Functions of poly-gamma-glutamic acid (γ-PGA) degradation genes in γ-PGA synthesis and cell morphology maintenance

Abstract

Poly-γ-glutamic acid (γ-PGA) is an important biopolymer with greatly potential in industrial and medical applications. In the present study, we constructed a metabolically engineered glutamate-independent Bacillus amyloliquefaciens LL3 strain with considerable γ-PGA production, which was carried out by single, double, and triple markerless deletions of three degradation genes pgdS, ggt, and cwlO. The highest γ-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 γ-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 γ-PGA degradation genes to improve γ-PGA production in a glutamate-independent strain and the first observation that γ-glutamyltranspeptidase (encoded by ggt) could be involved in the inhibition of cell elongation.

Construction of energy-conserving sucrose utilization pathways for improving poly-γ-glutamic acid production in Bacillus amyloliquefaciens

“…The plasmids for gene deletion (pKSV7-ΔsacA, pKSV7-ΔRBAM_031820 and pKSV7-ΔsacTPA) were constructed as previously reported protocols [14]. The gene deletion in this study was carried out following a previously reported marker-less gene deletion method [19, 20].…”

“…Therefore, it has been widely used in fields of foods, medicines, cosmetics and agriculture [11]. Bacillus amyloliquefaciens NK-1 strain is a glutamate-independent poly-γ-glutamic acid (γ-PGA) producing strain, and it can use sucrose as the carbon source [12–14]. The whole genome of this strain has been sequenced [15].…”

Enhancing poly-γ-glutamic acid production in Bacillus amyloliquefaciens by introducing the glutamate synthesis features from Corynebacterium glutamicum

“…The gene insertion plasmids pKSV7-P xyl and pKSV7-PO1 were constructed following the previously reported procedures [12]. The NADPH-dependent glutamate dehydrogenase gene from C. glutamicum ATCC13032 was codon optimized to match the Bacillus.…”

Mutations in genes encoding antibiotic substances increase the synthesis of poly-γ-glutamic acid inBacillus amyloliquefaciensLL3

“…Real‐time PCR analysis for the target genes was performed using the SYBR ® PremixEx Taq TM II (Takara, Dalian, China). Transcript levels of the target genes were normalized against the levels of rspU (Feng, Goa, Gu, Zang, Cao et al., ).…”