Systematic engineering of branch chain amino acid supply modules for the enhanced production of bacitracin from Bacillus licheniformis

Abstract

Bacitracin is a broad-spectrum cyclic peptide antibiotic mainly produced by Bacillus, precursor amino acid supply served as the critical role during its synthesis. In this study, we systematically engineered branch-chain amino acid (BCAA) supply modules for bacitracin production. Firstly, we demonstrated that Ile and Leu acted as limiting precursors for bacitracin synthesis, and that BCAA synthetic pathways were strengthened via simultaneous overexpression of, feedback-resistance acetolactate synthase IlvBN^fbr, 2-isopropylmalate synthetase LeuA^fbr and BCAA aminotransferase YbgE. Using this approach, bacitracin yield from strain DW-BCAA2 was 892.54 U/mL, an increase of 18.32% compared with that DW2 (754.32 U/mL). Secondly, the BCAA permeases, YvbW and BraB, which have higher affinities for Leu and Ile transportation, respectively, were both identified as BCAA importers, with their overexpression improving intracellular BCAA accumulations and bacitracin yields. Finally, the leucine-responsive family regulator, lrpC was deleted to generate the final strain DW-BCAA6, with intracellular concentrations of Ile, Leu and Val increased by 2.26-, 1.90- and 0.72-fold, respectively. The bacitracin yield from DW-BCAA6 was 1029.83 U/mL, an increase of 36.52%, and is the highest bacitracin yield reported. Equally, concentrations of other byproducts including acetic acid, acetoin and 2,3-butanediol were all reduced. Taken together, we devised an efficient strategy for the enhanced production of bacitracin, and a promising B. licheniformis DW-BCAA6 strain was constructed for industrial production of bacitracin.

Keywords: Bacillus licheniformis; Bacitracin; Branch-chain amino acid permease; Branch-chain amino acids; lrpC.

Fig. 1

Effects of Ile, Leu and Val additions on bacitracin production. A: Effects of 40 ​mg/L Ile, Leu and Val addition at different time points on bacitracin production, respectively. B: Effects of different concentrations of Ile and Leu additions at 18 ​h on bacitracin production, respectively. Each experiment was repeated at least three times, and 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

Metabolic engineering of BCAA synthetic, transportation and regulation modules for enhanced production of bacitracin in B. licheniformis DW2.

Fig. 3

Effects of strengthening BCAA synthetic pathways on bacitracin production. A: Effects of overexpression of ilvA, ilvBN, ilvCD, leuA, leuB, leuC, leuD on bacitracin yields, B: overexpression of the feedback resistant IlvBN^fbr and LeuA^fbr improved bacitraicn production, C: The concentrations of intracellular BCAAs, D: The concentrations of extracellular BCAAs. Each experiment was repeated at least three times, and 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. 4

Effects of BCAA aminotransferase overexpressions on bacitracin production. A: Effects of ilvE, ywaA and ybgE overexperessions on bacitracin yields, B: Effects of replacing the promoter of ybgE by PbacA on bacitracin production, C: The concentrations of intracellular BCAAs, D: The concentrations of extracellular BCAAs. Each experiment was repeated at least three times, and 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

Identification of BCAA permeases, YvbW and BraB, in B. licheniformis DW2. A: Effects of overexpression and deletion of yvbW and braB on bacitracin production, B: The concentrations of intracellular BCAAs, C: The concentrations of extracellular BCAAs, D: The concentrations of extracellular BCAAs of DW2/pHY300, E: The concentrations of extracellular BCAAs of DW2/pHY-YvbW, F: The concentrations of extracellular BCAAs of DW2/pHY-BraB. Each experiment was repeated at least three times, and 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. 6

Engineering BCAA permeases, YvbW and BraB, for enhancement production of bacitracin. A: Strengthening YvbW and BraB expressions improved bacitracin production. B: The concentrations of intracellular BCAAs, C: The concentrations of extracellular BCAAs. Each experiment was repeated at least three times, and 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

Effects of lrpC deletion on bacitracin synthesis. A: Deletion of lrpC improved bacitracin yield, B: The transcriptional level of BCAA transporter gene brnQ, C: The schematic diagram of GFP expression vector mediated by promoter PbrnQ, D: The green fluorescence intensities of recombinant strains. Each experiment was repeated at least three times, and 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. 8

Fermentation processes of B. licheniformis DW2 and DW-BCAA6. A: Bacitracin yields and cell biomasses of DW2 and DW-BCAA6 during bacitracin production, B: The concentrations of intracellular BCAAs, C: The concentrations of extracellular BCAAs, D: The concentrations of main byproducts, acetic acid, acetoin and 2,3-butanediol. Each experiment was repeated at least three times, and 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.