Enhancement Of MK-7 Production Via Mutation Breeding Of Bacillus Subtilis Natto And Get Insight Into The Metabolic Regulation Mechanism

Vitamin K₂is a necessary fat-soluble vitamin for human body.It can prevent and treat osteoporosis,cardiovascular disease and diabetes,etc.It has a good application prospect and development value in food health care and biomedical fields.As an important form of vitamin K₂,the menaquinone-7(MK-7)is attracting increasing attention due to its advantages of significant biological activity,long half-life of blood and high bioavailability over other forms of vitamin K₂.At present,MK-7 is mainly from traditional fermented food,but its small content is difficult to meet the needs of people for health and nutrition intervention;chemical synthesis is complicated in route and low in synthesis rate,and it will bring serious environmental pollution and food safety hazards;in contrast,it is more promising to produce MK-7 by fermentation of Bacillus subtilis natto with renewable biomass.How to improve the productivity of Bacillus subtilis natto and get insight into the metabolic regulation mechanism of MK-7 biosynthesis is the key to promote the industrial production of MK-7.In this paper,the productivity promotion and synthesis mechanism of MK-7 of Bacillus subtilis natto were studied by combining the screening and identification of strains,mutation breeding,extracellular secretion regulation and transcriptional analysis.The specific contents and results are as follows:Firstly,Strain BN0 selected from natto product was cultured in the laboratory.The results showed that the yield of MK-7 was 15.16 mg/L.As compared with most of the original MK-7 strains,it has good yield advantages.Through the phylogeny tree analysis,combined with the morphological and physiological and biochemical characteristics of the strain,BN0 was identified as Bacillus subtilis natto.Furthermore,the genetic background and gene function of the strain Bacillus subtilis natto were analyzed by genome sequencing.The data showed that the BN0 genome was composed of a 4119230 bp has a complete MK-7 biosynthesis pathway,which consists of two circular plasmids with the size of 64309 and5820 bp,respectively.Secondly,a strain with higher yield of MK-7 was successfully obtained by UV and ARTP combined mutation.The yield of mutant was 22.83 mg/L,which was 50.59%higher than that of the original strain BN0.In order to further improve the production and exocription rate of MK-7,surfactant and ultrasonic were used to treat the strain,and the yield was finally increased to 34.23 mg/L which was 1.26-fold higher than that of the original strain.Finally,in order to analyze the mechanism of high yield synthesis of MK-7 in mutant strain,the transcriptome of wild and mutant strains was analyzed.The results showed that there were a large number of up-regulated genes in the glycerol metabolism pathway and MEP pathway in MK-7 biosynthesis pathway in mutant strains,but the expression level of SA pathway and MK-7 pathway did not change significantly.In addition,the ABC transporter and MFS transporter genes and the gene involved in the Tat secretion pathway were significantly up-regulated,while most of the genes related to sporulation were down-regulated.It is suggested that the transcription proteins of the significant difference may be related to the extracellular secretion of MK-7,and the inhibition of sporogenesis can also promote the accumulation of secondary metabolites.The above data provide useful information and ideas for further improving the MK-7 production via rational engineering in the future.