Study On Secretome Of Bacillus Thuringiensis

Bacillus thuringiensis?Bt?is widely used as a biopesticide,which can produce the insecticidal crystal protein during sporulation.Its cell wall composition is quite simple as a Gram-positive bacterium.Proteins are secreted into the media without the need to penetrate the outer membrane,which makes it a good candidate host for the expression of exogenous proteins.However,Bt secretes proteolytic enzymes which may affect the production of target proteins.Therefore,we should study its secretory proteome.This work will lay the foundation for knockdown of proteolytic enzyme genes and for construction of Bt secreting expression host.T0 is marked as the turning point of exponential phase and stationary phase.T-1 is the point one hour before T0,T1 is the point one hour after T0,T2 is the point two hours after T0,and so on.In this study,the method of preparing Bt extracellular proteins sample was optimized and the interfering protein in the culture medium was removed by electrophoresis.The secretory proteins of Bt_HD73 in Luria Bertani?LB?medium in T-1,T1,T5,T14,T21 were detected by mass spectrometry after in-gel digestion.The Bt_HD73 genome sequence was used to identify the proteins,and then the types,contents,functions were analyzed.The results show that 177 extracellular proteins of Bt_HD73 are identified.Most of the extracellular proteins produced by HD73 in T-1 and T1 are enzymes,mainly hydrolases.The species of extracellular proteins in T5 are the richest,while they decrease significantly in T21.Most of the secretory proteins are phage proteins in T21.Further analysis of secretory proteases shows that proteases contents are relatively high,including HD73₂118?peptidase P60?,HD73₅638?peptidase M24?,HD73₀859?peptidase M24?,HD73₀744?peptidase M6?,HD73₁513?peptidase M6?and HD73₅219?leucyl aminopeptidase?.These proteolytic enzyme genes can be knocked out when constructing Bt secretory expression host to avoid the degradation of the target protein.The protease-deficient strain HD73-?0859 was successfully constructed and screened in this study.Signal peptide library is an important tool to improve the efficiency of secretory expression.The signal peptides and signal sequences of secretory proteins were predicted by bioinformatics software.A signal peptide library of this strain has been established,including Sec-type signal peptides,Tat-type signal peptides,lipoprotein signal peptides and signal sequences of non-classically secretory proteins.This research was aimed to investigate the types and contents of secretory proteins in the process of growth of Bacillus thuringiensis in rich medium,especially the extracellular proteases and signal peptides.This research laid the foundation for further studies on developing Bt as a candidate host for secretory expression,optimizing secretion expression and increasing secretion efficiency.It is of great significance for fully understanding the insecticidal mechanism and expanding the application of this bacterium.