Improving Heterologous Protein Expression And Poly-?-glutamic Acid Production By Regulating D-alanine Metabolic Nodes In Bacillus Licheniformis

D-alanine(D-Ala)is one of the important components of the tetrapeptide tail in peptidoglycans of bacterial cell wall,which plays an indispensable role in the normal growth of bacteria.However,the current research on D-alanine mainly focuses on the construction of food-grade expression systems,and there is little research on the relationship between D-alanine and bacterial growth.This study first successfully constructed a food grade expression system in Bacillus licheniformis with alanine racemase,and achieved high yield of a-amylase by regulating the expression level of alanine racemase.By knocking out the lactate dehydrogenase gene ldh,and overexpressing the alanine dehydrogenase gene ald,the aspartic acid decarboxylase gene panD,the alanine racemase gene dal and the alanine ligase gene ddl,high-level production of poly-y-glutamic acid(?-PGA)was achieved.Based on the preliminary investigation of the relationship between D-alanine and cell growth and product synthesis,the preliminary mechanism which promote cell growth and product synthesis by retrofiting D-alanine metabolic nodules was elaborated.It would make contribution to high yield of metabolites of Bacillus licheniformis.On the one hand,firstly,the genetic stability of exogenoxus plasmids in B.licheniformis was studied.Without the addition of antibiotics,the plasmid loss rate was as high as 90%,and the expression of foreign proteins was greatly affected.Secondly,the functional gene of alanine racemase was identified and the results showed that gene dal played the main function of coding alanine racemase.Subsequently,a food-grade expression system(BL10D/pP43 Dal)was successfully constructed in B.licheniformis by using alanine racemase as a selectable marker,and the genetic stability of the exogenous plasmid reached 100%.And then,the engineered strain BL10D/pP43SAT-P43Dal was constructed after a-amylase was cloned into this expression system to verify the ability of the foreign protein expression.The results showed that the a-amylase activity was 62.52 U/mL expressed by the engineered strain BL10D/pP43SAT-P43Dal,a decrease of 49.09%compared to that of the control strain BL10/pP43SAT(122.80 U/mL).Finally,the P43 promoter of gene dal was replaced successfully by the promoter Pdal from alanine racemase-encoding gene and the promoter Ptet from tetracycline-encoding gene to construct two other engineered strains BL10D/pP43SAT-PdalDal and BL10D/pP43SAT-PtetDal.It showed that the engineered strain BL10D/pP43SAT-PtetDal had the highest expression level of a-amylase and the enzyme activity was 155.45 U/mL,which was an increase of 26.59%compared to the control strain BL10/pP43SAT,followed by strains BL10/pP43SAT,BL10D/pP43SAT-PdalDal and BL10D/pP43SAT-P43Dal.At the same time,the amount of alanine racemase in the fermentation broth was detected by SDS-PAGE which showed that the amount of alanine racemase produced by the engineered strain BL10D/pP43SAT-P43Dal was the highest,and strain BL10D/pP43SAT-PdalDal,BL10D/pP43SAT-PtetDal and BL10/pP43SAT was lower.This result indicates that the synthesis of D-alanine makes a close relationship with the production of exogenous protein,and the synthesis of D-alanine contributes to the expression of exogenous protein.On the other hand,firstly,the effect of different concentrations of D-alanine on cell growth and y-PGA production during fermentation were detected.It showed that the microbial biomass and target product could be increased by adding low-concentration alanine(?0.2%).While high concentrations of alanine(>0.2%)was not conducive to cell growth and the synthesis of the desired product.Secondly,B.licheniformis WX-02 was used as the host strain,and the key genes ald,panD,dal,and ddl related to alanine metabolic pathway were intensified,and the lactate dehydrogenase gene ldh in the overflow metabolic pathway was deleted to constructe a series of engineered strains.The strains were investigated for their effects on the synthesis of y-PGA which showed that the overexpression of genes ald,panD,dal and ddl and loss of gene ldh increased the yield of y-PGA by 19.72%,42.97%,15.91%,60.90%and 4.79%,and increased the microbial biomass 15.58%,12.06%,18.34%,49.85%and 13.96%,respectively.On this basis,the effects of the regulation of the D-alanine metabolism node on the synthesis of other metabolites of B.licheniformis were further examined,such as exogenous proteins(nattokinase),antibiotic(bacitracin),pigment(Pucchrrimin)and biosurfactant(lichenysin),which showed that the increase in alanine synthesis could increase the production of these metabolites by more than 18%.This result shows that the enhancement of the synthesis of the D-alanine pathway contributes to the growth of bacterial cells and the high yield of metabolites.