Study On The Biosynthesis Of 4-hydroxybenzoate And Parabens In Microbulbifer

4-Hydroxybenzoate(4HBA)is a widely used organic synthetic raw material,mainly used in the synthesis of liquid crystalline polymers and 4-hydroxybenzoate esters.A variety of fields,such as chemical,food,cosmetics,medicine and so on,expect a substantial demand for 4-hydroxybenzoate esters as preservatives.Currently,4HBA is synthesized by petroleum products under the condition of high temperature and pressure with a large number of consumption of energy.Besides,this process generates quantities of phenol which is an environmental pollutant.At present,in order to solve the environmental pollution and resource depletion,biosynthesizing 4HBA and its derivatives by renewable resources is necessary.Thus,we isolated Microbulbifer sp.A4B-17 from marine environment.After a preliminary investigation,we found that it is able to naturally synthesize 4HBA and its esters.In order to transform the strain A4B-17 into an industrial strain,I have carried out a series of studies on genome sequencing,the expression of key genes,secondary metabolites analysis and so on.Firstly,inorganic medium was used as basic medium to detect the utilization of carbon source of strain A4B-17.The results manifested that strain A4B-17 could grow in different mediums with glucose,xylose,fructose,glycerol,sucrose,galactose,arabinose,mannitol,alginate or pyruvate.The average resultant quantity of 4HBA was about 10 ?mol/L.The maximum was 19 ?mol/L produced from sucrose and the minimum was 2 ?mol/L produced from glycerol.Last,the genome of strain A4B-17 was sequenced and annotated.The sequencing result showed that strain A4B-17 did not contain plasmid and had only one chromosome.The gene size is 5,040,512 bp with 48.73% GC content and 4,766 annotated genes.We found three metabolic pathways(EMP,HMP and ED)for utilization of sugars.Shikimate pathway which is a key rout for biosynthesis of aromatic compounds was found on the genome sequence.Then,we verified some key genes of these pathways by reverse transcription PCR method to determine whether or not they play a role in the synthesis of 4HBA and esters.These key genes are involved in the glucose transport,glucose degradation,DAHP synthesis,4HBA synthesis and parabens synthesis pathways.It is showed that these key genes had been expressed,which indicating that the metabolic pathway of genome prediction had physiological functions.Finally,we added alcohols such as methanol,ethanol,normal alcohol,butyl alcohol to the culturing media.And the results showed that synthetic amount of 4HBA was reduced and the 4HBA esters were increased.This indicated that A4B-17 had the ability to synthesize 4HBA and alcohols separately.The esterification reaction between them resulted in parabens synthesis.And the carboxylesterase gene also existed in genome information.In a summary,I illuminated strain A4B-17 for the utilization of carbon sources and synthesis of secondary metabolites;carried out the genome sequencing and annotation;investigated influences by adding various factors.These results will pave a foundation way for the development of the strain into an industrial microorganism.