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

Poly-gamma-glutamic acid (gamma-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 gamma-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 gamma-PGA hydrolases (ggt) were disrupted by integrating plasmids into the genomic DNA of B. licheniformis RK14-46 strain. Unexpectedly, we observed strong inhibition of gamma-PGA production following deletion of the pgdS gene, suggesting that pgdS is essential for gamma-PGA biosynthesis in strain RK14-46, and in its parent strain RK14. In contrast, gamma-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, gamma-PGA from the ggt-disrupted mutant (Delta ggt) maintained a larger molecular mass throughout the culture period, whereas that from the original RK14-46 strain had degraded after glucose consumption. gamma-PGA-containing culture supernatants from Delta ggt strain showed greater flocculation efficiency in sewage sludge than supernatants from the RK14-46 strain, reflecting greater production of gamma-PGA with larger molecular mass by the Delta ggt strain. This is the first report concerning the deletion of pgdS and ggt genes in B. licheniformis strain and the properties of gamma-PGA obtained from the mutant strain.