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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

Comparison of metabolic profiles of BSUL03 and BSUL05 in fed-batch fermentations. Time-course profiles of cell growth, isobutanol, glucose and acetate in fed-batch fermentations were compared between BSUL05 and the parental strain BSUL03. Symbols: square, glucose; circle, isobutanol; triangle, biomass; downtriangle, acetate.
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Figure 5: Comparison of metabolic profiles of BSUL03 and BSUL05 in fed-batch fermentations. Time-course profiles of cell growth, isobutanol, glucose and acetate in fed-batch fermentations were compared between BSUL05 and the parental strain BSUL03. Symbols: square, glucose; circle, isobutanol; triangle, biomass; downtriangle, acetate.

Mentions: Strain BSUL05 showed an improved isobutanol biosynthetic capability in batch fermentations. Here it was further assessed in fed-batch fermentations. At the same time, the parental strain BSUL03 was taken as control. As shown in Figure 5, both strains grew exponentially under the aerobic conditions. Though glucose and acetate were almost exhausted during this period, little isobutanol was detected in the fermentation broth. After entering the oxygen-limited period, cell growth of the two strains slowed down, whereas isobutanol biosynthesis began to accelerate. Despite of the almost identical cell growth tendency, the two strains presented different isobutanol biosynthetic behaviors. Isobutanol titer of BSUL03 reached the peak (3.2 ± 0.4 g/L) around 50 h and declined thereafter, whereas that of BSUL05 continuously increased to 5.5 ± 0.3 g/L at the end of fermentations. Meanwhile, isobutanol yield was up to 53% of the theoretical value to 0.31 ± 0.02 C-mol/C-mol, which was 1.9-fold of BSUL03. Contrastively, the final acetate concentration of BSUL05 decreased to 0.2 g/L, which was merely about 10% of that of BSUL03. These results demonstrate the better isobutanol biosynthetic performance of the rational improved BSUL05 than the parental strain.


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)

Comparison of metabolic profiles of BSUL03 and BSUL05 in fed-batch fermentations. Time-course profiles of cell growth, isobutanol, glucose and acetate in fed-batch fermentations were compared between BSUL05 and the parental strain BSUL03. Symbols: square, glucose; circle, isobutanol; triangle, biomass; downtriangle, acetate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Comparison of metabolic profiles of BSUL03 and BSUL05 in fed-batch fermentations. Time-course profiles of cell growth, isobutanol, glucose and acetate in fed-batch fermentations were compared between BSUL05 and the parental strain BSUL03. Symbols: square, glucose; circle, isobutanol; triangle, biomass; downtriangle, acetate.
Mentions: Strain BSUL05 showed an improved isobutanol biosynthetic capability in batch fermentations. Here it was further assessed in fed-batch fermentations. At the same time, the parental strain BSUL03 was taken as control. As shown in Figure 5, both strains grew exponentially under the aerobic conditions. Though glucose and acetate were almost exhausted during this period, little isobutanol was detected in the fermentation broth. After entering the oxygen-limited period, cell growth of the two strains slowed down, whereas isobutanol biosynthesis began to accelerate. Despite of the almost identical cell growth tendency, the two strains presented different isobutanol biosynthetic behaviors. Isobutanol titer of BSUL03 reached the peak (3.2 ± 0.4 g/L) around 50 h and declined thereafter, whereas that of BSUL05 continuously increased to 5.5 ± 0.3 g/L at the end of fermentations. Meanwhile, isobutanol yield was up to 53% of the theoretical value to 0.31 ± 0.02 C-mol/C-mol, which was 1.9-fold of BSUL03. Contrastively, the final acetate concentration of BSUL05 decreased to 0.2 g/L, which was merely about 10% of that of BSUL03. These results demonstrate the better isobutanol biosynthetic performance of the rational improved BSUL05 than the parental strain.

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