Research On Controlled Degradation Technollogy Of 5-Aminolevulinic Acid Dehydratase In Propionibacterium Freudennreichii

5-aminolevulinic acid is one of the five carbon amino acids of the non-protein class,which is present in many microbial and animal and plant cells.5-Aminolevulinic acid is a precursor of pyrrole compounds(the heme of hemoglobin,chlorophyll,and vitamin B12,etc.)of the important intermediates,microbial cells in animal and plant cells and synthesis.It plays an important role in the metabolism of organisms.It is characterized as non-toxic,but its stability is lower and easy to degrade at normal temperature,and has no residual characteristics,it is recognized as a kind of green environmental chemicals.The 5-ALA synthetic methods mainly include both chemical synthesis and microbial fermentation,among which,Chemical methods for synthesis of 5-ALA and reaction conditions to have more difficulty,cumbersome steps,by-products generated more,separation is difficult,for the purification process is very complex,and because chemical reagent cost is higher,is not easy to get,containing toxic,low production rate,so bio-fermentation has become the main trend of future research and development.The main biosynthesis of 5-ALA metabolic pathways are C4 and C5 way two pathways.Microorganism fermentation.it is through mutated strain or restructuring engineering strains,through the optimization of fermentation conditions to make it a substantial accumulation of 5-ALA.5-aminolevulinic acid market demand is large,in agriculture,medicine,food and other industries are widely used.In order to increase the content of 5ALA in propionate,by inserting the ssrA gene into the hemB gene of Propionibacterium freudenus,the protein can be rapidly degraded by ClpXP protease,thereby reducing the activity of the pigmented synthase in the organism,resulting in an increase in the content of ALA in Propionibacterium.The hemB1 and ssrA primers were designed by genetic engineering technology.The construction of pKHEM04-hemB1-ssrA vector was completed by the target fragment amplification and transformation and Blue-White Screening and Double enzyme digestion test.The recombinant vector was transformed into Propionibacterium competent cells and expressed successfully.A protein band of about 50 kDa was detected by SDS electrophoresis.The 5-ALA contents of pKHEM04-hemB1 and pKHEM04-hemB1-ssrA were 26 mg/L and 35 mg/L,and the contents were increased,achieve the purpose of the experiment.