Effect Of Sin R On The Synthesis Of Vitamin K₂ In Bacillus Subtilis Biofilm Formation

MK-7,a highly valued member of the fat-soluble vitamin K₂ family,is a polyene compound consisting of a 2-methyl-1,4-naphthoquinone ring structure with a side chain consisting of seven isoprene units.MK-7,a component of the microbial plasma membrane,is reported to be an important electron transport carrier in the respiratory chain.It has the advantages of long half-life and high biological affinity in human body,and plays an important role in preventing osteoporosis,cardiovascular disease and peripheral artery disease.Studies have shown that the formation of Bacillus subtilis biofilm can promote the synthesis of vitamin K₂.Therefore,in this paper,Bacillus subtilis 168 was used as the starting strain,and the biofilm formation and vitamin K₂ synthesis ability of the strain were improved by constructing sinR deletion mutant strain and changing different external growth factors,and the transcriptome differences between the original strain and the mutant strain were analyzed.It provides a theoretical basis for the industrialization of vitamin K₂.In this study,the main research contents and results are as follows:（1）Firstly,sinR knock out strains were constructed.Secondly,the biofilm formation ability,thallus morphology,vitamin K₂ synthesis ability and genes encoding biofilm extracellular matrix were analyzed by RT-q PCR.The results showed that the mutant strain showed stronger ability to form biofilms,and the biofilm surface of the mutant strain could form more folds after 24 hours of culture in the pore plate,and the rate of biofilm self-melting was slower than that of the original strain.In terms of morphology,irregular morphological changes gradually appeared after the third day of culture,and there were a lot of external polymer on the surface of the bacteria.In the synthesis of vitamin K₂,the mutant strain vitamin K₂ was 45%higher than the original strain.In the process of biofilm formation,sinR mainly affects biofilm formation of B.subtilis168 strain by regulating the expression of tas A.（2）To explore the influence of different external environment factors on the biofilm formation and vitamin K₂ production of mutant strain B.subtilis168-ΔsinR.The change of external environment can affect the growth state of bacteria,biofilm formation ability and vitamin K₂ synthesis ability.In this paper,the effects of different temperatures,p H and concentrations of metal ions(Mn²⁺,Fe²⁺,Ca²⁺)on biofilm formation ability and vitamin K₂ synthesis of original strain（B.subtilis168）and mutant strain（B.subtilis168-ΔsinR）were studied.The results showed that the mutant strain had stronger adaptability to temperature and acidity and alkalinity than the original strain during biofilm formation and vitamin K₂synthesis.A certain concentration of metal ions can promote the formation of biofilm and the synthesis of vitamin K₂.The optimal temperature for the mutant strain to form biofilm is 40℃,p H is 8.0,and the metal ion is Mn²⁺（0.01 mmol/L）.The optimum temperature for synthesizing vitamin K₂ by mutant strain is 40℃,p H is 8.0,and the metal ion is Mn²⁺（0.1mmol/L）,among which the amount of vitamin K₂synthesized by mutant strain is the highest when 0.1 mmol/L Mn²⁺is added.This study provides experimental basis and theoretical support for further exploring the mechanism of Bacillus subtilis biofilm formation and improving the synthesis yield of vitamin K₂.（3）In order to explore the molecular mechanism of sinR affecting biofilm formation and vitamin K₂ synthesis of Bacillus subtilis,the original strain and mutant strain were transcriptomically analyzed by high-throughput sequencing technology.The biological function and classified summary analysis of the differential genes between the two samples were carried out by GO and KEGG functional annotation.The results showed that there were 742 significant differential genes between the two samples,including 333 up-regulated genes and 409 down-regulated genes.Using KEGG database to annotate the metabolic pathway of the sequencing results of the two samples,the analysis results show that the significant metabolic differences between the two samples mainly focus on ABC transporter,two-component regulatory system,quorum sensing,glycolysis and gluconeogenesis,etc.Then,we analyzed the expression of key genes in the metabolic pathway of the molecular mechanism of Bacillus subtilis biofilm formation and the metabolic pathway of vitamin K₂ synthesis.