Functional Study Of Several Genes Involved In Fatty Acid Metabolism Of Pseudomonas Putida

Pseudomonas putida is a Gram-negative bacterium that can be used for controlling environmental pollution and to produce special industrial materials.However,researches on the mechanism aspects of its physiological metabolism are limited.This project uses biochemical,genetic and molecular biology techniques to study the function of the enzymes related to fatty acid metabolism of Pseudomonas putida.The following results have been observed:Pseudomonas putida F1 contains two genes encoding the fatty acyl-CoA synthetase in the same gene cluster,Pput?1340(PpfadD1)and Pput?1339(PpfadD2),respectively.The first part of the project was to identify and functionalize PpfadD1and PpfadD2.(1)Genetically complementations of the E.coli acyl-CoA synthetase mutant JW1794-1 with PpfadD1 and PpfadD2 showed that they were able to restore their growth on basal medium with oleic acid as the sole carbon source.At the same time,site-directed mutagenesis of amino acid residues in the ATP/AMP binding domain of Pp Fad D1 and Pp Fad D2,showed that both enzymes have similar structural features as E.coli Fad D.These results indicate that both Pp Fad D1 and Pp Fad D2 are acyl-CoA synthetases.(2)PpfadD1,PpfadD2 single and double mutant strain of Pseudomonas putida F1were constructed by homologous recombination.The growth of these mutant strains was tested on basal medium with different chain-length fatty acids as the sole carbon source.The results showed that mutating PpfadD1 significantly delayed the growth on various chain-length fatty acids,while the PpfadD1 and PpfadD2 double mutant exhibited the same phenotype but a greater degree,indicating that Pp Fad D1 is the enzyme responsible for the activation and utilization of exogenous fatty acids,while Pp Fad D2 plays a supporting role.PpfadD1 and PpfadD2 mutations were also constructed in a des AfabA Pseudomonas putida F1 mutant background by homologous recombination.Growth phenotypic analysis showed that oleic acid had difficulty in restoring the growth of the des AfabAfad D1 triple mutant.This again demonstrates that Pp Fad D1 is a key enzyme for the utilization of exogenous fatty acids by Pseudomonas putida F1.(3)Analysis of the culture medium of fatty acid composition on ¹⁴C acetate,the PpfadD1 single mutant and the PpfadD1PpfadD2 double mutant showed an increased fatty acid accumulation outside the cell in comparison to wide type,with the double mutant strain accumulating more fatty acids than the single mutant.In addition,studies have shown that the B.subtilis desaturase gene cannot restore growth of the des AfabAfad D1 triple mutant.These all indicate that Pp Fad D1 is a key enzyme for the utilization of endogenous fatty acids by Pseudomonas putida F1.(4)At the same time,it was found that the mutation of PpfadD1 also affected the motility of Pseudomonas putida F1.In Pseudomonas putida F1 has one gene encoding a protein homologous to E.coli 3-ketoacyl-ACP synthase I(Fab B):Pput?1693(PpfabB)and two genes encoding proteins homologous to E.coli 3-ketoacyl-ACP synthase ?(Fab F):Pput?3798(PpfabF1)and Pput?2422(PpfabF2).In the second part of this thesis,the identification and functional studies of these three homologous genes were carried out,and the following results were obtained:(1)Genetic complementation of E.coli fabB and fabF mutant showed that PpfabB can restore the synthesis of unsaturated fatty acids of E.coli fabB mutant,indicating that PpfabB has the activity of 3-ketoacyl-ACP synthase I.PpfabF1 was able to restore the synthesis of cis-octadecenoic acid in the mutant strain of E.coli fabF,indicating that Pp Fab F1 has the activity of 3-ketoacyl ACP synthetase ?.PpfabF2 not only restores the synthesis of unsaturated fatty acids in E.coli fabB mutant,but also compensates for the synthesis of cis-octadecenoic acid in the E.coli fabF mutant,indicating that Pp Fab F2 has both the activity of 3-ketoacyl ACP synthase I and 3-ketoacyl ACP synthase ?.(2)Pp Fab B,Pp Fab F1 and Pp Fab F2 proteins were purified by Ni-NTA affinity chromatography.In vitro reconstituted fatty acid synthesis also demonstrated that Pp Fab B is capable of extending unsaturated fatty acid synthesis with 3-ketoacyl-ACP synthase I activity.Pp Fab F1 is capable of extending the synthesis of saturated fatty acids with the activity of 3-ketoacyl ACP synthase ?.Pp Fab F2 is capable of extending the synthesis of saturated and unsaturated fatty acids with dual activities of3-ketoacyl ACP synthase I and ?.(3)PpfabF1,PpfabF2 single and double mutants and a single mutant of PpfabB were constructed by homologous recombination.The growth phenotype assay showed that the mutant strain of PpfabB was auxotrophic for unsaturated fatty acids,indicating that PpfabB is responsible for the synthesis of unsaturated fatty acids of Pseudomonas putida F1.The composition of the fatty acids of the PpfabFs mutant was determined,and it was found that the PpfabF1 mutant showed a significant decrease in the content of cis-octadecenoic acid,indicating that PpfabF1 is responsible for the synthesis of saturated fatty acids.In addition,studies have found that overexpression of PpfabF2can compensate for the loss of function of PpfabF1 and PpfabB.However,at the same expression level,the fatty acid content of the complementary strain of PpfabF2was lower than that of PpfabF1 and PpfabB itself,indicating that the elongation activity of Pp Fab F2 on fatty acids was lower than that of Pp Fab F1 and Pp Fab B.(4)Analysis of the transcriptional expression of PpfabB by gene fusion and DNA 5'-RACE technique revealed that while PpfabB and PpfabA can be co-transcribed in Pseudomonas putida F1,PpfabB also has its own promoter and can be independently transcribed.