Efficient Production Of Poly-γ-glutamis Acid Using Glycerol In Bacillus Licheniformis

Poly-y-glutamic acid(y-PGA)is a naturally occurring biopolymer with multiple potential applications.The high cost of y-PGA fermentation limits its industrialization application.In this study,to achieve high production of y-PGA by Bacillus licheniformis WX-02 through optimization of fermentation process using glycerol as the main carbon source.Furthermore,the glycerol pathway was examined in the WX-02 strain,and genetically engineered strains were constructed to highly efficient crude glycerol utilization for y-PGA production.Meanwhile,the simple,fast and precise analysis method of glycerol was established by high performance liquid chromatography with evaporative light scattering detector.External standard calibration curves were used for quantitation.Results showed that there are good linear correlations between the concentration logarithm and peak area logarithm of the glycerol with the correlation coefficients of 0.997,and the limit of detection(S/N>3)was 0.09 mg/mL,the relative standard deviation was 0.504%.The flask fermentation media and cultivation conditions of WX-02 were optimized.The optimal fermentation medium contains(g/L)glycerol 70.0,sodium glutamate 30.0,sodium nitrate 15.0,sodium citrate 12.0,ammonium chloride 8.0,K2HPO4·3H2O 1.0,MgSO4·7H2O 1.0,ZnS04·7H2O 1.0,CaCl2 1.0,MnSO4·H2O 0.15.The cultivation conditions were inoculum age 9 h and the inoculums amount 3%.Using the optimal fermentation process,the y-PGA yield,productiveity and glycerol utilization were achieved at 35.46 g/L,0.74 g/L/h and 67.3%,which were increased by 63.6%,64%and 20.7%,respectively as compared with those under the initial cultural conditions.Using preprocessed crude glycerol as the raw material,the y-PGA yield and productivity were 33.25 g/L,0.69 g/L/h,respectively in the optimal parameters.A series of gene deletion strains including the deletions of glpK,glpD in respiratory pathway and gldA,ybdH,dhaK in fermentation pathway were constructed,named WX-02△glpK,WX-02△glpD,WX-02△gldA,WX-02AybdH,WX-02△ybdH△gldA and WX-02△dhaK,respectively.The growth of these mutants on glycerol as the only carbon source under aerobic and anaerobic conditions was monitored.Under aerobic and anaerobic conditions,neither the △glpK mutant nor △glpD mutant were able to grow.The deletions of the gldA,ybdH and dhaK had no significant effects on cell growth under both conditions.These results indicated that glycerol is catabolized by respiratory pathway in the WX-02 strain.We constructed the overexpression strains of glpF,glpK,glpD,named WX-02/pHY-glpF,WX-02/pHY-glpK and WX-02/pHY-glpD,respectively.Compared to WX-02/pHY-300PLK strain,the glycerol consumption and y-PGA yield of WX-02/pHY-glpF had no significant difference.While the y-PGA yield of WX-02/pHY-glpK and WX-02/pHY-glpD increased by 19%and 17%respectively,and glycerol consumption of these two strains increased by 12%and 9.3%,respectively.Through homologous recombination,the recombinant strains of WX-02/P43glpFK and WX-02/PytzEglpFK were constructed with the constitutive promoter(P43)and the promoter(PytzE)of ytzE,respectively.Compared to the wild-type strain,the glycerol consumption of WX-02/P43glpFK strain was increased by 11.5%,its y-PGA production have no significant difference.While the y-PGA production and glycerol consumption of WX-02/PytzEglpFK were increased by 5.6%and 20%respectively compare with those of the wild-type strain.The point mutantion strain with His-230 of GlpK replaced by Arg in B.licheniformis was constructed,named WX-02/glpKH230R.The mutant was not able to grow on glycer-ol under the y-PGA fermentation conditions,which is different from the glpKH230R in B.subtilis that exhibited elevated activity.