Cloning And Expression Of 5-Aminolevulinic Acid Synthase Gene And Study On Biosynthesis Of 5-Aminolevulinic Acid

5-Aminolevulinate (ALA) is the first common precursor in the biosynthesis of tetrapyrrole compounds such as heme, porphyrins, chlorophyll and vitamin B₁₂, and also is a key metabolic intermediate to regulate the tetrapyrroles biosynthesis. ALA is widely existed in the cells of microorganisms, plants and animals. Recently, it has found that ALA can be used as biodegradable, herbicide and insecticide as well as growth-promoting factor in agriculture and is a new generation of photodynamic therapy in medicine, which has potential application for the treatment of various kinds of cancers, for example those developed in skin, oral, esophageal, colon, duodenal, pancreas and bladder, etc. Therefore, it is of great importance to enhance research and development of 5-aminolevulinic acid production process.In this work, genes of ALA sunthases were cloned from two kinds of microbes and the genetic engineering cells were constructed for ALA production. After detailed studies in the optimization of culture conditions, the ALA productivity increased dramatically.The major progresses in the research work are as followings:1) The genes (hemA) of 5-aminolevulinic acid synthase (ALAS) were cloned from Agrobacterium bacterium zju-0121 and Rhodobacter sphaeroides respectively. Through the comparison of gene sequences between this work and literature data, it was found that the hemA gene from Agrobacterium bacterium zju-0121 is a new one and that from Rhodobacter sphaeroides has been mutated in the 335th amino acid.2) Several plasmids were constructed with hemA gene from Agrobacterium bacterium zju-0121 and Rhodobacter sphaeroides respectively. According to the ALA productivity and ALAS activity, it was determined that E. coli BL21(DE3)(pET28-A.R-hemA) was the best strain for ALA production.3) The cultivation conditions of strain E. coli BL21 (DE3) (pET28-A.R-hemA) was evaluated at shake-flask scale. The effects of initial glucose concentration, dissolved oxygen, temperature, precursors and inhibitors of ALA dehydratase were examined, and the cultivation as well as induction conditions were optimized. The highest ALA productivity reached 1.3g/l, which is 16.2 times higher than that in initial conditions.4) The cultivation of recombinant engineer cell for ALA production was further studied in a 5 L fermentor. The effects of initial glucose concentration, dissolved oxygen, pH value as well as the culture strategy were studied. Under optimized conditions, the ALA productivity was as high as 3.1 g/L, which is 238.5% higher than that with shake-flask culture. The fermentation kinetics was studied based on the experimental data from 5 L fermentor. The equations for cell growth, substrate and precursors consumption and ALA formation were derived. The results indicated that model caltulations were in good agreement with the experimental data.5) Because ALA is a non-protein product, the function of expressed ALA sunthase is only a catalyst for ALA synthesis. A new strategy to induce gene expression was proposed and lactose was used as inducer instead of expensive and toxic stronger inducer of isopropyl-p-D- thiogalactopyranoside(IPTG). With fed-batch cultivation in the 5 L fermentor, the ALA productivity and ALAS activity increased by 43% and 15%, respectively.6) In order to facilitate the product separation and reduce the cost, it was found that D-glucose was a substitute of levulinic acid to inhibit activity of ALA dehydrates. Under favorable fed-batch cultivation process, the extracellular ALA productivity was 3.1 g/1.7) The effects of various factors on the ALA stability in fermentation broth were examined. The results revealed that the high concentration of ALA, high dissolved oxygen, high temperature, high pH value and light would cause the condensation reaction of ALA and were unfavorable for ALA stability. The experimental data were useful for the design of cultivation and product separation processes.