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.
[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
