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. 2023 Mar 15;26(4):106397. doi: 10.1016/j.isci.2023.106397

Figure 3.

Figure 3

Removing the growth dependence of B. licheniformis BLA3 on d-alanine through adaptive laboratory evolution

(A) Scheme of the laboratory evolutionary approach to remove the growth dependence of mutant strains on d-alanine.

(B) Characterization of the d-alanine dependence on evolved B. licheniformis BLA3-E1 by testing the growth performance in media containing various concentrations of d-alanine. Triplicate experiments were carried out for physiological measurements, and error bars represent standard deviations.

(C) Fed-batch fermentation of evolved B. licheniformis BLA3-E1.

(D) Mean fluorescence intensity of B. licheniformis BLA3 and evolved B. licheniformis BLA3-E1 incubated with 5-FAM labeled d-alanine. Fluorescence intensity represented the requirement of extracellular d-alanine for the biological process of cell wall synthesis. Triplicate experiments were carried out for physiological measurements, and error bars represent standard deviations.

(E) Flow cytometry analysis of B. licheniformis BLA3 and evolved B. licheniformis BLA3-E1 incubated with 5-FAM labeled d-alanine. The X axis is fluorescence intensity, and the Y axis is cell number. B. licheniformis BLA3 and BLA3-E1 are shown in blue and red, respectively.