Abstract:Polyglutamic acid, as a natural multifunctional polymer, has become a research hotspot in recent years. Microbial fermentation is currently an effective way to produce polyglutamic acid which is difficult to be synthesized by chemical methods. [Objective] To explore the roles of degS, degQ, degU, swrA, rocA, and putM genes in the polyglutamic acid synthesis of Bacillus subtilis and realize the regulation of the synthesis pathway through molecular modification. [Methods] The genetically engineered B. subtilis strains were constructed by knocking out degS, degQ, and degU or overexpressing swrA, rocA, and putM, respectively. The key nodes in the synthesis pathway were analyzed based on the content change of extracellular polyglutamic acid secreted by the engineered strains. [Results] In shake flask culture, the extracellular polyglutamic acid contents of the recombinant strains B. subtilis 168-swrA, 168-rocA, and 168-putM were 1.28, 1.47, and 1.37 times that of the original strain, respectively; the extracellular polyglutamic acid contents of B. subtilis 168-△degS, 168-△degQ, and 168-△degU were 1.01, 0.98 and 0.94 times that of the original strain, respectively. In static culture, B. subtilis 168-△degU could not form an intact biofilm, and the biofilm formation of B. subtilis 168-△degS, 168-△degQ, 168-swrA, 168-rocA, and 168-putM was 1.48, 1.31, 1.77, 2.59, and 2.16 times that of the original strain, respectively. The extracellular protein content was positively correlated with the biofilm formation. [Conclusion] The deletion of degS, degQ, and degU did not significantly affect the synthesis of polyglutamic acid, while the overexpression of swrA, rocA, and putM significantly improved the ability of B. subtilis to synthesize polyglutamic acid.
