Study On The Functions Of Fol Q Gene Of Lactobacillus Plantarum YML4-3 Strain In Folate Biosynthesis Pathway

The growth and development of organisms generally require the participation of folic acid.In nature,only fruits,vegetables and a few microorganisms have the ability to synthesize folic acid.Humans and animals can only ingest the necessary folic acid from the diet to meet their own growth and development needs and improve their naturalness.Folic acid production has become particularly important.Lactic acid bacteria(LAB)are often used to make folic acid fermented foods,but it is not clear which gene plays an important role in the folate synthesis pathway.Therefore,this study combines the genome sketch of Lactobacillus plantarum YML4-3 strain and the existing research on the prediction of folate synthesis pathway,and the L.plantarum YML4-3 strain by homologous recombination gene knockout and overexpression to explore the function and status of the fol Q gene in the folate synthesis pathway.Firstly,the YML4-3 strain was subjected to gene knockout using the temperature-sensitive plasmid p FED760 to obtain a ?fol Q strain,which was verified to be a single-copy gene by Southern Blot.The folate content of decanoyl monoglutamate produced by ?fol Q strain was significantly lower than that of wild-type strain YML4-3 at 24 h,and then fluctuated at other times.In addition,the fol Q gene complement mutant HBQ strain was obtained using the same methods.In this strain,the expression levels of several related enzyme genes in the folate synthesis pathway were increased to varying degrees,and the folic acid monoglutamic acid folate content was also increased to some extent before 108 h.When the folic acid content of the three strains was determined by liquid chromatography-mass spectrometry(LC-MS),it was found that the three strains showed a decrease in the content of folate polyglutamic acid folate during the growth process,monoglutamic acid folic acid is the opposite.By comparing the growth and the ability to inhibit bacteria of the three strains,there is not much difference.Secondly,the LAB constitutive plasmid p MG36 e was used to overexpress the fol Q gene to obtain the BDQ strain.The results showed that the expression of the fol Q gene was significantly increased,and the expression levels of other genes were also increased.The monoglutamate folate production rate of BDQ strain was faster than that of YML4-3 strain in the first 24 h,and the consumption rate was slower than that of YML4-3 strain within 24-48 h,and decreased after 48 h,while YML4-3 strain increased.Finally,since the ?fol Q strain still produces folic acid,the isozyme gene of the FOL Q enzyme——Xtp1,was found based on the genome sketch of the YML4-3strain.The purified Fol Q enzyme and Xtp1 enzyme were obtained through a series of operations using the p ET28 a protein expression vector to confirm that they were indeed isozymes.And this is the first time to extract DHNTPase from L.plantarum strains,laying the foundation for future experiments.This study systematically studied the biological function of the fol Q gene in the folate synthesis pathway from the perspective of gene,protein and enzyme engineering,and laid the foundation for finding the key enzyme genes and folic acid molecular synthesis mechanism in the folate synthesis pathway of L.plantarum.Later exploration of other genes of L.plantarum.in the folic acid synthesis pathway provides ideas,and is more conducive to the development of LAB folic acid fermented food.