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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

Relationships of cell growth, isobutanol production, intracellular ATP and pyruvate of different isobutanol-producing B. subtilis.  The experiments were carried out in LBGSM-I medium under microaerobic conditions. The plus symbol indicates that the strain was cultivated in the medium supplemented with 3 g/L sodium acetic acid.
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Figure 4: Relationships of cell growth, isobutanol production, intracellular ATP and pyruvate of different isobutanol-producing B. subtilis. The experiments were carried out in LBGSM-I medium under microaerobic conditions. The plus symbol indicates that the strain was cultivated in the medium supplemented with 3 g/L sodium acetic acid.

Mentions: Strain BSUL04 showed a 21% and 25% lower biomass (1.62 ± 0.03 g/L) and specific growth rate (0.29 ± 0.01 h-1) than BSUL03 (Figure 3), respectively, suggesting that cell growth was impaired by ldh disruption. In microaerobic batch fermentations, BSUL04 produced 2.11 ± 0.15 g/L isobutanol with a yield of 0.18 ± 0.02 C-mol/C-mol, which was 12.5% higher than that of BSUL03. As expected, lactate was undetected in BSUL04, however, ethanol increased by 67% to 1.82 ± 0.27 g/L and acetate even tripled to 10.65 ± 1.04 g/L (Table 2). Combining with the almost unchanged intracellular pyruvate pool (Figure 4), these phenomena implied that the carbon flux originally consumed by LDH was mainly splitted by the competing branches via PDHC and isobutanol biosynthetic pathway via ALS in BSUL04, in agreement with pyruvate node analysis.


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)

Relationships of cell growth, isobutanol production, intracellular ATP and pyruvate of different isobutanol-producing B. subtilis.  The experiments were carried out in LBGSM-I medium under microaerobic conditions. The plus symbol indicates that the strain was cultivated in the medium supplemented with 3 g/L sodium acetic acid.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475101&req=5

Figure 4: Relationships of cell growth, isobutanol production, intracellular ATP and pyruvate of different isobutanol-producing B. subtilis. The experiments were carried out in LBGSM-I medium under microaerobic conditions. The plus symbol indicates that the strain was cultivated in the medium supplemented with 3 g/L sodium acetic acid.
Mentions: Strain BSUL04 showed a 21% and 25% lower biomass (1.62 ± 0.03 g/L) and specific growth rate (0.29 ± 0.01 h-1) than BSUL03 (Figure 3), respectively, suggesting that cell growth was impaired by ldh disruption. In microaerobic batch fermentations, BSUL04 produced 2.11 ± 0.15 g/L isobutanol with a yield of 0.18 ± 0.02 C-mol/C-mol, which was 12.5% higher than that of BSUL03. As expected, lactate was undetected in BSUL04, however, ethanol increased by 67% to 1.82 ± 0.27 g/L and acetate even tripled to 10.65 ± 1.04 g/L (Table 2). Combining with the almost unchanged intracellular pyruvate pool (Figure 4), these phenomena implied that the carbon flux originally consumed by LDH was mainly splitted by the competing branches via PDHC and isobutanol biosynthetic pathway via ALS in BSUL04, in agreement with pyruvate node analysis.

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