Construction Of Engineering Strains For Over-Producing 5-Aminolevulinic Acid

5-aminolevulinic acid （ALA） is a kind of photodynamic compound. 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. In agriculture, it is a green environment-friendly herbicides, insecticides, pesticides and plant growth promoting agents. ALA has a very wide range of application prospects in the areas of bio-pesticides and fertilizers owing to its many advantages, such as good effect, no toxicity, degradable easily and so on. 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 ALA synthase gene （hemA） cloning, constructing highly efficient recombinant Escherichia coli and developing new and effective approaches for fermentation process to enhance the ALA productivity. Main results are as follows:（1） In order to enhance ALA productivity,1224 bp hemA coding ALA synthetase-hemA gene was cloned from the photosynthetic bacteria screened by this lab. Through the comparison of gene sequence with NC₀07493 USA Rhodobactet sphaeroides, it found that consistency was 99.92% and there was only a base pair mutant（the 1204th site mutated from C to T）. The amino acids sequence of hemA had 99% identity with the aboved strains（the 335th site mutated from Ala to Val）.（2） pET30a-hemA was constructed and transformed to E.coli BL21 （DE3）. hemA was overexpressed in suitably conditions. Analysis and detecting by SDS-PAGE, the protein bands of about 45 kDa expected molecular mass of the hemA was visualized clearly.（3） The medium and cultivation conditions of engineering bacteria were optimized. The results indicated that the best culture medium was TB, the best temperature was 16℃ It was also found that the addition of glycine and succinate was effective to improve ALA production. While the concentration of glycine and succinic acid up to 100 mM, the extracellular ALA concentration reached 538 mg/L（4） Considering the problem of limited steps, precursor substance supply and product excretion, ALA synthesis C4 pathway was constructed in recombinant Escherichia coli. On the basis of plasmid pETDuet-1, ALA synthase gene（hemA）, propionate CoA-transferase gene（Cat） and ALA excretion gene （YBi） were integrated. Through the co-expression of above three gene, the extracellular ALA concentration reached 1 124mg/L.