Optimization Of Conditions For 5-aminolevulinic Acid (ALA) Production In Escherichia Coil

5-aminolevulinic acid (ALA) is a kind of nonprotein amino acid, which has five carbons. It is an important precursor for pyrrole biosynthesis, such as chlorophyll, heme and vitamin B12. ALA has very important application in the field of agricultural that can be used as plant growth regulating factor, herbicides and pesticides. In the field of medicine, ALA can be used as a photodynamic diagnostic reagent and treatment drugs for tumor and skin cancer. Therefore, more and more attention will be paied on ALA, and it has important value of market development and application prospect.At present, chemical synthesis is a main method to produce ALA on industrial production. However, chemical synthesis is very complicated. There are some disadvantages, such as too much by-products, low yield and environmental pollution. Therefore more and more researchers have focused on the microbial synthesis of ALA. The main production has given priority to microbial synthesis via C4 pathway, but it needed to add glycine and succinic acid as substrates to the reaction. In addition, the production of ALA in the C4 pathway has a high cost, which is not suitable for large-scale industrial production. Our laboratory has producted ALA via C5 pathway innovatively, which added the only substrates such as glucose. We overexpressed the genes that coding two key enzyme in C5 pathway. The one hemA encodes glutamine-tRNA reductaseand the other one hemL encoding glutamine-1-half aldehyde transaminase gene. In addition, we remould ALA transportation system to transfer ALA. In the end, we constructed recombinant E. coli (DALA) to produce ALA. However tour laboratory constructed the recombinant strains producing the maximum yield of ALA is only about 4 g/L, and the recombinant plasmid is not stable to product ALA. Therefore, we need to further optimize the fermentation conditions in order to improve the yield of ALA, and structure the stability of ALA production strains at the same time.Firstly, my reseraches used the recombinant E. coli (DALA: DH5α/pUC19 hemAM -hemL + pCL1920 - rhtA) that our laboratory constructed to produce ALA via C5 way. I attemptted to use the calcium carbonate taht commonly used cheap industrial fermentation pH regulator instead of the laboratory normal pH regulator NaOH to adjust the pH of the fermentation process, and added tryptone in ALA fermentation medium to get higher yield of ALA. However we found that the calcium carbonate and tryptone can decrease the accumulation of ALA. Secondly, this researches optimized fermentation condition, such as the fermentation temperature, pH, dissolved oxygen, inoculation amount and medium composition optimization experiment, to determine that the recombinant E. coli (DALA) have adapted to the fermentation tank culture conditions. In a 5 L fermentation tank’s fermentation experiment, we set speed to 400 rpm/min and 1.5 VVM ventilationis, when recombinant strains are in the early stage of the fermentation stability, then plateau speed is 200 rpm/min,0.5 VVM, dissolved oxygen in the late fall of 0 %. The fermentation is in microaerobic conditions; The fermentation temperature is 37℃; Fermentation pH is 6.5 before the strain growth did not reach a plateau stage, when in the plateauing stage, pH is 6.0. The maximum yield of ALA is 5.3 g/L, which is 1.3 times higher than laboratory early optimized the fermentation conditions of 5 L fermentation tank fermentation to product of ALA. Then we have tried to ALA from the fermented liquid through a series of coarse separation purification steps to obtain crude product ALA. first of all, we acidificated ALA, in order to improve the stability of ALA, high-speed centrifugation and membrane treatment to remove impurities such as bacteria, reverse osmosis membrane in addition to get rid of water, active carbon decoloring, rotaryed evaporation concentration, freezed drying. In the end, we obtained ALA crude product.Fermentation strains containing the plasmid is not stable, easy to lost. Then it often causes the problem, which product production is not stable. In this reseraches, by using laboratory building FRT/FLP site specific restructuring method, we can integrate the ALA C5 pathway of two key genes hemAM-hemL into the host which knock out the arcA and recA genes of DH5a. By detecting copy number, we can construct an industrial production of ALA strains that does not rely on plasmid. The stable strain which has the highest 6 gene copy number can produce ALA to 0.6 g/L via fermentation experiments.To sum up, this thesis in the laboratory building of ALA recombinant strains (DALA). Through various means to optimize the fermentation conditions, the yield of ALA is improved. Then we also carried on the crude extraction of ALA. These tests will provide guidance for the optimized fermentation conditions and downstream purification in the future.Through the FLP/FRT site specificity restructuring method random integration in the E. coli genome, we constructed a stability stain for ALA production, which will provide a good foundation for industrial production of ALA.