Improving The Production Of 4-aminobenzoic In Engineered Escherichia Coli By Combinatorial Regulation

p-aminobenzoic acid(PABA)is one of important chemicals for organic synthesis and has been extensively used in pharmaceutical and dye industry.In recent years,PABA has received increasing attention as a potential component of high-strength polymer and can also be applied to the surface treatment of materials to give the surface antibacterial property.At present,PABA is produced by chemical synthesis with serious pollution,thus bring the great concern to environmental preotection.Hence,it is necessary to develop the green biosynthesis of PABA and its downstream products for the future application.In this thesis,to produce PABA via the biosynthesis,the synthetic pathway of PABA was constructed and optimized by combinatiral regulation of key enzymes for obtaining the cost-effective Escherichia coli cell factories.Three genes of pabA,pabB and pabC involved in PABA synthesis in was combinatirally regulated with three kinds of constitutive promoters with low,medium and high strength,respectively,to improve the production of PABA.At the same time,in order to reduce the accumulation of by-product tyrosine,chorismate mutase/prebenzoate dehydrogenase(TyrA),a key enzyme in tyrosine synthetic pathway,is weakly regulated with low-strength promoter.This regulation resulted in not only reducing accumulation the the by-product tyrosine,but also increasing the accumulation of PABA significantly.Finally,0.67 g/L of PABA was produced in the shak-flask fermentation experiment.The optimal temperature for microbial production of PABA was also confirmed to 35? by shak-flask fermentation.After further condition optimization,the engineered E.coli produced 6.4 g/L PABA under 5 L fed-batch fermentation.In addition,according to a report in the literature,the new synthetic pathway for microbial production of PABA was explored.By phylogenetic tree analysis of key genes,two functional genes from P proteobacteria was confirmed to directly converse 3-Deoxy-D-arabino-heptulosonic acid 7-phosphate in the shikimate pathway to produce PABA.To sum up,the maximum titer of PABA produced by E.coli was achieved via combinatorial regulation of key enzymes,and the new pathway for PABA production was verified.This study can provide the important reference for improving microbial productionof PABA as well as many other biobased products..