The Secection Of 5-Aminolevulinic Acid Producing Strain And The Cloning And Expression Of Its Synthase Gene

5-aminolevulinic acid （ALA） is a kind of hydrocarbons which contain oxygen and nitrogen. It is the biosynthetic precursor of pyrrolidine. ALA is a porphyrin, （ferrous） heme, chlorophyll, as well as the structure of vitamin B12 analogues. The physiological activity of ALA is very active and ALA has a very wide range of applications. It is a green agricultural pesticide which has no toxicity, is degradable easily. It is applied in pharmaceutical as curative drug and diagnostic reagent for against cancer, and has the role of accelerating the production haemachrome and hair. Biosynthesis ALA by microbial fermentation has some advantage, for example, easy to train, non-toxic, low cost etc. In view of these superiorities and wide application of ALA, biosynthesis of 5-aminolevulinic acid were studied in this paper, mainly including the screening of photosynthetic bacteria, the identification of molecular biology, the selection of culture conditions, mutation breeding and ALA synthase gene （hemA） cloning.Using separation culture medium as a specific model, five photosynthetic bacteria were selected, and were named respectively G-sludgeâ… , G-sludgeâ…¡, G-5, G-7, G-14. By studying the result of growth and ALA product of these strains, a high-yield strain named G-sludgeâ…¡was got and its ALA product was about 4.5mg/L. For identification the strain, 16sRNA had been amplified by PCR genomic DNA from G-sludgeâ…¡as template. By analysis the sequence of PCR products, the 16S rRNA sequence of G-sludgeâ…¡was most closely related to Rhodobacter sphaeroides with 99% nucleotide being identical.The growth and ALA metabolism characteristics of G-sludgeâ…¡were studied. The optimal mediums and culture conditions were confirmed: sodium acetate 4g/L, panceas peptone 10g/L, inoculum 10%, 50mL of medium （pH 7.0） in 250mL Erlenmeyer flask, glycine 100mmol/L, succinate 60mmol/L, aerobic dark, 30℃, 180r/min, for 96 hours. ALA final yield can be achieved 24.73mg/L.In order to increase the product of ALA, mutation breeding was performed with the ultraviolet treatment and lithium chloride. A high yield product ALA mutant was obtained and producted 24.26mg/L of ALA.In order to enhance ALA productivity, the gene coding ALA synthetase was needed cloned into expression plasmid and was expressed in E.coli. A PCR was performed genomic DNA from G-sludgeâ…¡as template and 30bp DNA designed according to the sequence of ALAs of Rhodobacter sphaeroides in GENEBANK as prime, and the 1230bp target DNA was obtained. Through ligation and transformation, hemA-PEASY-T1 was constructed and was identified by digesting with EcoRI and HindIII. The Results of restriction enzyme digesting and plasmid sequencing indicated that the hemA was cloned triumphantly. Through the comparison of gene sequence with NC₀07493 USA Rhodobacter sphaeroides, it found that consistency was 99.92% and there was only a base pair mutant. The amino acids sequence of hemA has 99% identity with the aboved strains.The expression vector, pET30a-hemA, was constructed and transformed to E.coli BL21 （DE3）. hemA was overexpressed in 37℃culture temperature. Analysis and detecting by SDS-PAGE, the protein bands of about 50kD expected molecular mass of the hemA was visualized clearly.