Effect Of Mutation And Key Gene Regulation On Vitamin K2 Synthesis

Vitamin K2 is an essential liposolubility vitamin,it can be quickly absorbed by the intestines in the human body,which can increase the density of the body's bones and prevent osteporosis.At the same time,people can also reduce the incidence of heart disease and cardiovascular sclerosis with a high intake of vitamin K2 effectively.Due to the problems of environmental pollution caused by chemical synthesis of vitamin K2,it is restricted from direct use in food in many regions.However,the synthesis of vitamin K2 by microbial fermentation has become the preferred method for the synthesis of vitamin K2 due to its advantages such as easy availability of fermentation raw materials,mild fermentation conditions,relatively low environmental pressure,and the fact that this kind of vitamin K2 comes from organisms themselves,and the biogenic VK2 has high biological activity and high biocompatibility.However,the production of vitamin K2 by microbial fermentation has the problem of low strain activity,which restricts the industrial application of the production of vitamin K2 by microbial fermentation.In order to solve the above problems,the following researches are carried out in this paper:(1)Mutagenesis and breeding of vitamin K2 dominant strainsThe primary strain Bacillus subtilis 168(B-S-1)was mutated by plasma at ARTP,combine the structural analoges-resistant primary screening and 24-well plate fermentation were performed for re-screening.A dominant vitamin K2 mutant strain Bacillus subtilis(B-S-2)was obtained,and the vitamin K2 yield reached 11.30±0.58 mg/L.The stability tests showed that B-S-2 of the K2 dominant mutant obtained by mutagenesis maintained high stability in terms of yield.(2)The influence of key gene regulation of dominant strains on vitamin K2 synthesis abilityThrough knockout branch gene dhbB,express speed limit vitamin K2 synthetic metabolic pathways of MEP key gene ispG,ispH,ispA synthesis pathway,and connected to the master side chain combination key genes in menA,construct recombinant strains BS9,vitamin K2 production reached 28.36±0.96 mg/L,that's 8.03 times more than the original strain.(3)Optimization of flask fermentation process of vitamin K2 dominant strain BS9After single factor experiment,determine the optimal carbon and nitrogen source and inorganic salts concentration range,using the response surface design,combined with statistical software for data processing and forecasting response value,the optimal fermentation process as follows:0.69 g/L KH2PO4,glycerine 66.01 g/L,yeast powder 25.12 g/L,under the condition of this vitamin K2 to predict the maximum yield of 33.58 mg/L.To verify the optimal process parameters,the vitamin K2 yield was 33.26±1.23 mg/L after repeated experiments in three groups,which was close to the predicted value of the response surface experiment.(4)Two-stage speed regulation strategy and establishment of fermentation kinetics modelIn order to study the ability of bacillus subtilis to produce vitamin K2 by rocking bed rotation,four different rocking bed rotation rates were set,namely 0 r/min,100 r/min,200 r/min and 300 r/min.According to the changes of specific growth rate of bacteria and specific synthesis rate of vitamin K2 at different rotational speeds,a two-stage rotational speed strategy was proposed:the rotational speed was adjusted at 300 r/min at 0-30 h,after 30 h fermentation,the rotational speed was adjusted at 0 r/min;after 168h fermentation,the yield of vitamin K2 reached 44.35±0.59 mg/L,that's 12 times more than the original strain.