The Study Of Functions On The Key Genes FolP And FolE Of Folate Biosynthesis Of Lactobacillus Plantarum YM-4-3 Strain

Folate has important physiological functions.With the gradual clarity of folate biosynthesis pathway,the study of the key genes of folate biosynthesis or changing the metabolism of folate in plants and microorganisms by genetic engineering has been paid much attention in order to improve natural folate production and fulfil the human needs.At present,a lot of studies were limited to the way to improve the yield of folate by overexpressing the key genes of folate biosynthesis.However,the study of these genes' function by gene knockout technique is very few.In this study,the genes of folE,folKE and folP of Lactobacillus plantarum YM-4-3 strain were knocked out by homologous recombination gene knockout technique,and the results of their effects on the folate synthesis and growth of L.plantarum were studied.The result of sequence alignment and splicing of the key genes folE,folK,folP,folB and folQ,which are in the biosynthesis pathway of folate,found that these genes were in the form of gene cluster in the genome.The knockout strains ?folE,?folKE and ?folP were successfully constructed by temperature sensitive type plasmid pFED760 by homologous recombinant.And the results were further verified by PCR and DNA southern blot,and it was proved that all of three genes were single copy.In the following experiments,the strains of gene knockout were studied.By observing the growth state of bacteria,the ?folP strain was grown by precipitation while ?folE and ?folKE strains were uniform.The results analyzing by scanning electron microscop y and transmission electron microscopy shown that compared with the wide YM-4-3 strain,the cells of ?folE strain did not change.However,the morphology of ?folKE and ?folP strains cells changed a lot.The adhesion between cells and cells were enhanced and the cytoplasm were separated.Besides,some of the cells were irregular rod like,even a depression.The results also found that the leakage of contents of cells.The dynamic monitoring of the growth state of the knockout strains and the wildtype strain showed that the biomass and growth rate of the knockout strains were significantly lower than that of the wild type strain,and the growth cycle was longer,the acid production capacity was weaker.Through analysising of the folate production,we found that the folate content of each strain'fermentation fluctuated with time.And the folate content of the knockout strains' fermentation broth were significantly different from that of the wild type fermented broth.The results proved that folE,folKE and folP genes played important role in the folate biosynthesis.While folKE and folP genes are related to the cell morphology.The results even further proofed that folE gene and folK gene encoded protein alone.The gene complement mutant strain H-folP and gene overexpression strain B-folP were successfully constructed with the expression vector pMG36 e,and the quantitative real-time PCR results showed that the expression of folP gene was 177.87 times and 40.28 times as high as that of the wildtype strain,respectively.However,the total folate content in fermentation broth of H-folP strain and B-folP strain were lower than that of wildtype strain.Therefore,it was speculated that the folP gene has feedback inhibition.The function of the key genes of folate biosynthesis pathway folE,folKE and folP genes were studied in this experiment systematically,which laid a theoretical foundation for the study of molecular regulation mechanism of L.plantarum folate biosynthesis pathway,And provided new ideas for exploring other folate biosynthesis pathways of L.plantarum.