Physicochemical Regulation And Proteomics Study Of Extracellular Secretion Of Vitamin K₂ From Flavobacterium

Vitamin K2 is an important vitamin that is indispensable in the human body.The low yield and high cost of microbial fermentation to produce vitamin K2 have been the bottleneck restricting the synthesis of vitamin K2 through biological method.Flavobacterium is a Gram-negative bacterium,and the cell envelope acts as a permeation barrier inside and outside the cell.Its narrow pore protein channel and low-flow lipopolysaccharide can reduce the diffusion rate of small hydrophilic molecules and lipophilic molecules across the membrane.Although vitamin K2 is a fat-soluble vitamin,it has a large molecular weight and is difficult to diffuse freely across the membrane.After intracellular synthesis,it mainly binds to the cell membrane and participates in electron transport.It is rarely secreted extracellularly,and the intracellular accumulation is not only toxic to cells but also causes feedback inhibition to limit yield,therefore it is difficult to break through the limit of the intracellular vitamin K2 production.In order to further strengthen the synthesis performance of vitamin K2,the transmembrane transport of vitamin K2 is taken as the entry point,and the membrane permeability is changed by the physical and chemical synergistic regulation method,which can improve the metabolic flux of vitamin K2 produced and the yield of vitamin K2 by the fermentation of Flavobacterium,stimulate the extracellular secretion of vitamin K2.For exploring the mechanism of transmembrane transport of vitamin K2,the proteins associated with vitamin K2 tolerance in Flavobacterium were screened out by proteomics and bioinformatics analysis.The results obtained are as follows:(1)Using Flavobacterium sp.CK as a starting strain to study mutagenesis method of low energy nitrogen ion beam,nitrosoguanidine and strong magnetic field.Finally the high-efficiency mutagenized strain Flavobacterium sp.N3-13 was obtained by compound mutagenesis.The yield of its fermentation synthesis intracellular VK2 was increased by 121.4%compared with the original bacteria,its good genetic stability was also confirmed.(2)The effect of steady-state magnetic field on the fermentation process of Flavobacterium sp.N3-13 was studied.It was found that the medium magnetic field has threshold and time window effects on Flavobacterium.After treatment for 48 h at 100 mT,the biomass and vitamin K2 production reached the maximum,which was 86.8%and 71.3%higher than that of the control group,respectively.At the same time,the medium magnetic field greatly changes the vitality and morphology of the cells.The cells become more slender,and the metabolism of ATP and G6PDH is more vigorous.(3)The effects of several typical surfactants on the cell viability,morphology,cell membrane permeability and extracellular secretion of vitamin K2 of Flavobacterium sp.N3-13 were studied.Tween80,Triton X-100 and POE surfactants can promote the leakage of vitamin K2 into the extracellular and increase the production of vitamin K2.POE has the greatest potential to promote extracellular secretion of vitamin K2.Tween80 is a heterogeneous surfactant that has weak binding ability to phospholipids flowing in cell membranes and has little effect on cell morphology and cell membrane permeability;Triton X-100 and POE are homogeneous surfactants,they are mutually soluble with phospholipid molecules in cell membrane,causing the cell membrane to shrink or even cleave.The toxic effect of TritonX-100 on N3-13 cells is greater than that of POE.The optimal conditions of the surfactant system were discussed.The results showed that 1.5%POE and 1%TritonX-100 were added at 24 h of N3-13 fermentation,and the yield of total VK2 was 30.48±1.43 mg/L after fermentation on the 8th day,an increase of 316.9%compared to the absence of surfactant.The yield of extracellular VK2 was 23.68±1.09 mg/L.(4)The effect of ultrasound on the growth and fermentation of vitamin K2 by Flavobacterium sp.N3-13 was studied.Ultrasonic process conditions were further optimized by analyzing the effects of ultrasound on cell viability and cell membrane permeability.The results showed that Flavobacterium sp.N3-13 was sonicated for 30 s at 40 kHz and 80%(×130 W)for 30 s,and the ultrasound was superimposed twice,with a gap time of 5 s.The yield of total VK2 was 31.44±1.08 mg/L when fermented to the 8th day,which was 330.1%higher than that without ultrasound.The yield of extracellular VK2 was 25.74±0.92 mg/L.The main mechanism to confirm the change of cell membrane permeability is that the cavitation effect causes the cell membrane to damage and produce reversible perforation,thereby improving the permeability of the cell membrane and promoting the extracellular leakage of VK2.(5)The effect of synergistic effect of ultrasound and surfactant on the synthesis of vitamin K2 by Flavobacterium sp.N3-13 was studied.The results showed that the combination of low-intensity ultrasound and POE(Polyoxyethylene 20 oleyl ether))changed the morphology of N3-13 cells,increased the esterase activity and cell membrane permeability of N3-13 cells,and alleviated the damage of cell membrane during POE treatment alone.Extracellular protein production and extracellular vitamin K2 production are significantly enhanced.The optimized leakage conditions were as follows:N3-13 cells were inoculated into a fermentation medium supplemented with 0.87%POE,and sonicated at 20 kHz,83%(×130 W)for 38 s when fermented to 48 h,ultrasound was superimposed twice,with a gap time of 5 s per treatment,the extracellular VK2 yield obtained was 42.09±0.97 mg/L.(6)Analysis of differentially expressed proteins of the resistant strain Flavobacterium sp.r11 and the original strain Flavobacterium sp.CK by the iTR'AQ quantitative proteomics method.The SusC/RagA family TonB associated with substance transport outer membrane protein,TonB protein,TonB periplasmic protein;cytochrome c oxidase associated with cellular metabolism and AmpC,MprB,QseC,C coN,CcoO,CcoP protein in EnvZ/OmpR two-component signaling system and MK-4 stress-related PDI proteins were separated and screened.Screening of these proteins can supply the identified information and biological targets for genetically engineered cell membrane permeability of Flavobacterium and promoted extracellular secretion of vitamin K2.