Study On The Non-Classical Secretion Of 1,4-?-Glucan Branching Enzyme In Escherichia Coli

GBE(1,4-?-glucan branching enzyme)is a glycosyltransferase that hydrolyzes ?-1,4-glycosidic bonds in starch molecular and synthesizes ?-1,6-glycosidic bonds to improve its branching degree,which plays an important role in the application of enzymatically modified starch.According to the literatures,most of the microbial-derived GBE are intracellular enzymes.In the previous study,the laboratory constructed Escherichia coli intracellular expression systems of Gt-GBE and Ro-GBE,respectively.And then it was found that both the GBEs do not contain any classical signal peptide sequence but could be secreted into medium.The understanding of the secretion mechanism of the two GBEs without signal peptide can provide a new idea for the secretory expression of heterologous proteins,and can also provide theoretical guidance for strategies to promote the expression of GBE secretion.Therefore,in this paper,the secretion mechanism of the two GBE without signal peptides was studied.The main research results are as follows:Firstly,the signal peptide-less secretion phenomenon of the two GBEs were analysed and compared.It was found that they could be secreted in a large amout in E.coli,which did not depend on the inducer IPTG,and the secretion rate of Gt-GBE was more than Ro-GBE.The two GBEs were then analysed by using multiple bioinformatic prediction site,and the results showed that they should be intracellular proteins without signal peptide.Therefore,it was speculated that their secretion belongs to non-classical secretion.To investigate whether these signal peptide-free proteins could be secreted by the classical secretory pathway,the two GBEs were analysed by subcellular localization.It was found that their secretion was "two-step transmembrane transport" but did not belong to the Sec pathway,which was the major pathway of "two-step transmembrane transport".Though the N-terminus of Ro-GBE contains a double arginine structure,which was the feature of the Tat pathway,but the Tat pathway was excluded by site-directed mutagenesis.Meanwhile,by exchanging and truncating the N-termini of the two GBEs,which may be the important role in protein secretion,it was found that the N-terminus not only influenced their secretion but also affected the expression.Secondly,since protein secretion is a process of transmembrane transport,the effect of cell membranes on secretion cannot be ignored.Through the determination of the permeability of E.coli cell membrane during fermentation using lactate dehydrogenase as an indicator,it was found that the permeability of the E.coli cell membrane producing Gt-GBE and Ro-GBE was significantly greater than that of the E.coli containing the empty plasmid.And the result was also the same with the method of flow cytometer.Because E.coli is a gram-negative bacterium having two layers of cell membranes,to further analyze the effects of the two GBEs on their cell membranes,their influence on the permeability of the cell inner membrane and the outer membrane were measured,respectively.The results showed that the non-classical secreted proteins Gt-GBE and Ro-GBE both significantly increased the permeability of the inner membrane and outer membrane of the cells.Thirdly,based on the experimental conclusions above,a hypothetical non-classical secretion model of the GBEs was constructed,and it was pointed out that the GBEs were secreted extracellularly by increasing the membrane permeability of E.coli.Therefore,attempts have been made to add several media additives that could affect the membrane permeability of E.coli in order to further promote the secretion of the GBEs.The results showed that both glycine and Triton X-100 could increased the secretion of the GBEs.The promotion of the additives to the secretion of the GBEs further validated the reliability of the above model.In summary,the non-classical secretion may be due to their overexpression in E.coli,causing an increase in cell membrane permeability,which allows them to be secreted in a "two-step transmembrane transport" manner.In addition,the secretion can be promoted by medium additives,and the N-termini also have a significant effect on the secretion.