Enhancement of precursor amino acid supplies for improving bacitracin production by activation of branched chain amino acid transporter BrnQ and deletion of its regulator gene lrp in Bacillus licheniformis

Abstract

Bacitracin, a new type of cyclic peptide antibiotic, is widely used as the feed additive in feed industry. Branched chain amino acids (BCAAs) are the key precursors for bacitracin synthesis. In this research, soybean meal was served as the raw material to supply precursor amino acids for bacitracin synthesis, and enhanced production of bacitracin was attempted by engineering BCAA transporter BrnQ and its regulator Lrp in the bacitracin industrial production strain Bacillus licheniformis DW2. Firstly, our results confirmed that Lrp negatively affected bacitracin synthesis in DW2, and deletion of lrp improved intracellular BCAA accumulations, as well as the expression level of BCAA transporter BrnQ, which further led to a 14.71% increase of bacitracin yield, compared with that of DW2. On the contrary, overexpression of Lrp decreased bacitracin yield by 12.28%. Secondly, it was suggested that BrnQ acted as a BCAA importer in DW2, and overexpression of BrnQ enhanced the intracellular BCAA accumulations and 10.43% of bacitracin yield. While, the bacitracin yield decreased by 18.27% in the brnQ deletion strain DW2△brnQ. Finally, BrnQ was further overexpressed in lrp deletion strain DW2△lrp, and bacitracin yield produced by the final strain DW2△lrp::BrnQ was 965.34 U/mL, increased by 22.42% compared with that of DW2 (788.48 U/mL). Collectively, this research confirmed that Lrp affected bacitracin synthesis via regulating the expression of BCAA transporter BrnQ and BCAA distributions, and provided a promising strain for industrial production of bacitracin.

Keywords: BCAA transporter BrnQ; Bacillus licheniformis; Bacitracin; Branched chain amino acid; Regulator Lrp.

Fig. 1

Effects of lrp deletion and overexpression on bacitracin production in B. licheniformis DW2. Data are represented as the means of three replicates and bars represent the standard deviations, ∗, P < 0.05; and ∗∗, P < 0.01 indicate the significance levels between recombinant strains and control strain.

Fig. 2

The concentrations of extracellular and intracellular precursor amino acids in DW2 and DW2△lrp during bacitracin fermentation. A: Intracellular at 24 h; B: Intracellular at 36 h; C: Intracellular at 48 h; D: Extracellular at 24 h; E: Extracellular at 36 h; F: Extracellular at 48 h.

Fig. 3

Effects of lrp deletion and overexpression on the transcriptional levels of genes in bacitracin synthesis and BCAA transportation. A: Transcriptional levels of bacitracin synthetase genes; B: Transcriptional levels of BCAA transporter genes.

Fig. 4

Effects of brnQ deletion and overexpression on bacitracin production. Data are represented as the means of three replicates and bars represent the standard deviations, ∗, P < 0.05; and ∗∗, P < 0.01 indicate the significance levels between recombinant strains and control strain.

Fig. 5

The concentrations of extracellular and intracellular precursor amino acids in DW2 and DW2::BrnQ. A: Intracellular at 24 h; B: Intracellular at 36 h; C: Intracellular at 48 h; D: Extracellular at 24 h; E: Extracellular at 36 h; F: Extracellular at 48 h.

Fig. 6

Overexpression of BrnQ in lrp deletion strain DW2△lrp to further improve bacitracin yield. Data are represented as the means of three replicates and bars represent the standard deviations, ∗, P < 0.05; and ∗∗, P < 0.01 indicate the significance levels between recombinant strains and control strain.

Fig. 7

The concentrations of extracellular and intracellular precursor amino acids in DW2 and DW2△lrp::BrnQ at 36 h. A: Intracellular at 36 h; B: Extracellular at 36 h.

Fig. 8

Fermentation process curves of DW2 and DW2△lrp::BrnQ during bacitracin production.