食品与发酵工业 >

2021 , Vol. 47 >Issue 14: 23 - 30

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.025877

研究报告

不同培养条件对枯草芽胞杆菌BS168-ΔsinR生物膜形成及维生素K2产量的影响

  • 吴静 ,
  • 李伟 ,
  • 冯静静 ,
  • 周梦洁 ,
  • 胡汶松 ,
  • 汪剑 ,
  • SOKHNA MBACKE Gningue ,
  • 吴佳雯 ,
  • 赵礼军 ,
  • 徐文瀚 ,
  • 薛正莲 ,
  • 王洲 ,
  • 刘艳
展开
  • 1(安徽工程大学 生物与食品工程学院,安徽 芜湖,241000)
    2(安徽省工业微生物分子育种工程实验室,安徽 芜湖,241000)
硕士研究生(刘艳教授为通讯作者,E-mail:liuyan@ahpu.edu.cn)

收稿日期: 2020-10-19

  修回日期: 2021-01-22

  网络出版日期: 2021-08-20

基金资助

国家自然科学基金(31871781;31772081);安徽高校自然科学研究重点项目(KJ2018A0106);芜湖市科技计划重点项目(2020yf62);国家级大学生创新创业项目(201910363042;201810363046);安徽省大学生创新创业计划项目(201710363178)

收起

Effect of different cultivated conditions on biofilm formation and menaquinone synthesis of Bacillus subtilis BS168-ΔsinR

  • WU Jing ,
  • LI Wei ,
  • FENG Jingjing ,
  • ZHOU Mengjie ,
  • HU Wensong ,
  • WANG jian ,
  • SOKHNA MBACKE Gningue ,
  • WU Jiawen ,
  • ZHAO Lijun ,
  • XU Wenhan ,
  • XUE Zhenglian ,
  • WANG Zhou ,
  • LIU Yan
Expand
  • 1(College of Biological and Chemical Engineering, Anhui Polytechnic University, Wuhu 241000, China)
    2(Anhui Provincial Engineering Laboratory for Molecular Breeding of Industrial Microbial, Wuhu 241000, China)

Received date: 2020-10-19

  Revised date: 2021-01-22

  Online published: 2021-08-20

Fold

摘要

枯草芽胞杆菌(Bacillus subtilis)是合成维生素K2的微生物之一,维生素K2的合成量又与细菌生物膜有重要关系。其中sinR是生物膜形成环节中的关键调节基因,我们用同源重组的方法构建枯草芽胞杆菌sinR缺失突变体(ΔsinR)菌株,再以BS168-ΔsinR菌株为出发菌株,采用微孔板法-结晶紫染色测定生物膜形成量,核酸定量仪测定胞外蛋白,苯酚硫酸法测定胞外多糖, HPLC测定维生素K2的生物合成量。比较分析了不同温度、pH、以及不同浓度的金属离子(Mn2+、Fe2+、Ca2+)对枯草芽胞杆菌BS168及BS168-ΔsinR生物膜形成能力以及维生素K2合成量的影响。结果表明,重组菌BS168-ΔsinR生物膜形成能力和维生素K2合成能力均优于原始菌BS168,且重组菌在生物膜形成、维生素K2合成过程中对温度、酸碱性的适应能力也强于原始菌。一定浓度的金属离子可以促进生物膜的形成及维生素K2的合成。2株菌生物膜形成的最佳条件为温度37~40 ℃、pH 8.0、Mn2+浓度0.01 mmol/L、Fe2+浓度1 μmol/L、Ca2+浓度0.01 mmol/L,维生素K2合成的最佳条件是温度40 ℃、pH 7.0~8.0、Mn2+浓度0.1 mmol/L、Fe2+浓度50 μmol/L、Ca2+浓度1 mmol/L。该研究为深入探究sinR基因参与枯草芽胞杆菌生物膜形成的机制及提高维生素K2合成产量提供实验基础和理论支撑。

关键词： 枯草芽胞杆菌; sinR; 生物膜; 培养条件; 维生素K2产量

本文引用格式

吴静 , 李伟 , 冯静静 , 周梦洁 , 胡汶松 , 汪剑 , SOKHNA MBACKE Gningue , 吴佳雯 , 赵礼军 , 徐文瀚 , 薛正莲 , 王洲 , 刘艳 . 不同培养条件对枯草芽胞杆菌BS168-ΔsinR生物膜形成及维生素K2产量的影响[J]. 食品与发酵工业, 2021 , 47(14) : 23 -30 . DOI: 10.13995/j.cnki.11-1802/ts.025877

Abstract

Bacillus subtilis is one of the most important strains for synthesizing vitamin K2. The production of vitamin K2 is closely related to the constitution of biofilm of this strain. sinR is a key regulatory gene during the formation of biofilm. Firstly, a mutant strain with sinR deletion (ΔsinR) was constructed by homologous recombination. Secondly, the effects of different cultivated factors including temperature, pH and metal ions (Mn2+, Fe2+, Ca2+) on biofilm formation and vitamin K2 synthesis of Bacillus subtilis BS168 and BS168-ΔsinR were compared and analyzed. The biomass of biofilm, extracellular protein, extracellular polysaccharide vitamin K2 yield of ΔsinR were measured by microplate-crystal violet staining method, nucleic acid analyzer, phenol-vitriolic colorimetry and HPLC, respectively. The results showed that the ability of biofilm formation and vitamin K2 synthesis of recombinant bacteria BS168-ΔsinR was better than that of the original bacteria. In addition, the adaptability of the engineered bacteria to temperature, acid and alkalinity was also stronger in the process of biofilm formation and vitamin K2 synthesis. A certain concentration of metal ions could promote the formation of biofilms and the synthesis of vitamin K2.The optimum conditions for biofilm formation of BS168 and BS168-ΔsinR were at 37-40 ℃, pH 8.0, 0.01 mmol/L Mn2+, 1 μmol/L Fe2+, 0.01 mmol/L Ca2+. The optimum conditions for vitamin K2 synthesis were at 40 ℃. pH 7.0-8.0, 0.1 mmol/L Mn2+, 50 μmol/L Fe2+, 1 mmol/L Ca2+. This study provides experimental basis and theoretical support for further exploring the mechanism of how sinR participating in biofilm formation and vitamin K2 synthesis in Bacillus subtilis.

Key words： Bacillus subtilis; sinR; biofilm; cultivated condition; vitamin K2 production

参考文献

[1] COLLINS M D, JONES D.Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implication[J].Microbiological Reviews, 1981, 45(2):316-354.
[2] KISHI S, SAITO K, KATO Y, et al.Redox potentials of ubiquinone, menaquinone, phylloquinone, and plastoquinone in aqueous solution[J].Photosynthesis Research, 2017, 134(2):193-200.
[3] WANG H, LIU H, WANG L, et al.Improvement of menaquinone-7 production by Bacillus subtilis natto in a novel residue-free medium by increasing the redox potential[J].Applied Microbiology and Biotechnology, 2019,103(18):7 519-7 535.
[4] CUI S X, LYU X Q, WU Y K, et al.Engineering a bifunctional Phr60-Rap60-Spo0A quorum-sensing molecular switch for dynamic fine-tuning of menaquinone-7 synthesis in Bacillus subtilis[J].ACS Synthetic Biology, 2019, 8(8):1 826-1 837.
[5] CUI S X, XIA H Z, CHEN T C, et al.Cell membrane and electron transfer engineering for improved synthesis of menaquinone-7 in Bacillus subtilis[J].iScience, 2020, 23(3):100 918.
[6] MAHDINIA E, DEMIRCI A, BERENJIAN A.Effects of medium components in a glycerol-based medium on vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors[J].Bioprocess and Biosystems Engineering, 2018, 42(2):223-232.
[7] BERENJIAN A, MAHANAMA R, TALBOT A, et al.Designing of an intensification process for biosynthesis and recovery of menaquinone-7[J].Applied Biochemistry & Biotechnology, 2014, 172(3):1 347-1 357.
[8] BERENJIAN A, CHAN N L C, MAHANAMA R, et al.Effect of biofilm formation by Bacillus subtilis natto on menaquinone-7 biosynthesis[J].Molecular Biotechnology, 2013, 54(2):371-378.
[9] MAHDINIA E, DEMIRCI A, BERENJIAN A, et al.Optimization of Bacillus subtilis natto growth parameters in glycerol-based medium for vitamin K (Menaquinone-7) production in biofilm reactors[J].Bioprocess & Biosystems Engineering, 2018, 41(2):195-204.
[10] MAHDINIA E, DEMIRCI A, BERENJIAN A.Strain and plastic composite support (PCS) selection for vitamin K (Menaquinone-7) production in biofilm reactors[J].Bioprocess and Biosystems Engineering, 2017, 40(10):1 507-1 517.
[11] GIDI P, SHIRA O B, URI G.Menaquinone and iron are essential for complex colony development in Bacillus subtilis[J].PLoS One, 2017, 8(11):86-99.
[12] KIRBY D T, SAVAGE J M, PLOTKIN B J.Menaquinone (vitamin K2) enhancement of Staphylococcus aureus biofilm formation[J].Journal of Biosciences & Medicines, 2014, 2(1):26-32.
[13] CHOPRA L, SINGH G, KUMAR JENA K,et al.Sonorensin:A new bacteriocin with potential of an anti-biofilm agent and a food biopreservative[J].Scientific Reports, 2015, 5(1):134-146.
[14] 周万龙, 焦学, 孙亚杰, 等.1株海洋来源鞘氨醇单胞菌胞外多糖的含量测定[J].中国海洋药物, 2017, 36(2):27-34.
ZHOU W L, JIAO X, SUN Y J, et al.Determination of extracellular polysaccharides produced by marine derived Sphingomonas sp.WG[J].Chinese Journal of Marine Drugs, 2017, 36(2):27-34.
[15] 严为留, 张伟国, 钱和.响应面法优化维生素K2(MK-7)发酵条件[J].工业微生物, 2015, 45(3):14-23.
YAN W L, ZHANG W G, QIAN H.Optimization of fermentation conditions for vitamin K2(MK-7) by response surface methodology[J].Industrial Microbiology, 2015, 45(3):14-23.
[16] MORIMATSU K, HAMANAKA D, TANAKA F, et al.Effect of temperature fluctuation on biofilm formation with bacterial interaction between Salmonella enterica and Pseudomonas putida[J].九州大学大学院农学研究院纪要, 2012, 58(1):125-129.
[17] 冯静静, 杨自名, 吴静, 等.培养条件及表面活性剂的添加对纳豆芽孢杆菌生物膜形成的影响[J].食品与发酵工业, 2020, 46(3):90-96.
FENG J J, YANG Z M, WU J, et al.The effects of culture conditions and surfactants on the biofilm formation of Bacillus subtilis natto[J].Food and Fermentation Industries, 2020, 46(3):90-96.
[18] MHATRE E, TROSZOK A, GALLEGOS-MONTERROSA R, et al.The impact of manganese on biofilm development of Bacillus subtilis[J].Microbiology, 2016, 162(8):1 468-1 478.
[19] HAYRAPETYAN H, MULLER L, TEMPALAARS M, et al.Comparative analysis of biofilm formation by Bacillus cereus reference strains and undomesticated food isolates and the effect of free iron[J].International Journal of Food Microbiology, 2015, 200(4):72-79.
[20] BANIN E, VASIL M L, GREENBERG E P.From the cover:Iron and Pseudomonas aeruginosa biofilm formation[J].Proceeding of National Academy of Sciences, 2005, 102(31):11 076-11 081.
[21] TSCHLER A H, LIE L, THOMPSON C M, et al.Discovery of calcium as a biofilm-promoting signal for Vibrio fischeri reveals new phenotypes and underlying regulatory complexity[J].Journal of Bacteriology,2018, 200(15):16-18.
[22] CHU F, KEARNS D B, BRANDA S S, et al.Targets of the master regulator of biofilm formation in Bacillus subtilis[J].Molecular Microbiology, 2010, 59(4):1 216-1 228.
[23] KEARNS D B, CHU F, BRANDA S S, et al.A master regulator for biofilm formation by Bacillus subtilis[J].Molecular Microbiology, 2005, 55(3):739-749.
[24] MILTON M E, DRAUGHN G L, BOBAY B G, et al.The solution structures and interaction of sinR and sinI:Elucidating the mechanism of action of the master regulator switch for biofilm formation in Bacillus subtilis[J].Journal of Molecular Biology, 2019, 432(2):343-345.
[25] MA Y, MCCLURE D D, SOMERVILLE M V, et al.Metabolic engineering of the MEP Pathway in Bacillus subtilis for increased biosynthesis of menaquinone-7[J].ACS Synthetic Biology, 2019, 8(7):1 620-1 630.
[26] MAHDINIA E, ALI D, BERENJIAN A.Implementation of fed-batch strategies for vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors[J].Applied microbiology and biotechnology, 2018, 14(9):9 147-9 157.
[27] MAHDINIA E, DEMIRCI A, BERENJIAN A.Enhanced vitamin K (Menaquinone-7) production by Bacillus subtilis natto in biofilm reactors by optimization of glucose-based medium[J].Current Pharmaceutical Biotechnology, 2018, 19(11):917-924.
[28] LIU Y, DING X M, XUE Z L, et al.Site-directed mutagenesis of UbiA to promote menaquinone biosynthesis in Elizabethkingia meningoseptica[J].Process Biochemistry, 2017, 58(2):186-192
Options
文章导航

/

〈 〉

技术支持：北京玛格泰克科技发展有限公司