Mechanism Study Of Para-nitrophenol Degradation And Its Effects On Glucose Metabolism In Pseudomonas Putida DLL-E4

Para-nitrophenol?PNP?is an important chemical raw material among the nitrophenol,which can be used in the production of pesticides,pharmaceuticals,dyes and other substances.PNP can accumulate in the nature a long time and can cause serious pollution to the environment.Nowadays many PNP-degrading bacteria have been isolated,and the PNP-degrading pathway have been studied extensively.However,the study of carbon cataboite interactions between PNP degradation and other carbon source metabolic and the temperature-sensitive regulation of PNP degradation is still unknown.In this study,we use Pseudomonas putida DLL-E4,a PNP degrading microorganism,as raw material,and observe the metabolism when DLL-E4 grown on 0.25%Glucose plus 1mM PNP in minimal salt medium?MSM?.There was a series of interesting phenomena,such as glucose can not be fully utilized,the cell growth is badly and the degradation of PNP is accelerated.It is believed that there is carbon catabolite interactions of DLL-E4 in the process of simultaneous metabolism of glucose and PNP.Studying this metabolism process,finding that PNP was a key factor affecting glucose metabolism in strain DLL-E4,and could make the metabolic medium acidified.Gluconic acid?GA?and 2-ketoglugonate?2KG?was identified using GC-MS in the fermentation broth which DLL-E4 simultaneousmetabolism glucose and PNP.In order to know about the reasons of the generation and accumulation of organic acids,the glucose dehydrogenase gene?gcd?and gluconate dehydrogenase gene?gad?were knocked out from the DLL-E4 genome by homologous recombination.These mutant strains were cultured under different condition of metabolic,to study the three peripheral pathways of glucose catabolism in DLL-E4.In the presence of PNP,the metabolic pathway of glucokinase was significantly inhibited,indicating that most of the glucose was catalyzed by Gcd and then metabolized.Analyzing the pH tolerance of the peripheral glucose metabolic pathways in the presence of 1 mM PNP or 0.9 mM NO2-in the culture medium,it was found that the bacteria no longer used carbon source for growth when the pH value of the culture medium decreased to about 6.0.All results showed that most of used glucose was catalyzed by Gcd and Gad to GA and 2KG in the process of simultaneous metabolism of glucose and PNP.However,the metabolic process of GA and 2KG was affected due to the presence of PNP and N02-,organic acid was accumulation in the extracellular.When the pH of the culture medium was as low as 4.4,metabolism of bacteria stopped,glucose in this medium could not be used again,and most of the consumption of glucose was converted into organic acids accumulated in the extracellular instead of produce energy.By transposon tagging method for DLL-E4,the temperature-sensitive mutant MT54 is obtained.Compared with the wild strain,it was found that MT54 lost the ability of PNP degradation at 37?,while the ability of hydroquinone degradation was not affected.In order to know about the mechanism of temperature-sensitive degradation of MT54,the genomic difference of DLL-E4 and MT54 were analyzed.It was found that the DW66₀143,DW66₀153 and pnpB genes of MT54 were inserted by exogenous large fragments,which could cause gene inactivation.Studying of the mutations of these three genes in DLL-E4 genome,the results showed that the inactivation of pnpB gene was the cause of temperature-sensitive degradation of PNP by MT54.The interactions between PnpA and PnpB protein was studied by using bacterial adenylate cyclase two-hybrid system.It was found that there was no obvious inteaction between PnpA and PnpB protein.To study the effect of PnpA activity when PnpB was added,keeping the amount of PnpA in the reaction system,the amount of PnpB was gradually increased.The results indicating that there was product inhibition of p-benzoquinone in PnpA.Based on the above results,the following conclusions could be drawn:In MT54,PnpB could not be transcribed because of the insertion mutation of exogenous fragments,resulting that PNP metabolites could not be converted into HQ timely.The accumulation of p-benzoquinone formed the PnpA product inhibition.In addition,the activity of low temperature enzyme PnpA was weak at 37?,made MT54 basically lost the ability of PNP degradation at 37?.