The Study Of Function On The Key Genes Fol B And Fol K Of Folate Biosynthesis Of Lactobacillus Plantarum YM-4-3

Folic acid is an essential element in the human diet and involves many metabolic pathways,mainly in carbon transfer reactions such as purine and pyrimidine biosynthesis and amino acid interconversion.Studies have shown that animal cells can not autonomously synthesize folic acid and can only be obtained through food intake.Therefore,once the intake of folic acid is too small,it will cause the organism to have a corresponding folic acid deficiency disease.As the folate biosynthetic pathway is gradually clear,in order to increase the natural folic acid production and thus meet human needs,genetic engineering methods to change the metabolism of folic acid in plants or microorganisms have received considerable attention.Among them,it has been reported that the fol B and fol K genes may play an important role in the biosynthesis of folic acid,but the study on the mechanism and specific effects of the two genes on the biosynthesis of folic acid in bacteria has not yet been clarified.In this study,the key genes fol E,fol K,fol P,fol B and fol Q of Lb.plantarum YM-4-3 folic acid biosynthetic pathway were sequenced and spliced.The genes were clustered in the genome.In the form of an open reading frame for the genes fol B and fol K,which were translated into amino acid sequences,NCBI sequence alignments were performed on the proteins expressed in these two genes.The properties and functions of the proteins were predicted and analyzed by GO analysis.Two genes are functionally annotated.It was found that both were closely related to the biosynthesis of folic acid in Lb.plantarum.Later,in this study,we used the constitutive expression plasmid pMG36 e to overexpress the fol B and fol K genes of Lb.plantarum YM-4-3 to study its effect on the synthesis and growth of folic acid from Lb.plantarum.After PCR amplification of the fol B and fol K of Lb.plantarum YM-4-3,the recombinant plasmid was over-expressed by restriction enzyme ligation and transformed into the Lb.plantarum YM-4-3 strain by electroporation.H-fol B and H-fol K strains over-expressing fol B and fol K genes;Quantitative PCR results showed that the expression level of fol Bgene in H-fol B strain was 33 times that of wild type strain YM-4-3,The expression of fol K gene in H-fol K strain was 88 times higher than that of wild-type strain YM-4-3;however,the folic acid content in the fermentation broth of H-fol B strain and H-fol K strain was lower than that of wild-type strain,and The growth rate of H-fol B strain was much slower than that of the wild-type strain.However,from the standpoint of single cells,the content of folic acid in the single cell of H-fol B strain is much higher than that of the wild-type strain.From the configuration of folic acid,the content of purine diglutamate and purine hexaglutamate was the lowest;at 12 hours,the percentage of purine monoglutamate in total folic acid was lower than other times,which may be because The bacteria started producing purine polyglutamic acid,but purine polyglutamic acid was unstable and converted to purine monoglutamate over time.To further determine the effect of the two genes on the biosynthesis of folic acid in Lb.plantarum,knockout of the fol B and fol K genes of Lb.plantarum YM-4-3 was also performed by a gene knockout technique of homologous recombination.First,the homologous segments of the target gene were amplified by PCR(about 1000 bp each gene sequence),and overlapping PCR was used to connect the upstream and downstream homology arms to obtain an approximately 2,000 bp band(UP-DOWN).Using the temperature-sensitive plasmids pFDE760 and UP-DOWN to construct the recombinant plasmid is also knocked out of the vector,knocked into E.coli DH5? by heat transfer,coated,and cultured at 28°C to obtain a recombinant plasmid,and then transformed into a recombinant plasmid by electroporation.In the case of Lb.plantarum YM-4-3,knockouts were finally screened by a series of manipulations such as temperature changes.However,it was further confirmed by designing the corresponding primers and sending the company to verify the success of the knockout.This experimental system studies the functions of the fol B and fol K genes,key genes in the folic acid biosynthesis pathway,and lays a theoretical foundation for the study of the molecular regulation mechanism of the biosynthesis pathway of folic acid for Lb.plantarum,providing a theoretical basis for the development of functionalfoods rich in folic acid.