Improvement Of Bacillus Licheniformis WX-02 For Efficient Expression Of Heterologous Proteins

According to genome sequence and protease annotation results of B.licheniformis WX-02,I had constructed 10 knockout vectors.This study has knocked 10 genes that may be relevant to exocytosis expression(hag,mpr,vpr,aprX,epr,bpr,wprA,aprE,amyL,bprA),and got a series of deletion mutants BL1-10.Gelatin zymography detected BL9,the result shows that there are four protease bands,accounting for about 55%of the wild-type extracellular protease activity.BL10's gelatin zymography result shows no extracellular protease activity at all.According to the results and other relevant protease reported in the articles,speculated that in the process BprA secreted to extracellular,it can self-cut into three other smaller molecular weight proteases.In these four proteases,the minimum molecular weight of about 30KDa is the mature protein of BprA.Its protease activity is largest of the 4.This research constructed an expression vector pP43SAT to constitutive secretion-expression of alpha-amylase,to detection of the engineered host's secretion-expression ability.In producing amylase medium,BL9(pP43SAT)of the highest enzyme activity reached 94.48 U/mL;BL10(pP43SAT)of the highest enzyme activity reached 106.02 U/mL.The enzyme activity of BL10(pP43SAT)increased by 12.22%than BL9(pP43SAT).BL10(pP43SAT)'s unit cell enzyme activity is 1.13 times than WX-02(amyL;pP43SAT),the latter's unit cell enzyme activity and BL9(pP43SAT)almost the same.The results confirmed removal of BprA protease is conducive to the expression of alpha-amylase,speculated that BprA may partially degraded alpha-amylase or inhibit its synthesis and secretion.This study constructed an expression vector pP43SNT to constitutive secretion-expression of nattokinase,which was applied to investigate the effect of the improved host on secretion of heterologous protein.The engineered bacteria BL10(pP43SNT)was applied to express NK,it was cultured in LB 28 h,analysised by SDS-PAGE electrophoresis and measured with its fibrinolytic activity.The results confirmed exogenous protein NK can efficient secretory expression in the mutant.The study shows that the signal peptide of Vpr can efficiently mediate heterologous protein secreted to extracell,while it is the first time of nattokinase expressed in Bacillus licheniformis.Cultured in LB medium for 24 h,BL10(pP43SNT)'s fibrinolytic activity reached 6.00 FU/mL.When optimization of the component on the basis of LB,used 1%soy peptone,1%yeast extract and 1%maltose as the fermentation medium,cultured BL10(pP43SNT)32 h,the enzyme activity reached 11.37 FU/mL.When soybean was used as substrate,solid fermentation for 44 h,BL10(pP43SNT)firinolytic activity reached 433.73 FU/g(dry weight),higher than all of the results reported in the literature;BL10(pP43SNT)increased by 12%than WX-02(pP43SNT).Meanwhile,there is a little gamma-PGA of BL10(pP43SNT)(6.70 g/kg)in the solid fermentation process,and low gamma-PGA production is conducive to the separation and purification of nattokinase.