Fementation Optimization Of ?-PGA Synthesis Of Glutamate-independent Bacillus Licheniformis Knoncking Out Sporulation Genes

Poly-?-glutamic acid(y-PGA)is an easily dealed,environmental friendly polymers,is connected using glutamic acid monomer with amide bond by microbial synthesis.Because of its excellent characteristics ofphysics,chemistry and biology,it can be wiedly used in medical treatment,agriculture,food industry,daily necessities,environmental protection and many other fields.At present,microorganisms used industrial production of y-PGA are mostly the genus of Bacillus,when lacking of nutrients in the late period of fermentation,bacteria will stop synthetizeing ?-PGA.Most bacteria synthetize ?-PGA with sodium glutamate exogenously adding,but the cost of sodium glutamate is very expensive,leading to not being suitable to apply to industry.In this study,I use Bacillus licheniformis WX-02 whose sporulation genes are knocking out to synthetize ?-PGA without adding sodium glutamate,increasing the production of y-PGA and cut down the cost via fermentation optimization.Later I had studied the transcription quantification of y-PGA synthesis genes when WX-02?spoOF and WX-02 AspolV were cultured in medium which glucose was acted as main carbon.The results showed the gene transcription level of pgsB,psgC,swrA,degQ of WX-02?spoOF is higher than WX-02?spo?.spo0F?spo?AC?spo? are three key genes in the early,middle and late periods of spouration of Bacillus licheniformis WX-02,any one of three genes knocking out can not form spores.In this study,I used Bacillus licheniformis WX-02?spo0F,WX-02?spo?AC,WX-02?spo? whose sporulation genes was knocking out to check out the capacity of y-PGA synthesis in three different medium.It concluded that spore deficient strain can synthetize large amounts of y-PGA without adding sodium glutamate into the medium.When glucose acted as main carbon source without adding sodium glutamate in the common medium,the ?-PGA yield of WX-02?spo0F was the highest(19.53 g/L),increased by 105.36%when compared with the wild strain WX-02(9.51 g/L).While in the high stress medium they-PGA yield of WX-02?spo0F reached the maximum(39.95 g/L),increase up 31.03%compared with the y-PGA yield of WX-02(30.49 g/L).When glycerol acted as main carbon source without adding sodium glutamate in the medium,the y-PGA yield of WX-02?spo? was the maxium(18.21 g/L)5 increasing 78.18%compared with ?-PGA yield of WX-02(10.22g/L).In summary,when glucose was acted as the main carbon source without sodium glutamate WX-02?spo0F showed more strong capacity of ?-PGA synthesis than any other strain.While glycerol was acted as main carbon source WX-02?spo? showed more strong capacity of y-PGA synthesis.According to fermentation curves of WX-02 and engineering strains,spoOF had a great effect on the bility of using glucose,spo?AC showed great influence on the growth of strains,but spo? had a little effect on these but it is not obvious.In order to study the difference about the capacity of ?-PGA synthesis of WX-02? spoOF and WX-02 ? spo?,when glucose or glycerol was acted as main carbon source,testing the concentration of acetoin and 2,3-butanediol at the ending point of fermentation.The result showed that when glucose was acted as carbon source in the medium,the concentration of acetoin of WX-02? spoOF and WX-02? spo? is 3.51 g/L?3.56 g/L,the concentration of 2,3-butanediol is 10.19 g/L,10.25 g/L,it make no difference on the concentration of acetoin and 2,3-butanediol between WX-02? spoOF and WX-02? spo?.While glycerol was acted as carbon source,the concentration of acetoin of WX-02? spoOF and WX-02? spo? is 1.72 g/L,2.11 g/L,the concentration of 2,3-butanediol is 7.95 g/L,7.72 g/L,the difference is significant.The reason is probably because more resources were used to synthetize secondary metabolites while the sources used to synthetize y-PGA were reduced.To improve the ?-PGA yield of WX-02?spo0F and WX-02?spo? in fermentation medium without sodium glutamate,the composition of medium and culture condition of WX-02?spo0F were optimized(g/L):glucose 90,sodium citrate 20,NH4(SO4)2 10,sodium nitrate 20,K2HPO4·3H2O 0.8,MgSO4·7H2O 1.0,ZnSO4·7H2O 1.2,CaCl2 0.4,MnS04 H20 0.2,pH 7.2,culture temperature 37?,liquid loading volume 50mL/250mL,inoculation volume 3%,speed 180r/min,seed age is 9 hour,the final y-PGA yield was 27.68 g/L,compared with the y-PGA production of WX-02 increased by 41.73%.Thecomposition of medium and culture condition of WX-02?spo? were optimized(g/L):glycerol 60,sodium citrate 12,NH4Cl12,sodium nitrate 10,K2HPO4·3H2O 1.0,MgSO4·7H2O 1.2,ZnSO4·7H2O 1.2,CaCl2 1,MnSO4 H2O 0.2,pH value 7.2,culture temperature 37?,liquid loading volume 50 mL/250 mL,inoculation volume 3%,speed 180 r/min,seed age is 8 hour.The final y-PGA yield was 25.47 g/L,compared with WX-02 increased by 39.87%.