Poly-gamma-glutamic acid (gamma-PGA) is an important biopolymer with greatly potential in industrial and medical applications. In the present study, we constructed ametabolically engineered glutamate-independent Bacillus amyloliquefaciens LL3 strain with considerable gamma-PGA production, which was carried out by single, double, and triple markerless deletions of three degradation genes pgdS, ggt, and cwlO. The highest gamma-PGA production (7.12 g/L) was obtained from the pgdS and cwlO double-deletion strain NK-pc, which was 93 % higher than that of wild-type LL3 strain (3.69 g/L). The triple-gene-deletion strain NK-pgc showed a 28 % decrease in gamma-PGA production, leading to a yield of 2.69 g/L. Furthermore, the cell morphologies of the mutant strains were also characterized. The cell length of cwlO deletion strains NK-c and NK-pc was shorter than that of the wild-type strain, while the ggt deletion strains NK-g, NK-pg, NK-gc, and NK-pgc showed longer cell lengths. This is the first report concerning the markerless deletion of gamma-PGA degradation genes to improve gamma-PGA production in a glutamateindependent strain and the first observation that gamma-glutamyltranspeptidase (encoded by ggt) could be involved in the inhibition of cell elongation.