Study On 5-aminolevulinic Acid Transporters In Escherichia Coli

5-aminolevulinic acid(abbreviated as ALA),a precursor for synthesizing four-pyrrole compounds in living organisms,has been widely used in agriculture and medical photodynamics therapy.At present,production of ALA by microbial fermentation has made significant achievement.However,excessive ALA accumulation in cells resulted in oxidative damage to biological macromolecules.Accelerating ALA excretion with the aid of an ALA transporter is a direct method to reduce ALA concentration in vivo.Few research on ALA transporters was done instead,and no feasible method is developed for searching ALA transporters.Over-production of ALA may change the expression level of some transporters,and transcriptomics analyzes transcription level of all genes under a certain physiological condition.In the present thesis,comparative transcriptomic study was carried out to compare expression differences between ALA overproducing strain and corresponding control strain,so as to find ALA transporters.Major research content and results are shown below:(1)A plasmid stability system was constructed in a chassis strain.Gene A on genome was knocked out and another copy was constructed on the plasmid.(2)A control strain producing almost no ALA was constructed.A mutation was introduced into the active site of levulinic acid synthase(ALAS),which resulted in no ALAS activity without affecting its soluble expression.Cells harboring mutant-carrying plasmid showed no obvious growth defect.(3)A method for detecting ALA in vivo was established.Samples were taken at four time points,when cells have significant change of ALA concentration in vivo,.RNA were extracted and transcriptomic sequencing was performed.By analyzing transcriptomic data,together with E.coli TransportDB database,transporter genes with potential ALA transfer function were selected.(4)The role of these putative ALA transporters was evaluated by studying the effect of overexpressing or defect of these genes on ALA accumulation.The results showed that EamA,RhtA、EmrYK、G1tIJKL、FliYYecSCand glnHPQ are involved in ALA efflux.The present study provided several novel targets for metabolic engineering of recombinant strains for ALA overproduction.The method established in this thesis will also benefit other transporters mining study.