Knockout of pgdS and ggt gene changes poly-γ-glutamic acid production in Bacillus licheniformis RK14-46

Poly-gamma-glutamic acid (γ-PGA) is a water-soluble, nontoxic biocompatible polymer, which is extensively used in medicines, foodstuffs, cosmetics, and in water treatment. We previously isolated a novel γ-PGA producing strain Bacillus licheniformis RK14 from soil and developed a hyper-producing mutant strain RK14-46 by an ethyl methanesulfonate (EMS) treatment. In this study, endo-type (pgdS) and exo-type γ-PGA hydrolases (ggt) were disrupted by integrating plasmids into the genomic DNA of B. licheniformis RK14-46 strain. Unexpectedly, we observed strong inhibition of γ-PGA production following deletion of the pgdS gene, suggesting that pgdS is essential for γ-PGA biosynthesis in strain RK14-46, and in its parent strain RK14. In contrast, γ-PGA production increased by the deletion of the ggt gene and reached 39 g/L in the presence of 90 g/L glucose and elevated oxygen supply. Furthermore, γ-PGA from the ggt-disrupted mutant (Δggt) maintained a larger molecular mass throughout the culture period, whereas that from the original RK14-46 strain had degraded after glucose consumption. γ-PGA-containing culture supernatants from Δggt strain showed greater flocculation efficiency in sewage sludge than supernatants from the RK14-46 strain, reflecting greater production of γ-PGA with larger molecular mass by the Δggt strain. This is the first report concerning the deletion of pgdS and ggt genes in B. licheniformis strain and the properties of γ-PGA obtained from the mutant strain.