Enhanced dipicolinic acid production during the stationary phase in Bacillus subtilis by blocking acetoin synthesis

Figures & data

Fig. 1. Central carbon metabolic pathways with DPA biosynthetic pathways in B. subtilis.

Notes: G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate; Pyr, pyruvate; AcCoA, acetyl-CoA; Cit, citrate; αKG, α-ketoglutarate; Suc, succinate; Mal, malate; OAA, oxaloacetate; Glu, glutamate; Asp, aspartate; ASA, aspartate semialdehyde; Lys, lysine.

Table 1. Strains used in this study.

Strain	Genotype
OA105	168, ΔSPβ, ΔPBSX, Δskin, Δpps, Δpks, Δpro1–7, trp+
OA105ΔalsSD	OA105, ΔalsSD
OA105-DPA	OA105, PspoVG-spoVFAB
OA105ΔalsSD-DPA	OA105-DPA, ΔalsSD

Table 2. Primer list for DPA-producing strain construction.

Primer	Sequence (5ʹ -> 3ʹ)
CmF2	GGATAGACTCCACCAGAAGAGC ATCATCGGCAATAGTTACCC
CmR2	CCAGGATGTAGTATCCTTCCG CGGCGTAGAGGATCTGGAGC
spoVGpro-F	CGGAAGGATACTACATCCTG GCGTTAGTCGAGATCGAAGTTA
spoVGpro-R	TATTTTCCCTCCTTTTCGCC CTATATAAAAGCATTAGTGTATC
spoVFA-F1	GAAGCTTAATGCTGAGCTTAG
spoVFA-R1	GCTCTTCTGGTGGAGTCTATCC TATTTACTCTTGTCGAGGAGGC
spoVFA-F2	GGCGAAAAGGAGGGAAAATA ATGTTAACCGGATTGAAAATTGC
spoVFA-R2	TTCAGTGATACGTGCCAGATG

Fig. 2. Batch cultures of B. subtilis OA105 and OA105-ΔalsSD strains.

Notes: (A) OD₆₀₀, (B) glucose concentration, (C) acetoin concentration and (D) acetate concentration; open circles, OA105; closed circles OA105-ΔalsSD. All cultures were performed in triplicate, and the average ± standard deviation is shown.

Table 3. Comparison of the specific rates between wild-type and ΔalsSD strains during the stationary phase.

Strain (genotype)	Specific glucose consumption rate (mmol g^−1h^−1)	Specific production rate (mmol g^−1h^−1)
		Acetoin	Acetate
OA105(wild-type)	2.66 ± 0.05	1.48 ± 0.37(0.56 ± 0.14)	0.00 ± 0.01(0.00 ± 0.00)
OA105ΔalsSD(ΔalsSD)	1.02 ± 0.04	0.08 ± 0.09(0.08 ± 0.09)	0.60 ± 0.10(0.59 ± 0.10)

Note: The values in parentheses represent the yield of products (mol/mol of glucose).

Fig. 3. Batch cultures of B. subtilis OA105-DPA and OA105ΔalsSD-DPA strains.

Notes: (A) OD, (B) glucose concentration, (C) acetoin concentration and (D) acetate concentration (E) DPA concentration; Open circles, OA105-DPA; closed circles OA105ΔalsSD-DPA. All cultures were performed in triplicate, and average ± standard deviation is shown.

Table 4. Comparison of the specific rates of DPA production between wild-type and ΔalsSD strains during the stationary phase.

Strain	Specific glucose consumption rate (mmol g^−1 h^−1)	Specific production rate (mmol g^−1 h^−1)
		Acetoin	Acetate	DPA
OA105-DPA (PspoVG-spoVGBA)	2.19 ± 0.14	1.27 ± 0.05(0.58 ± 0.02)	0.00 ± 0.00(0.00 ± 0.00)	0.06 ± 0.00(0.03 ± 0.00)
OA105ΔalsSD-DPA (PspoVG-spoVGBA, ΔalsSD)	1.21 ± 0.10	0.09 ± 0.05(0.07 ± 0.04)	0.29 ± 0.30(0.24 ± 0.26)	0.11 ± 0.07(0.09 ± 0.06)

Note: The values in parentheses represent the yield of products (mol/mol of glucose).

Fig. 4. Total carbon recovery of OA105-DPA and OA105ΔalsSD-DPA during the growth phase (GP) and the stationary phase (SP).

Notes: The evolved CO₂ was present in the unidentified fraction.

Fig. 5. Concentrations of intracellular metabolite in the TCA cycle at the stationary phase in DPA-producing B. subtilis.

Notes: Intracellular pool sizes of metabolites in glycolysis (A) and the TCA cycle (B) are shown. Open and closed bars represent the intracellular pool sizes in OA105-DPA and OA105ΔalsSD-DPA, respectively. The average ± standard deviation of triplicate cultures is shown. nd. denotes not detected.