set the stage for Bacillus licheniformis′s expression system to be enhanced and perfected.

参考文献

[1] WOESE C R, KANDLER O, WHEELIS M L.Towards a natural system of organisms:Proposal for the domains Archaea, Bacteria, and Eucarya[J].Proceedings of the National Academy of Sciences of the United States of America,1990,87(12):4576-4579.
[2] HOLMES M L, PFEIFER F, DYALL-SMITH M L Analysis of the halobacterial plasmid pHK2 minimal replicon[J].Gene,1995,153(1):117-121.
[3] KONIECZNY I, BURY K, WAWRZYCKA A, et al.Iteron plasmids[J].Microbiology Spectrum,2014, 2(6).DOI:10.1128/9781555818982.ch2.
[4] ABELES A L, REAVES L D, AUSTIN S J.A single DnaA box is sufficient for initiation from the P1 plasmid origin[J].Journal of Bacteriology,1990, 172(8):4386-4391.
[5] MUROTSU T, MATSUBARA K, SUGISAKI H, et al.Nine unique repeating sequences in a region essential for replication and incompatibility of the mini-F plasmid[J].Gene,1981,15(2-3):257-271.
[6] GERMINO J, BASTIA D.The replication initiator protein of plasmid R6K tagged with beta-galactosidase shows sequence-specific DNA-binding[J].Cell,1983,32(1):131-140.
[7] LACKS S A, LOPEZ P, GREENBERG B, et al.Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1[J].Journal of Molecular Biology,1986, 192(4):753-765.
[8] LIN L S, MEYER R J.Directly repeated, 20-bp sequence of plasmid R1162 DNA is required for replication, expression of incompatibility, and copy-number control[J].Plasmid,1986, 15(1):35-47.
[9] WASCHKAU B, WALDECK J, WIELAND S, et al.Generation of readily transformable Bacillus licheniformis mutants[J].Applied Microbiology and Biotechnology,2008, 78(1):181-188.
[10] MURAS A, ROMERO M, MAYER C, et al.Biotechnological applications of Bacillus licheniformis[J].Critical Reviews in Biotechnology,2021, 41(4):609-627.
[11] KOBAYASHI M, KUBOTA M, MATSUURA Y.Refined structure and functional implications of trehalose synthase from Sulfolobus acidocaldarius[J].Journal of Applied Glycoscience,2003, 50(1):1-8.
[12] 刘宗利,王乃强,刘峰, 等.海藻糖在日化领域的应用研究进展[J].精细与专用化学品,2015,23(2):18-20.
LIU Z L, WANG N Q, LIU F, et al.Study on the application of trehalose in the daily chemical field[J].Fine and Specialty Chemicals,2015, 23(2):18-20.
[13] 赵鑫馨.地衣芽孢杆菌强启动子开发及人工进化[D].无锡:江南大学,2021.
ZHAO X X.Development and artificial evolution of strong promoters of Bacillus licheniformis[D].Wuxi:Jiangnan University,2021.
[14] XIAO F X, LI Y R, ZHANG Y P, et al.Construction of a novel sugar alcohol-inducible expression system in Bacillus licheniformis[J].Applied Microbiology and Biotechnology,2020,104(12):5409-5425.
[15] BUSTIN S A, VLADIMIR B, GARSON J A, et al.The MIQE guidelines:minimum information for publication of quantitative real-time PCR experiments[J].Clinical Chemistry, 2009, 55(4):611-622.
[16] JOSHUA C J, PEREZ L D, KEASLING J D.Functional characterization of the origin of replication of pRN1 from Sulfolobus islandicus REN1H1[J].PloS One,2013, 8(12):e84664.
[17] KOWALCZYK L, RAJEWSKA M, KONIECZNY I.Positioning and the specific sequence of each 13-mer motif are critical for activity of the plasmid RK2 replication origin[J].Molecular Microbiology,2005, 57(5):1439-1449.
[18] SONG C W, CHELLADURAI R, PARK J M, et al.Engineering a newly isolated Bacillus licheniformis strain for the production of (2R,3R)-butanediol[J].Journal of Industrial Microbiology & Biotechnology,2020, 47(1):97-108.
[19] BRÁZDA V, LAISTER R C, JAGELSKÁ E B, et al.Cruciform structures are a common DNA feature important for regulating biological processes[J].BMC Molecular Biology,2011, 12:33.
[20] 陆一鸣,李由然,许银彪, 等.启动子工程提高海藻糖生产用酶在地衣芽孢杆菌中的表达[J].基因组学与应用生物学,2022,41(8):1703-1712.
LU Y M,LI Y R,XU Y B,et al.Promoter engineering improves the expression of trehalose production enzyme in Bacillus licheniformis[J].Genomics and Applied Biology,2022,41(8):1703-1712.
文章导航

/