Construction Of An Efficient Prokaryotic Expression System For 5-Aminolevulinate Acid And Its Applications

5-aminolevulinate acid(ALA) is the first common precursor in the biosynthesisof tetrapyrrole compounds such as heine, porphyrins, chlorophyll and vitamin B₁₂,and also is a key metabolic intermediate to regulate the tetrapyrroles biosynthesis.ALA is widely present in microorganism, plant and animal. Recently, it has beenfound that ALA can be used as biodegradable, herbicide and insecticide as well asgrowth-promoting factor in agriculture. Moreover, ALA can be used for preventingand curing plant virus diseases. ALA, a new generation of photodynamic therapy inmedicine, has potential application for the treatment of various kinds of cancers, andits outstanding advantages include less side effect, better curative effect, penetrabilityand lower price, etc.Therefore, it is of great importance to emphasize research anddevelopment of 5-aminolevulinic acid producing process.On this study, genes of ALA synthases were cloned from Rhodoblastus acidoph-ilus and the genetic engineering cells were constructed for ALA production. To increa-se the amount of ALA production, the culture conditions were optimized, Furthermore,we found that exogenous ALA can influence of the physiological index in tobacco andpepper, such as chlorophyls, output and the ability of living in the adversity.The major progress in this work are as follows:1. A specific primer for 5-aminolevulinic acid sythase (ALAS) gene was design-ned based on the sequences comparasion of Rhodo lastus acidophilus with other phot-osynthetic bacteria, The ALAS gene was further subcloned into an expression vectorpQE30, namely ALAS-pQE30. For the production of ALA, the recombinant ALASwas overexpressed in Escherichia coli strains JM109, M15 and BL21 (DE3), respect-ively. The optimal strain for producing ALA is M15.2.The various of condition including strain, medium, substrate of ALA synthesi-ze (glycine and succinic acid) and ALA dehydratase inhibitor (levulinic acid) were op-timized for attainning the maximum yield of ALA in E. coli. The highest ALA produc-tion was found with E.coli M15,mediumA supplied with 0.08 mol·L^-1 glycine, 0.1 mol·L^-1 succinic acid,20 mM levulinic acid increasing intermittently and 0.1 mmol·L^-1.The activity of ALAS was up to 333U/min·mg of protein in these condition. Meanwh-ile, the output of ALA was reached to 5.379g/L after 20h incubate,which is the highestyield of ALA up to date by biofermentation.3.After destroying the enzyme activity treating fermentation broth with high tem-perature and SDS, ALA stability is increasing. ALA is most stable after treating with1% SDS in-20℃.4.The content of chlorophyll and the weight of Specific Leaf Area in the tobaccoand pepper leaves were increasing after plant were treated with exogenous ALA.Moreover, the activity of POD and CAT were increased.5.The growth ability of tobacco and pepper under the frost and drough stress wasimproved after the plants were treated with exogenous ALA.