Study On Chorismate Lyase In Microbulbifer A4B-17

The Microbulbifer sp.A4B-17 strain was isolated from the ocean.we found that it can make use of renewable resources such as carbohydrates to synthesize 4-hydroxybenzoic acid(4HBA)and its esters.4-Hydroxybenzoate(4HBA)is an important chemical compound used for synthesis of liquid crystal.4-Hydroxybenzoic acid esters are widely used in food,beverages,cosmetics,medicine and many other areas because of its good sterilization ability and stability.At present,the production process of hydroxybenzoic acid in China mainly uses petroleum products benzene and propylene.On the one hand,the whole production process of high temperature and pressure conditions are very difficult to control.On the other hand,the production process consumes a lot of energy,so that greatly increased production costs.In addition,there are environmental pollutants such as phenol in the production process.Production of 4HBA from renewable resources is an effective mean to solve problems such as environmental pollution and petroleum shortage.The purpose of this study is to understand the A4B-17 strain of the use of renewable resources on the synthesis mechanism of 4-hydroxybenzoic acid,and determine the key genes in the biosynthesis pathway,which will construct A4B-17 strain to be "cell factories" of 4-hydroxybenzoic acid with environmental protection and efficient production.In this study we have conducted the sequence of A4B-17 strain's genome and give some analysis of the genome.Genome sequencing showed that the genome size of A4B-17 strain is 5035829 bp,with a total of 4604 genes.We have found 3 metabolic pathways for the synthesis of benzoic acid and esters.They are the glycolytic pathway,the ED pathway,and the pentose phosphate pathway.According to the annotation information of genome,we have speculated that the GM004533 gene encodes the enzyme of the chorismate lyase,which catalyzes chorismate lyase into the 4-hydroxybenzoic acid.we have cloned and expressed the gene of chorismate lyase in Escherichia coli.The recombinant pET-21a(+)-GM004533 was successfully constructed by cloning the chorismate lyase lyase gene into the vector pET-21a(+),and then transformed into E.coli BL21(DE3)for protein expression.SDS-PAGE showed that it had higher expression level,and the enzyme activity was detected using HPLC.Secondly,we have looked into the unique cultural medium of the A4B-17 strain and detected the expression of Chorismate lyase lyase gene in different cultural medium by using fluorescence quantitative PCR.The A4B-17 strain is able to grow on 10 different carbon sources(glucose,xylose,sucrose,fructose,galactose,glycerol,mannitol,sodium pyruvate,sodium alginate,Arabia sugar).Because it is the only carbon source c?ltural medium,it takes about 3 days to grow to be stable.Under different c?lture conditions,the expression of mRNA in the glucose medium was highest.In conclusion,All these results will pave a foundation way for the development of the strain into an industrial microorganism.