The Preliminary Study On Limiting Factors For 5-Aminolevulinate Production Using The Recombinant Engineering Strain

5-aminolevulinic acid (ALA), an intermediate in the biosynthesis of hemes, chlorophylls and vitamin B12 ,has been used extensively as a selective and biodegradable herbicide and insecticide in agriculture. It can improve the crops to resist cold temperature and tolerant salt. In addition, ALA can be used in the diagnosis of tumor and cancer treatment. So far, the ALA production has been applied from either the mutants of the some photosynthetic bacteria species or the Escherchia coli overexpressing 5-aminolevulinic acid synthase (ALAS) which catalyzes the succinyl-CoA and glycine to condense ALA. In the organisms, the biosynthesis of ALA was inhibited by heme feedback. In this study, the active ALAS from Saccharomyces cerevisiae was overexpressed and had the ability to catalyze the formation of ALA. The contents of ALA in vivo and invitro were determined. The limiting factors on the ALA content catalyzed by the recombinant ALAS were investigated preliminarily . The rapid ALA purification procedure were estabilished. All results in this work were described as followed:1. The primer and anti-sense primer were designed deliberately based on the published sequence of hem1 gene from yeast, which encodes the assumed mature ALAS from the first amino acid residue Asn63. The PCR reaction was performed including the designed primers, and yeast genome as the template. A main band about 1500bp was shown by agarose gel electrophoresis. The amplified PCR product was inserted in the vector pMAL-2x, to yield the recombinant expression vector, named pMAL-ALAS. The fragment and its correct insertion was determined by sequencing.2. The recombinant plasmid and the control vector are transformed into Escherichia coli expression strains respectively. The colonies carrying the constructed plasmid displayed strong red fluorescent upon UV light radiation, denoting the downstream oxidative intermediates porphyrins were accumulated. The retardation of growth for the strains expressing yeast ALAS was observed, resulting from the accumulating porphyrins which destroyed the cells membranes and thus caused the cells growth slowly. The molecular weight of subunit of solubly expressed recombinant ALAS was estimated about 49kDa, as shown by SDS-PAGE. The optimum of inducer IPTG for maximum ALAS activity was 0.1 mmol/L.3. Several factors for limiting ALA biosynthesis from the recombinant strains were examined. The pH value was most considering factor that affecting the ALA production. At pH 7.0, the ALAS activity was identified to maximum, and at pH 6.5, the rate of the ALA production-the growth of recombinant in E.coli BL21(DE3) was up to maximum. Exogenous pre-substrate succinate influenced the ALA production and at the concentration of 20mmol/L up to maximum .The additional glycine at the concetration of 20mmol/L stimulated the biosynthesis of ALA, whereas that at 80mmol/L inhibited the cells growth.4. The national-made large-dimensions resin I was effective for absorption for porphyrins except ALA, and resin II was specific absorbed for ALA. The capillary electrophoresis measurements confirmed that the high purity of ALA after cooling-drying was obtained through the two-step purification .To summarize, the paper reported the research on the clone of hem1 gene from Saccharomyces cerevisiae encoding 5-aminolevulinic acid synthase, the construction of expression vector and optimized some limiting factors for ALA production, and the establishment for rapid and cheap ALA purification. All results provided the basis for the further industrial ALA production.