| As a rich aromatic organic carbon in nature,the catabolism of lignin is closely related to the global carbon cycle and the utilization of biological resources.Microorganisms with lignin degradation ability have evolved in nature,but the degradation efficiency of lignin is low due to its complex structure.Microorganisms secrete lignin depolymerase to the extracellular layer to depolymerize lignin.Therefore,the key factors affecting the degradation efficiency are the catalytic efficiency and secretion efficiency of depolymerase.Previous studies showed that dye-decolorizing peroxidase(DypB3152)encoded by Pseudomonas putida A514 had lignin depolymerization activity,and could be secreted into periplasmic space and extracellular space.However,sequence analysis showed that the Nterminal and C-terminal did not contain typical signal peptides.Previous periplasmal proteome experiments showed that DypB3152 was significantly associated with elements of the type VI secretion system(T6SS),while effector proteins secreted by type VI did not require signal peptides.Therefore,the hypothesis that DypB3152 secretion is related to type VI secretion system was proposed in this project.To test this hypothesis,firstly,the CRISPR/Cas9n-λ-Red gene editing system was optimized and modified based on the CRISPR/Cas9 system.Secondly,the gene editing system was used to knock out key genes of T6SS one by one and superposition,as well as complement.vgrG elements were involved in DypB3152 periplasmic secretion by the enzyme activity and protein quantification of the mutant.Then,we observed by scanning electron microscopy that P.putida A514 can synthesize the outer membrane vesicles(OMV).Protein quantitative experiments showed that OMV could be secreted into the extracellular by enclosing periplasmic DypB3152.Finally,we expressed DypB3152-membrane protein fusion and displayed it on the surface of OMV to promote the contact between the enzyme and the lignin substrate and increase the enzymatic reaction.At the same time,its extracellular expression was increased by about 5 times through the deletion of membrane protein gene and the optimization of culture conditions.In conclusion,this study analyzed the extracellular secretion process of P.putida A514 DypB3152,which is conducive to the understanding of the secretion mechanism of lignin depolymerase and provides new ideas for lignin resource utilization. |
