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Rational improvement of the engineered isobutanol-producing Bacillus subtilis by elementary mode analysis.

Li S, Huang D, Li Y, Wen J, Jia X - Microb. Cell Fact. (2012)

Bottom Line: Moreover, this mutant produced approximately 70 % more isobutanol to the maximal titer of 5.5 ± 0.3 g/L in fed-batch fermentations.EMA was employed as a guiding tool to direct rational improvement of the engineered isobutanol-producing B. subtilis.The consistency between model prediction and experimental results demonstrates the rationality and accuracy of this EMA-based approach for target identification.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.

ABSTRACT

Background: Isobutanol is considered as a leading candidate for the replacement of current fossil fuels, and expected to be produced biotechnologically. Owing to the valuable features, Bacillus subtilis has been engineered as an isobutanol producer, whereas it needs to be further optimized for more efficient production. Since elementary mode analysis (EMA) is a powerful tool for systematical analysis of metabolic network structures and cell metabolism, it might be of great importance in the rational strain improvement.

Results: Metabolic network of the isobutanol-producing B. subtilis BSUL03 was first constructed for EMA. Considering the actual cellular physiological state, 239 elementary modes (EMs) were screened from total 11,342 EMs for potential target prediction. On this basis, lactate dehydrogenase (LDH) and pyruvate dehydrogenase complex (PDHC) were predicted as the most promising inactivation candidates according to flux flexibility analysis and intracellular flux distribution simulation. Then, the in silico designed mutants were experimentally constructed. The maximal isobutanol yield of the LDH- and PDHC-deficient strain BSUL05 reached 61% of the theoretical value to 0.36 ± 0.02 C-mol isobutanol/C-mol glucose, which was 2.3-fold of BSUL03. Moreover, this mutant produced approximately 70 % more isobutanol to the maximal titer of 5.5 ± 0.3 g/L in fed-batch fermentations.

Conclusions: EMA was employed as a guiding tool to direct rational improvement of the engineered isobutanol-producing B. subtilis. The consistency between model prediction and experimental results demonstrates the rationality and accuracy of this EMA-based approach for target identification. This network-based rational strain improvement strategy could serve as a promising concept to engineer efficient B. subtilis hosts for isobutanol, as well as other valuable products.

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Related in: MedlinePlus

Simulated flux-fold changes of the central metabolism of isobutanol-producing B. subtilis under the optimal conditions. The chart represents the simulated fold change of the average flux through the central metabolism under optimal conditions considering the physiological states of different isobutanol-producing B. subtilis. All fluxes are given as relative molar flux normalized to 1 mol of glucose. Fold change of each reaction in the mutants is calculated by comparing to the corresponding flux in BSUL03. The dot lines indicate the multiple steps; the olive lines indicate the obviously increased flux by inactivating the targets (represented by red ×); the negative flux indicates a reaction occurs in reverse direction. Metabolites abbreviations: Gluc, Glucose; G6P, D-Glucose 6-phosphate; F6P, D-Fructose 6-phosphate; F16P, D-Fructose 1,6-bisphosphate; DHAP, Dihydroxyacetone phosphate; G3P, Glyceraldehyde 3-phosphate; 3PG, 3-Phospho-D-glycerate; PEP, Phosphoenolpyruvate; PYR, Pyruvate; AcCoA, Acetyl-coenzyme A; Cit, Citrate; ICit, Isocitrate; alKG, 2-Oxoglutarate; SuccCoA, Succinyl-CoA; Succ, Succinate; Fum, Fumarate; Mal, L-Malate; OxA, Oxaloacetate; RL5P, Ribulose-5-phosphate; R5P, alpha-D-Ribose 5-phosphate; X5P, Xylulose-5-phosphate; G3P, Glyceraldehyde 3-phosphate; S7P, Sedoheptulose 7-phosphate; E4P, D-Erythrose 4-phosphate; Isb, Isobutanol; Lac, L-Lactate; Ac, Acetate.
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Figure 2: Simulated flux-fold changes of the central metabolism of isobutanol-producing B. subtilis under the optimal conditions. The chart represents the simulated fold change of the average flux through the central metabolism under optimal conditions considering the physiological states of different isobutanol-producing B. subtilis. All fluxes are given as relative molar flux normalized to 1 mol of glucose. Fold change of each reaction in the mutants is calculated by comparing to the corresponding flux in BSUL03. The dot lines indicate the multiple steps; the olive lines indicate the obviously increased flux by inactivating the targets (represented by red ×); the negative flux indicates a reaction occurs in reverse direction. Metabolites abbreviations: Gluc, Glucose; G6P, D-Glucose 6-phosphate; F6P, D-Fructose 6-phosphate; F16P, D-Fructose 1,6-bisphosphate; DHAP, Dihydroxyacetone phosphate; G3P, Glyceraldehyde 3-phosphate; 3PG, 3-Phospho-D-glycerate; PEP, Phosphoenolpyruvate; PYR, Pyruvate; AcCoA, Acetyl-coenzyme A; Cit, Citrate; ICit, Isocitrate; alKG, 2-Oxoglutarate; SuccCoA, Succinyl-CoA; Succ, Succinate; Fum, Fumarate; Mal, L-Malate; OxA, Oxaloacetate; RL5P, Ribulose-5-phosphate; R5P, alpha-D-Ribose 5-phosphate; X5P, Xylulose-5-phosphate; G3P, Glyceraldehyde 3-phosphate; S7P, Sedoheptulose 7-phosphate; E4P, D-Erythrose 4-phosphate; Isb, Isobutanol; Lac, L-Lactate; Ac, Acetate.

Mentions: To ensure the validity of the chosen targets, intracellular flux distributions of the parental strain BSUL03 and its corresponding mutants were further simulated. When considering lactate production, the theoretical isobutanol yield of BSUL03 sharply decreased from 0.64 to 0.20 C-mol/C-mol under the optimal conditions (Figure 1). Pyruvate node analysis showed that pyruvate flux split ratio of LDH to acetolactate synthease (ALS) was 3.7:1, much higher than the values of LDH to the other branches. As the effect of gene knockout could be simulated by removing the corresponding reactions from the stoichiometric matrix, the phenotype of the specific mutant could be analyzed by the remaining EMs. As shown in Figure 2, the relative flux of the suppositional LDH-deficient strain BSUΔldh was obviously changed in tricarboxylic acid (TCA) cycle, PPP and isobutanol biosynthetic pathway under the optimal conditions. Compared to BSUL03, the twofold relative flux through ALS of BSUΔldh increased the theoretical isobutanol yield to 0.41 C-mol/C-mol (Figure 1). Additionally, pyruvate node analysis also showed that the flux drained off pyruvate was remarkably redistributed among the remaining branches. In particular, the flux fraction through pyruvate dehydrogenase complex (PDHC) was significantly increased by 10-fold (4.0% → 44.3%), higher than the 1.5-fold increase through ALS (19.9% → 49.4%). Synchronously, acetate secretion was enhanced by the excessive carbon flux through PDHC (Figure 2). In the suppositional LDH- and PDHC-deficient strain BSUΔldhΔpdhC, ALS occupied 94% of pyruvate flux and became the dominant pyruvate gainer at optimal performance according to pyruvate node analysis. The relative flux through the objective reaction was approximately tripled in comparison with that of BSUL03 (Figure 2). As a result, this in silico strain obtained a 2-fold increase of the theoretical isobutanol yield of 0.59 C-mol/C-mol (Figure 1).


Rational improvement of the engineered isobutanol-producing Bacillus subtilis by elementary mode analysis.

Li S, Huang D, Li Y, Wen J, Jia X - Microb. Cell Fact. (2012)

Simulated flux-fold changes of the central metabolism of isobutanol-producing B. subtilis under the optimal conditions. The chart represents the simulated fold change of the average flux through the central metabolism under optimal conditions considering the physiological states of different isobutanol-producing B. subtilis. All fluxes are given as relative molar flux normalized to 1 mol of glucose. Fold change of each reaction in the mutants is calculated by comparing to the corresponding flux in BSUL03. The dot lines indicate the multiple steps; the olive lines indicate the obviously increased flux by inactivating the targets (represented by red ×); the negative flux indicates a reaction occurs in reverse direction. Metabolites abbreviations: Gluc, Glucose; G6P, D-Glucose 6-phosphate; F6P, D-Fructose 6-phosphate; F16P, D-Fructose 1,6-bisphosphate; DHAP, Dihydroxyacetone phosphate; G3P, Glyceraldehyde 3-phosphate; 3PG, 3-Phospho-D-glycerate; PEP, Phosphoenolpyruvate; PYR, Pyruvate; AcCoA, Acetyl-coenzyme A; Cit, Citrate; ICit, Isocitrate; alKG, 2-Oxoglutarate; SuccCoA, Succinyl-CoA; Succ, Succinate; Fum, Fumarate; Mal, L-Malate; OxA, Oxaloacetate; RL5P, Ribulose-5-phosphate; R5P, alpha-D-Ribose 5-phosphate; X5P, Xylulose-5-phosphate; G3P, Glyceraldehyde 3-phosphate; S7P, Sedoheptulose 7-phosphate; E4P, D-Erythrose 4-phosphate; Isb, Isobutanol; Lac, L-Lactate; Ac, Acetate.
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Figure 2: Simulated flux-fold changes of the central metabolism of isobutanol-producing B. subtilis under the optimal conditions. The chart represents the simulated fold change of the average flux through the central metabolism under optimal conditions considering the physiological states of different isobutanol-producing B. subtilis. All fluxes are given as relative molar flux normalized to 1 mol of glucose. Fold change of each reaction in the mutants is calculated by comparing to the corresponding flux in BSUL03. The dot lines indicate the multiple steps; the olive lines indicate the obviously increased flux by inactivating the targets (represented by red ×); the negative flux indicates a reaction occurs in reverse direction. Metabolites abbreviations: Gluc, Glucose; G6P, D-Glucose 6-phosphate; F6P, D-Fructose 6-phosphate; F16P, D-Fructose 1,6-bisphosphate; DHAP, Dihydroxyacetone phosphate; G3P, Glyceraldehyde 3-phosphate; 3PG, 3-Phospho-D-glycerate; PEP, Phosphoenolpyruvate; PYR, Pyruvate; AcCoA, Acetyl-coenzyme A; Cit, Citrate; ICit, Isocitrate; alKG, 2-Oxoglutarate; SuccCoA, Succinyl-CoA; Succ, Succinate; Fum, Fumarate; Mal, L-Malate; OxA, Oxaloacetate; RL5P, Ribulose-5-phosphate; R5P, alpha-D-Ribose 5-phosphate; X5P, Xylulose-5-phosphate; G3P, Glyceraldehyde 3-phosphate; S7P, Sedoheptulose 7-phosphate; E4P, D-Erythrose 4-phosphate; Isb, Isobutanol; Lac, L-Lactate; Ac, Acetate.
Mentions: To ensure the validity of the chosen targets, intracellular flux distributions of the parental strain BSUL03 and its corresponding mutants were further simulated. When considering lactate production, the theoretical isobutanol yield of BSUL03 sharply decreased from 0.64 to 0.20 C-mol/C-mol under the optimal conditions (Figure 1). Pyruvate node analysis showed that pyruvate flux split ratio of LDH to acetolactate synthease (ALS) was 3.7:1, much higher than the values of LDH to the other branches. As the effect of gene knockout could be simulated by removing the corresponding reactions from the stoichiometric matrix, the phenotype of the specific mutant could be analyzed by the remaining EMs. As shown in Figure 2, the relative flux of the suppositional LDH-deficient strain BSUΔldh was obviously changed in tricarboxylic acid (TCA) cycle, PPP and isobutanol biosynthetic pathway under the optimal conditions. Compared to BSUL03, the twofold relative flux through ALS of BSUΔldh increased the theoretical isobutanol yield to 0.41 C-mol/C-mol (Figure 1). Additionally, pyruvate node analysis also showed that the flux drained off pyruvate was remarkably redistributed among the remaining branches. In particular, the flux fraction through pyruvate dehydrogenase complex (PDHC) was significantly increased by 10-fold (4.0% → 44.3%), higher than the 1.5-fold increase through ALS (19.9% → 49.4%). Synchronously, acetate secretion was enhanced by the excessive carbon flux through PDHC (Figure 2). In the suppositional LDH- and PDHC-deficient strain BSUΔldhΔpdhC, ALS occupied 94% of pyruvate flux and became the dominant pyruvate gainer at optimal performance according to pyruvate node analysis. The relative flux through the objective reaction was approximately tripled in comparison with that of BSUL03 (Figure 2). As a result, this in silico strain obtained a 2-fold increase of the theoretical isobutanol yield of 0.59 C-mol/C-mol (Figure 1).

Bottom Line: Moreover, this mutant produced approximately 70 % more isobutanol to the maximal titer of 5.5 ± 0.3 g/L in fed-batch fermentations.EMA was employed as a guiding tool to direct rational improvement of the engineered isobutanol-producing B. subtilis.The consistency between model prediction and experimental results demonstrates the rationality and accuracy of this EMA-based approach for target identification.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.

ABSTRACT

Background: Isobutanol is considered as a leading candidate for the replacement of current fossil fuels, and expected to be produced biotechnologically. Owing to the valuable features, Bacillus subtilis has been engineered as an isobutanol producer, whereas it needs to be further optimized for more efficient production. Since elementary mode analysis (EMA) is a powerful tool for systematical analysis of metabolic network structures and cell metabolism, it might be of great importance in the rational strain improvement.

Results: Metabolic network of the isobutanol-producing B. subtilis BSUL03 was first constructed for EMA. Considering the actual cellular physiological state, 239 elementary modes (EMs) were screened from total 11,342 EMs for potential target prediction. On this basis, lactate dehydrogenase (LDH) and pyruvate dehydrogenase complex (PDHC) were predicted as the most promising inactivation candidates according to flux flexibility analysis and intracellular flux distribution simulation. Then, the in silico designed mutants were experimentally constructed. The maximal isobutanol yield of the LDH- and PDHC-deficient strain BSUL05 reached 61% of the theoretical value to 0.36 ± 0.02 C-mol isobutanol/C-mol glucose, which was 2.3-fold of BSUL03. Moreover, this mutant produced approximately 70 % more isobutanol to the maximal titer of 5.5 ± 0.3 g/L in fed-batch fermentations.

Conclusions: EMA was employed as a guiding tool to direct rational improvement of the engineered isobutanol-producing B. subtilis. The consistency between model prediction and experimental results demonstrates the rationality and accuracy of this EMA-based approach for target identification. This network-based rational strain improvement strategy could serve as a promising concept to engineer efficient B. subtilis hosts for isobutanol, as well as other valuable products.

Show MeSH
Related in: MedlinePlus