Breeding Of 5-aminolevulinic Acid-producing Photosynthetic Bacteria,expression And Enzymatic Properties Analysis Of HemA

5-Aminolevulinic acid(5-ALA)is an uncoded amino acid containing five carbon atoms and found commonly in animals,plants,microorganisms and algae.5-ALA has extremely important application value,and which is not only the synthetic substrate of vitamin B12,chlorophyll and heme but also the important intermediate of tetrahydropyrrole compounds.Also,5-ALA can be used as plant growth regulator,insecticide,herbicide in agricultural production and photosensitizer for cancer treatment in clinical medicine.However,as an intermediate for synthesizing tetrahydropyrrole,5-ALA has less content in the organism.Though 5-ALA is mainly synthesized by chemical approach,this method generally has low yield and is environmental friendliness.Therefore,the heterologous expression of 5-aminolevulinic acid synthase(ALAS)gene has been gradually developed with highly efficient synthesis of 5-ALA.However,it is hard to verify the ability of 5-ALA production of such many microorganisms one by one,consequently the variety of strain that product 5-ALA is still less and making the production of 5-ALA difficult to break through.To develop a high-yield 5-ALA microbial resources,the photosynthetic bacteria from nature were screened,enriched and purified to obtain a strain with high-yield of 5-ALA,then which were identified by way of morphology and molecular biology.RrHemA of the high-yield strains were cloned and preliminarily identified with aspects of structure,function and expression.Furthermore,to enhance5-ALA production capacity of the recombinant strain,culture conditions were optimized.The main results are as follows:(1)Through multiple separation,screening and purification,8 photosynthetic bacteria were obtained from collected water samples,and named as A1?A8.The ability of these 8photosynthetic bacteria producing 5-ALA was determined.A3 was found to be the dominant strain and had the highest 5-ALA production with a yield of 3.62 mg/L,and was so far at the upper-middle level of the found wild strain yields found.Based on the morphology characteristics of colonies and bacterial cells,and the results of 16S r DNA sequence analysis,A3 was identified to be an Alpha-proteobacteria,belongs to Rhodospirillales of Rhodospirillaceae of Rhodospirillum Molisch,and named as Rhodospirillum rubrum-A3.(2)Two RrHemA were cloned from Rhodospirillum rubrum-A3,named RrHemA1and RrHemA2,respectively.Results of bioinformatics analysis showed that ORF of RrHemA1 and RrHemA2 were 1227 bp and 1278 bp,encoding a protein with length of 408aa and 425 aa,and the relative molecular masses were 45417.84 k Da and 46482.87 k Da respectively.RrALAS1 and RrALAS2 proteins had no transmembrane domain and signal peptide,and they were non-secreted proteins.Analysis of the functional domain showed that RrALAS1 had a strictly conserved aminotransferase domain,thus RrALAS1 was speculated to have strong catalytic ability.(3)The pQE30b(+)-RrHemA1 and pQE30b(+)-RrHemA2 prokaryotic expression vectors were successfully constructed and transferred to E.coli M15 for expression analysis.Results of expression analysis showed that the optimal expression conditions of recombinant RrHemA gene were:0.5 mmol/L IPTG,cultured at 28?for 8 h.After purification,RrALAS1 and RrALAS2 were obtained with concentrations of 1.6682 mg/m L and 0.7440 mg/m L,respectively.Enzymatic analysis showed that the optimum temperature of the reaction of RrALAS1 protein was 37?,the optimum pH was 7.8,K_m^S-CoA was 0.1650 mmol/L.The activity of RrALAS1 was significantly inhibited by SDS,EDTA,Zn²⁺,Cu²⁺and Co²⁺,and Enhanced by Mg²⁺.The optimum temperature and pH of RrALAS2 could not be determined,due to its low activity.(4)The fermentation conditions for recombinant strain E.coli-M15/pQE30(+)-RrHemA producing 5-ALA was optimized using single factor experiment and Box-Behnken response analysis.Results showed that the optimal culture conditions for producing 5-ALA were:pH 6.5,temperature 28?,cultivated 8 h,nitrogen source 1.0 g/L ammonium bicarbonate,carbon source 1.5 g/L xylose,and the best combination of precursor for 5-ALA synthesis was 0.5 g/L glycine,5 g/L succinic acid,1.19 g/L cysteine and 1.19 g/L?-ketoglutarate.Under this condition,production of 5-ALA increased from1.09 g/L to 2.63 g/L.