Construction Of Recombinant Yeast For Production Of 5-Aminolevulinate Acid And Optimization Of Fermentation Process

5-Aminolevulinic acid(ALA),a kind of amino acid derivative widely existed in the cells of animals,plants and microorganisms,is an important intermediate for the synthesis of pyrrole compounds.ALA has been widely used in the fields of agriculture and medicine.ALA can be used as herbicide,pesticides and plant growth regulator in the agricultural filed.At the same time it is a green and environment friendly product due to its properties of easy decomposition and no residue in the environment.ALA can also be used for the treatment of various cancers,such as skin cancer,bladder cancer and rectal cancer.Compared to other agents,ALA treatment has the advantages of good curative effect,little side effects and the accurate and stable treatment;so it is believed that ALA had great potential in the field of cancer therapy.Therefore,further development and application of ALA will have immeasurable economic and social benefits.At present,the large scale production methods of ALA mainly include chemical synthesis and microbial fermentation.However,the chemical synthesis has disadvantages,such as complicated steps,serious pollution,and low yield recovery.As environmental awareness strengthens,the chemical synthesis of ALA is expected to be replaced by microbial fermentation in the near future.But at present,the ALA yield by microbial fermentation is very low,which can not meet the requirements of industrial production.As the development of genetic transformation in microbial strains as well as the optimization of fermentation process with a series of achievements in recent years,the industrial production of ALA by microbial fermentation is expected to be realized.In this subject,the key gene of 5-aminolevulinic acid synthesis from Saccharomyces cerevisiae was integrated into the genome of Pichia pastoris GS115 through homologous recombination and regulated by constitutive expression pattern.The recombinant GS115 strain,UJNPA,with high yield of ALA was successfully constructed.The yield of ALA was two times higher than that of the wild GS115 strains.The recombinant vector is pGAPZA and the promoter is GAP.Taking constructed strain,UJNPA,as the starting strain,effects of six factors(succinicacid,glycine,malonic acid,garlic extract and levulinic acid)was investigated with regarde to the ALA production.Through the single factor experiment and response surface experiment,the optimazed parameters of fermentation conditions are: succinic acid 6.11 g/L,glycine 1.67g/L,malonic acid 2.46 g/L,garlic extract liquid 7.24mL/L,levulinic acid 1.5 g/L,glycerol 8mL/L;and the greastest ALA accumulation was 1783.33 mg/L under the optimal conditions.Under the above experimental conditions and feeding 2%(v/v)methanol as auxiliary carbon source at 12 h interval,the maximum ALA yield(2256.0mg/L)was achieved.