The Expression Of Genes Involved In Polymyxin E Biosynthesis In Paenibacillus Polymyxa And Construction Of Negative Regulator AbrB Mutant Strain

Polymyxin E belongs to cationic polypeptides antibiotic and consists offourtypesofdirectprecursoraminoacidsincluding L-2,4-diaminobutyric acid?L-Dab?,L-Leu,D-Leu and L-Thr.It has been reported that several genes,including pmxABCDE,spo0A,abrB,ectB,sfp,is related to polymyxin biosynthesis.In this dissertation,the expression of these genes and the construction of negative regulator abrB mutant strain had been studied.The results shows that the polymyxin E production is associated with the variation trend of the expression of genes involved in polymyxin E biosynthesis,especially spo0A?abrB and pmxA.Addition of direct precursor amino acids to high concentration significantly affected the expression of genes involved in polymyxin E biosynthesis and inhibited polymyxin E production.L-Dab,L-Leu and D-Leu repressed the polymyxin synthetase genes expression.L-Thr affected the expression of not only pmxA,but also regulatory genes spo0A and abrB.As the precursor of L-Dab,L-Asp also repressed the expression of ectB,pmxA and pmxE.Moreover,L-Asp affected the expression of spo0A and abrB.The electrotransformation method for P.polymyxa C12 was established.The results showed that the optimum method is OD₆₀₀00 0.8,electric field intensity 18 KV/cm,plasmid DNA 1?g,recovery incubation time 4h.To knockout abrB,the recombinant plasmid pMD19-H1KmH2 was constructed.H1KmH2 was amplified by PCR and transformed into P.polymyxa C12.abrB mutant strain were obtained by H1KmH2 transformation.