Enhancement Of Poly-?-glutamic Acid Biosynthesis By Alkaline PH Stress Treatment In Bacillus Licheniforamis

Poly-y-glutamic acid(y-PGA)is a biopolymer produced by microorganism.It possesses multiple good properties of edibility,biodegradability,flocculation and absorption ability.Hence,it has been applied in various fields,such as environmental protection,cosmetic,food additives,pharmaceuticals and agriculture.At present,mass exogenous glutamates are needed to be added into the medium for high y-PGA production,and high glutamate content resulted in high cost,which hindered the industrial production of y-PGA.In this study,fermentation parameters were optimized for high production of y-PGA in medium without glutamate using Bacillus licheniformis WX-02,and key genes involved in high production of y-PGA under alkaline stress were investigated.B.licheniformis WX-02 was used as ?-PGA producing strain in the glutamate-free medium,and the alkaline stress intensity was optimized.The results showed that the initial pH of 9.0 was the optimal pH.Based on this condition,the medium components were also investigatedto obtain the optimal compositions as:glucose 80 g/L,sodium citrate 30 g/L,NH4Cl 8 g/L,NaN03 15 g/L,K2HPO4·3H20 1 g/L,MgS04 7H20 1 g/L,ZnS04·7H20 1 g/L,CaCl2 1 g/L,MnS04·H20 0.15 g/L.The y-PGA yield of the alkaline stress group reached 33.56 g/L,increased by 52.89%than that of the control group(21.95 g/L).On a 50-L bioreactor,the maximum y-PGA yield of alkaline stress group reached 36.26 g/L,which was 79%higher than that of control group.Moreover,transcriptional levels of ?-PGA synthetase genes(pgsB and pgsC)and their transcriptional regulators(degU and swrA)were measured.Compared with the control group,the transcriptional levels of pgsB,pgsC,degU and swrA of treatment group were increased to 19.9-fold,32.2-fold,4.0-fold and 7.3-fold respectively.Acetoin and 2,3-butanediol were main byproducts during fermentation.Under alkaline stress,the yield of acetoin and 2,3-butanediol reached 9.56 g/L and 8.01 g/L,respectively,showing 46.28%and 21.96%decrease than that of the control group,respectively.Glutamic acid was the precursor for the biosynthesis of y-PGA,thus,the transcriptional levels of genes related to biosynthesis of glutamic acid were measured.Under alkaline stress,the transcriptional level of citrate synthase gene citZ and isocitrate dehydrogenase gene icd was increased to 2.09-fold and 1.69-fold,respectively.Compared to controlled group,the transcriptional level of a-oxoglutarate dehydrogenase gene odhB and dihydrolipoamide succinyltransferase gene ogdH in treatment group were decreased to 44%and 66%,respectively.These results indicated that the synthesis of glutamic acid was enhanced under alkaline stress.Meanwhile,the RQ value of treatment group was much higher than that of control group.In addition,transcriptional levels of anaerobic regulators fnr was increased to 4.96-fold compared to control group,indicating that the nitrate respiration was enhanced under alkaline stress.Under the alkaline stress,the synthesis of glutamate was enhanced,and the production of acetoin and 2,3-butanediol was decreased,as well as the improved nitrate respiration in low oxygen environment to provide more energy for cell toconsume,which were the main reason for improved y-PGA production under alkaline stress.Based on above results,motA,yhbJ,degU and swrA were selected to investigate the mechanism of improved ?-PGA production under alkaline stress.Knockout of motA and yhbJ showed little effect on y-PGA production,indicating that motA and yhbJ were not the key genes.While the mutant strain with enchanced expressed degU produced more y-PGA(32.79 g/L)than control strain(27.06 g/L)in normal condition(non-alkaline stress),and the y-PGA yield of these two strain had no significant difference in alkaline stress condition,which indicated that degU was one of the key genes involved in high y-PGA production under alkaline stress.The y-PGA production of swrA knockout strain and swrA enchanced expressed strain was 1.30 g/L and 2.31 g/L,respectively,which was much lower than that of control strain.The results showed that SwrA had major effect on the biosynthesis of y-PGA.However,the relation between swrA and of y-PGA was still unclear at this point.