Construction of a Mutant Bacillus Subtilis Strain for High Purity Poly-γ-Glutamic Acid Production

Abstract Purpose To construct a Bacillus subtilis strain for improved purity of poly-γ-glutamic acid. Results The construction of strain GH16 was achieved by knocking out five extracellular protein genes and an operon from Bacillus subtilis G423. Then we analyzed the protein content in the γ-PGA produced by the resultant strain GH16/pHPG which decreased by 6.08%. Subsequently the fla-che operon, PBSX and the yrpD, ywoF and yclQ genes were knocked out successively and the mutant strain GH17, GH18, and GH19 was obtained. Ultimately, the protein content was reduced by 43.9%. In addition, the polysaccharide content in the γ-PGA was decreased from 2.21–1.93% due to the epsA-O operon was knocked. Conclusion γ-PGA has potential applications as a drug carrier, sustained-releasing agent and medical composite in medicine. To our knowledge, this is the first report of engineered Bacillus subtilis strains which can produce γ-PGA with a purity higher than 97%. Our results confirmed that this upstream strategy significantly enhanced specific protein purity by the removal of extracellular protein genes in Bacillus subtilis, and it is promising in other protein purification.