| Pesudomonas putida is widely found in nature.It is an opportunistic pathogen,a common infectious disease in surgery,and a fish pathogen in the aquaculture industry.Because P.putida can grow in soils and sediments contaminated by high concentrations of heavy metals and organic pollutants,it has a high application prospect in bioremediation and biodegradation.In addition,the strain has extensive use value in the production of special industrial materials.Bacterial fatty acid metabolism,as a basal metabolism.It is provides energy for organisms and provides precursors for other important metabolic pathways.However,studies on fatty acid metabolism of P.putida have not been reported.Therefore,this study used bioinformatics,biochemistry,genetics and molecular biology techniques to study the function of 3-ketoacyl-ACP reductase(Fab G)in the fatty acid synthesis pathway of P.putida F1.Obtain the following results:Bioinformatics analysis showed that there are six homologous genes encoding3-ketoacyl-ACP reductase in the genome of P.putida F1: Pput?0620(Ppfab G1),Pput?2199(Ppfab G2),Pput?2972(Ppfab G3),Pput?3177(Ppfab G4),Pput?3800(Ppfab G5)and Pput?3860(Ppfab G6).The results of in vivo genetic complementation experiments showed that Ppfab G1,Ppfab G3 and Ppfab G5 genes could restore the growth of E.coli fab G temperature-sensitive mutant CL104 at non-permissive temperature.The Ppfab G3 and Ppfab G5 genes complemented better,while the Ppfab G1 gene complemented the growth.It was preliminarily proved that the proteins encoded by Ppfab G1,Ppfab G3 and Ppfab G5 have the function of 3-ketoacyl ACP reductase,but the catalytic activity of PpFabG1 is weak.3-ketoacyl-ACP reductase belongs to the superfamily of short-chain alcohol dehydrogenase/reductase(SDRs)and has a similar catalytically active core sequence(Ser-Tyr-Lys).This study will conserve three of Ppfab G3 and Ppfab G5.The sites Ser,Tyr and Lys were site-directed to Ala,respectively,and the p BAD24 M complementary series vector was constructed and complemented with CL104.The results showed that mutations in PpFabG3 S140 A could not restore CL104 growth at unlicensed temperatures,andmutations in PpFabG5 Y154 A could not restore CL104 growth at unlicensed temperatures.The importance of conserved site integrity for 3-ketoacyl ACP enzyme activity is illustrated.In order to further determine the function of PpFabGs protein of P.putida,the six enzyme proteins of PpFabGs were isolated and purified by nickel column affinity chromatography,and the fatty acid synthesis system was reconstituted in vitro.The results showed that PpFabG1,PpFabG3 and PpFabG5 had 3-ketoacyl-ACP reductase activity in the initiation and extension reactions of fatty acid synthesis in vitro,but the PpFabG1 enzyme activity was weak.PpFabG6 is only catalytically active in the initial reaction.In order to further study the function of Ppfab Gs,the suicide vector p K18 mobsac B vector was constructed and transformed into S17-1.Mutant strains of five other genes except Ppfab G5 were directly obtained by homologous recombination technology.In addition,by constructing the conditional mutant strain ?Pp G5 X,experiments showed that?Pp G5 X could not grow without the addition of an inducer,demonstrating that Ppfab G5 is an essential gene of P.putida.Physiological analysis of the mutant strain showed that the swimming motility of ?Pp G1(?Ppfab G1)was slightly weakened compared with the wild type,the iron carrier secretion of ?Pp G3(?Ppfab G3)was decreased,and the formation of biofilm of ?Pp G6(?Ppfab G6)was decreased,indicating that PpFabGs There are also differences in protein function.This study demonstrated that the proteins encoded by the Ppfab G1,Ppfab G3 and Ppfab G5 genes have 3-ketoacyl-ACP reductase activity,and PpFabG6 can only function as a 3-ketoacyl-ACP reductase in the initial reaction of fatty acid synthesis.PpFabGs have The function of 3-ketoacyl-ACP reductase also plays a different role in various physiological functions of P.putida. |
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