Activating a dormant metabolic pathway for high-temperature l-alanine production in Bacillus licheniformis

Abstract

l-Alanine is an important amino acid widely used in food, medicine, materials, and other fields. Here, we develop Bacillus licheniformis as an efficient l-alanine microbial cell factory capable of realizing high-temperature fermentation. By enhancing the glycolytic pathway, knocking out the by-product pathways and overexpressing the thermostable alanine dehydrogenase, the engineered B. licheniformis strain BLA3 produced 93.7 g/L optically pure l-alanine at 50°C. Subsequently, d-alanine dependence of an alanine racemase-deficient strain is relieved by adaptive laboratory evolution, implying that a dormant alternative pathway for d-alanine synthesis is activated in the evolved strain. The d-amino acid aminotransferase Dat1 is shown to be a key enzyme in the dormant alternative pathway. Molecular mechanism of the d-alanine dependence is revealed via mutational analysis. This study demonstrates a novel technology for high-temperature l-alanine production and shows that activating dormant metabolic pathway(s) is an effective strategy of metabolic engineering.

Keywords: Applied microbiology; Microbial biotechnology; Microbiology.

Graphical abstract

Figure 1

Redirection of carbon metabolism in B. licheniformis to obtain efficient alanine producer (A) Alanine synthesis pathway and main by-product pathways in B. licheniformis. The overexpressed enzymes were marked in green, while the knockout enzymes were marked in blue. (B) Fed-batch fermentation of B. licheniformis BLA1. (C) The final titers and yields of fed-batch fermentations of B. licheniformis overexpressing alanine dehydrogenases from different sources. (D) Fed-batch fermentation of B. licheniformis BLA2.

Figure 2

Efficient production of optically pure l-alanine achieved by knocking out alanine racemase and glycerol by-product biosynthesis pathway (A) Effects of individual and simultaneous knockout of two alanine racemase alr1 and alr2 on the optical purity of l-alanine produced by fed-batch fermentation of B. licheniformis. (B) Characterization of the d-alanine dependence of B. licheniformis BLA3 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 B. licheniformis BLA3. (D) Fed-batch fermentation of B. licheniformis BLA4.

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.

Figure 4

Identification of the alternate d-alanine synthetic pathway involving d-amino acid aminotransferase Dat1 (A–C) Characterization of the d-alanine dependence on B. licheniformis BLA3Δdat1, BLA3-E1Δdat1, and BLA3-Pdat1 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. (D) Fed-batch fermentation of B. licheniformis BLA3-Pdat1.

Figure 5

Identification of the effects of alsT5, dltB, and mtnW on the exogenous d-alanine dependence of the alr1/alr2 double-knockout B. licheniformis (A–D) Characterization of the d-alanine dependence on B. licheniformis BLA3ΔalsT5, BLA3-E1ΔalsT5, BLA3ΔdltB, and BLA3ΔmtnW 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.

Figure 6

Schematic of evolved strain BLA3-E1 removing the growth dependence on exogenous d-alanine The elimination of alanine racemase in strain BLA3 blocks the endogenous d-alanine synthesis pathway, which results in the growth dependence on exogenous d-alanine. The evolved strain BLA3-E1 produces additional endogenous d-alanine by activating dormant alternative d-alanine synthesis pathway, improves the utilization of exogenous d-alanine by enhancing the expression of d-alanine transporter and reduces the consumption of d-alanine by blocking the incorporation of d-alanine into teichoic acid.