| Poly-?-glutamic acid(?-PGA)is a multi-functional biopolymer.Different molecular weight?-PGAs have different uses in food,medicine,environmental protection,cosmetics and agriculture because of their different properties.However,studies on the controllable degradation of the biopolymer are limited.The aim of this work is to achieve production of?-PGA with a wide variation of molecular weight through screening,manipulating expression and fermentation optimization of?-PGA depolymerase in Bacillus licheniformis WX-02.In Bacillus,PgdS,?-glutamyltransferase(GGT)and Cw1O have been reported to be capable of degrading?-PGA.First,in order to investigate the effects of these three degrading enzymes on the synthesis of?-PGA by B.licheniformis WX-02,ggt,cwl O,pgd S deletion and overexpression strains were constructed in B.licheniformis WX-02,respectively.The results of shake flask fermentation showed that the average molecular weight of the pgd S gene deletion strain WX-02?pgd S increased by 32.00%(P<0.05),and the overexpression of pgd S decreased the molecular weight of?-PGA by 92.20%(P<0.01).However,the deletion or overexpression of the two degrading enzyme genes cwl O and ggt had no significant effect on the molecular weight and yield of?-PGA.The above results indicate that PgdS is a degrading enzyme mainly affecting the molecular weight of?-PGA synthesized by WX-02.Signal peptides and promoters are two key factors that influence the expression and secretion of extracellular proteins.This study investigated the effects of different PgdS expression and secretion levels on the molecular weight of?-PGA.Twelve PgdS expression vectors were constructed with 8 signal peptides(Sac C,Vpr,Bpr A,GGT,Apr E,PgdS,YvpA?Sac B)and 4 promoters(P43,Pbac A,Pbpr A?Ppgd S),respectively.The vectors constructed above were electroporated into B.licheniformis WX-02?pgd S,respectively.Analysis of extracellular PgdS content by SDS-PAGE revealed that different signal peptides resulted in different levels of PgdS expression and signal peptide intensity was SPsac B>SPyvp A>SPpgd S>SPapr E>SPggt>SPbpr A>SPvpr>SPsac C.According to SDS-PAGE and transcript level results,the promoter strength was P43>Ppgd S>Pbac A>Pbpr A.The results of shake flask fermentation indicated that 12 strains of engineered bacteria produced?-PGA with different molecular weights,and the molecular weight distribution was 6.82×10~4-1.78×10~6Da,which was lower than the molecular weight of the control strain by 14.2%-96.6%.It was shown that the molecular weight of?-PGA can be controlled by regulating the secretion ability and transcription level of PgdS.To further elucidate the relationship between PgdS expression level and?-PGA molecular weight,different strength promoters and signal peptides were selected for expression of PgdS,and 9 strains were constructed,which were SP41(p HYPbpr A-SPsac C),SP45(p HYPbpr A-SPapr E),SP48(p HYPbpr A-SPsac B),SP31(p HYPbac A-SPsac C),SP35(p HYPbac A-SPapr E),SP38(p HYPbac A-SPsac B),SP21(p HYPpgd S-SPsac C),SP25(p HYPpgd S-SPapr E)and SP28(p HYPpgd S-SPsac B).The activity and protein content of PgdS in 9 strains were determined.The results showed that the activity and expression of PgdS were positively correlated with the strength of promoter and signal peptide.The higher the activity/expression of PgdS,the lower the molecular weight of?-PGA.By analyzing the correlation,the Pearson correlation coefficient was-0.983,indicating that the molecular weight of?-PGA was significantly negatively correlated with the expression level of PgdS(P<0.01).By comparing the molecular weight and the yield of?-PGA,it was found that the yield of?-PGA increased with the decrease of molecular weight,and the Pearson correlation coefficient was-0.988,P<0.01.To further analyze how reducing the molecular weight of?-PGA increases?-PGA production,we compared the k _La and dissolved oxygen(DO)of the three engineered and control strains in a 1-L quadruple can.The results show that the molecular weight reduction can reduce the oxygen mass transfer coefficient,increase the oxygen and substance transfer rate,and promote the bacterial metabolism and?-PGA synthesis.Finally,in order to investigate whether the one-step method of the above-described construction efficiently synthesizes a system of a specific molecular weight?-PGA has the universality of industrial production.Three recombinant B.licheniformis(SP12,SP14,SP18)were fermented and amplified in a 3-L fermenter.The fermentation results showed that the yield of all engineering bacteria?-PGA was higher than that of the control bacteria,and the molecular weight was lower than that of the control bacteria.The molecular weight of SP18?-PGA was 7.83×10~4 Da,the highest yield was 39.13g/L,and the?-PGA yield was 1.30 g/L/h,which was 34.93%higher than the control.The?-PGA produced by this system has the highest growth efficiency compared to other glutamate-independent strains.Collectively,we have developed an efficient one-step system for the production of specific molecular weight?-PGA by regulating the expression level of PgdS.This system not only has great potential for industrial production of different molecular weight?-PGA,but also can guide other biopolymer synthesis of different molecular weights. |
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