Study On The Function Of Anti-terminator GlpP In Bacillus Subtilis And The Construction Of Glycerol Induced Expression System

Bacillus subtilis is an important Gram-positive model strain.The expression system of B.subtilis is widely used in the fields of enzyme engineering,metabolic engineering,and molecular biology research.B.subtilis can efficiently utilize glycerol which is strictly regulated by the inhibition of carbon metabolism.The anti-termination protein GlpP originated from B.subtilis(BsGlpP) plays a regulatory role for enzymes in the glycerol metabolism pathway.It can specifically bind with glycerol-3-phosphate(G3P) and disrupt the terminator structure to allow continuation of the transcription process analyzed the mechanism of GlpP through structural analysis.The glycerol-induced expression system(GIES)was constructed based on this anti-terminator element.Furthermore,the high efficiency auto-inducible expression of exogenous proteins can be realized using the mixture medium of glucose and glycerol.The specific content is as follows:(1)Construction and characterization of GIES system.The expression system was constructed using a promoter P_glpD regulated by BsGlpP and a reporter gene sfGFP.The addition of different carbon sources for cultivation proves that the system is specifically induced by glycerol and the expression level of foreign proteins is higher.The equal good performance of this expression system on plasmids with different replicators indicates that it has strong robustness in different genetic environments.(2)Analyzing the mechanism of BsGlpP by molecular dynamics simulation.3D model of BsGlpP binding with the G3P and the ligand binding pocket were obtained by homology modeling and molecular docking.Alanine scanning mutation result showed that R14,R104 and R157 are the key residues for ligand binding.Comparing the conformation of BsGlpP on the bound and unbound state through molecular dynamics simulation showed that the relative position change of H_G and H_F from"parallel"to"crossing"is essential for the activation of BsGlpP.(3)Realizing the high efficiency auto-inducible expression of proteins in GIES with the mixture of glucose and glycerol in medium.For the strict carbon metabolism inhibition effect of B.subtilis,glycerol was not used until the glucose ran out in the medium.Auto-induction of gene downstream of anti-terminator was achieved when the glycerol was catalyzed to be G3P.Further studies have shown that changing the amount of glucose can control the time for gene expression in this auto-induction method;while changing the amount of glycerol can control the level of gene expression.(4)Using the auto-induction culture of GIES to achieve high efficiency expression of nattokinase in a 5 L bioreactor.1%glycerol and 0.1%glucose were verified to be the best ratio for the expression of nattokinase in shake flask.On this basis,batch fermentation and fed-batch fermentation were carried out in a 5 L bioreactor.The batch fermentation result shows that the enzyme activity of nattokinase peaked at 10.25 U·m L^-1,6 times the level of shake flask;The enzyme activity of fed-batch fermentation peaked at 11.90 U·m L^-1,6.90times the level of shake flask.