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Metabolic engineering of Bacillus subtilis for chiral pure meso-2,3-butanediol production.

Fu J, Huo G, Feng L, Mao Y, Wang Z, Ma H, Chen T, Zhao X - Biotechnol Biofuels (2016)

Bottom Line: Next, both pta and ldh gene were deleted to decrease the accumulation of the byproducts, acetate and l-lactate.We further introduced the meso-2,3-BD dehydrogenase coding gene budC from Klebsiella pneumoniae CICC10011, as well as overexpressed alsSD in the tetra-mutant (ΔacoAΔbdhAΔptaΔldh) to achieve the efficient production of chiral meso-2,3-BD.This work offered a novel strategy for the production of chiral pure meso-2,3-BD in B. subtilis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education); SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072 People's Republic of China.

ABSTRACT

Background: 2,3-Butanediol (2,3-BD) with low toxicity to microbes, could be a promising alternative for biofuel production. However, most of the 2,3-BD producers are opportunistic pathogens that are not suitable for industrial-scale fermentation. In our previous study, wild-type Bacillus subtilis 168, as a class I microorganism, was first found to generate only d-(-)-2,3-BD (purity >99 %) under low oxygen conditions.

Results: In this work, B. subtilis was engineered to produce chiral pure meso-2,3-BD. First, d-(-)-2,3-BD production was abolished by deleting d-(-)-2,3-BD dehydrogenase coding gene bdhA, and acoA gene was knocked out to prevent the degradation of acetoin (AC), the immediate precursor of 2,3-BD. Next, both pta and ldh gene were deleted to decrease the accumulation of the byproducts, acetate and l-lactate. We further introduced the meso-2,3-BD dehydrogenase coding gene budC from Klebsiella pneumoniae CICC10011, as well as overexpressed alsSD in the tetra-mutant (ΔacoAΔbdhAΔptaΔldh) to achieve the efficient production of chiral meso-2,3-BD. Finally, the pool of NADH availability was further increased to facilitate the conversion of meso-2,3-BD from AC by overexpressing udhA gene (coding a soluble transhydrogenase) and low dissolved oxygen control during the cultivation. Under microaerobic oxygen conditions, the best strain BSF9 produced 103.7 g/L meso-2,3-BD with a yield of 0.487 g/g glucose in the 5-L batch fermenter, and the titer of the main byproduct AC was no more than 1.1 g/L.

Conclusion: This work offered a novel strategy for the production of chiral pure meso-2,3-BD in B. subtilis. To our knowledge, this is the first report indicating that metabolic engineered B. subtilis could produce chiral meso-2,3-BD with high purity under limited oxygen conditions. These results further demonstrated that B. subtilis as a class I microorganism is a competitive industrial-level meso-2,3-BD producer.

No MeSH data available.


Related in: MedlinePlus

Meso-2,3-BD production from glucose using BSF9 in fed-batch fermentation. Filled square glucose concentration; Filled circle 2,3-BD concentration; Filled triangle AC concentration; Filled diamond cell density, optical density at 600 nm (OD600). Cultivation was carried out at an initial pH of 6.5; during the fermentation processes, the pH was uncontrolled. Agitation speed was 300 rpm, and aeration rate was 0.02 vvm
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Fig5: Meso-2,3-BD production from glucose using BSF9 in fed-batch fermentation. Filled square glucose concentration; Filled circle 2,3-BD concentration; Filled triangle AC concentration; Filled diamond cell density, optical density at 600 nm (OD600). Cultivation was carried out at an initial pH of 6.5; during the fermentation processes, the pH was uncontrolled. Agitation speed was 300 rpm, and aeration rate was 0.02 vvm

Mentions: Under the optimized fermentation conditions, a final titer of 103.7 g/L meso-2,3-BD (purity >99 %) and 1.1 g/L AC as the main byproducts were achieved by BSF9 (Fig. 5). The meso-2,3-BD productivity was 0.459 g/(L h), which was almost twice as much as that in flask experiments. No more than 0.2 g/L succinate or acetate was produced. No pyruvic, α-ketoglutaric acid, diacetyl, and lactate were detected. During the fermentation, the biomass increased slowly in the first 50 h and was maintained at the level of approximately 5.2 g/L CDW until the end. This low oxygen level resulted in a low biomass and a slow glucose consumption rate, which might account for the unsatisfactory meso-2,3-BD productivity. However, no more than 1.1 g/L AC was produced under this condition, which not only caused a high yield of 0.487 g meso-2,3-BD/g glucose (97.4 % of the theoretical maximum yield), but also would be beneficial for the downstream purification process.Fig. 5


Metabolic engineering of Bacillus subtilis for chiral pure meso-2,3-butanediol production.

Fu J, Huo G, Feng L, Mao Y, Wang Z, Ma H, Chen T, Zhao X - Biotechnol Biofuels (2016)

Meso-2,3-BD production from glucose using BSF9 in fed-batch fermentation. Filled square glucose concentration; Filled circle 2,3-BD concentration; Filled triangle AC concentration; Filled diamond cell density, optical density at 600 nm (OD600). Cultivation was carried out at an initial pH of 6.5; during the fermentation processes, the pH was uncontrolled. Agitation speed was 300 rpm, and aeration rate was 0.02 vvm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4837526&req=5

Fig5: Meso-2,3-BD production from glucose using BSF9 in fed-batch fermentation. Filled square glucose concentration; Filled circle 2,3-BD concentration; Filled triangle AC concentration; Filled diamond cell density, optical density at 600 nm (OD600). Cultivation was carried out at an initial pH of 6.5; during the fermentation processes, the pH was uncontrolled. Agitation speed was 300 rpm, and aeration rate was 0.02 vvm
Mentions: Under the optimized fermentation conditions, a final titer of 103.7 g/L meso-2,3-BD (purity >99 %) and 1.1 g/L AC as the main byproducts were achieved by BSF9 (Fig. 5). The meso-2,3-BD productivity was 0.459 g/(L h), which was almost twice as much as that in flask experiments. No more than 0.2 g/L succinate or acetate was produced. No pyruvic, α-ketoglutaric acid, diacetyl, and lactate were detected. During the fermentation, the biomass increased slowly in the first 50 h and was maintained at the level of approximately 5.2 g/L CDW until the end. This low oxygen level resulted in a low biomass and a slow glucose consumption rate, which might account for the unsatisfactory meso-2,3-BD productivity. However, no more than 1.1 g/L AC was produced under this condition, which not only caused a high yield of 0.487 g meso-2,3-BD/g glucose (97.4 % of the theoretical maximum yield), but also would be beneficial for the downstream purification process.Fig. 5

Bottom Line: Next, both pta and ldh gene were deleted to decrease the accumulation of the byproducts, acetate and l-lactate.We further introduced the meso-2,3-BD dehydrogenase coding gene budC from Klebsiella pneumoniae CICC10011, as well as overexpressed alsSD in the tetra-mutant (ΔacoAΔbdhAΔptaΔldh) to achieve the efficient production of chiral meso-2,3-BD.This work offered a novel strategy for the production of chiral pure meso-2,3-BD in B. subtilis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education); SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072 People's Republic of China.

ABSTRACT

Background: 2,3-Butanediol (2,3-BD) with low toxicity to microbes, could be a promising alternative for biofuel production. However, most of the 2,3-BD producers are opportunistic pathogens that are not suitable for industrial-scale fermentation. In our previous study, wild-type Bacillus subtilis 168, as a class I microorganism, was first found to generate only d-(-)-2,3-BD (purity >99 %) under low oxygen conditions.

Results: In this work, B. subtilis was engineered to produce chiral pure meso-2,3-BD. First, d-(-)-2,3-BD production was abolished by deleting d-(-)-2,3-BD dehydrogenase coding gene bdhA, and acoA gene was knocked out to prevent the degradation of acetoin (AC), the immediate precursor of 2,3-BD. Next, both pta and ldh gene were deleted to decrease the accumulation of the byproducts, acetate and l-lactate. We further introduced the meso-2,3-BD dehydrogenase coding gene budC from Klebsiella pneumoniae CICC10011, as well as overexpressed alsSD in the tetra-mutant (ΔacoAΔbdhAΔptaΔldh) to achieve the efficient production of chiral meso-2,3-BD. Finally, the pool of NADH availability was further increased to facilitate the conversion of meso-2,3-BD from AC by overexpressing udhA gene (coding a soluble transhydrogenase) and low dissolved oxygen control during the cultivation. Under microaerobic oxygen conditions, the best strain BSF9 produced 103.7 g/L meso-2,3-BD with a yield of 0.487 g/g glucose in the 5-L batch fermenter, and the titer of the main byproduct AC was no more than 1.1 g/L.

Conclusion: This work offered a novel strategy for the production of chiral pure meso-2,3-BD in B. subtilis. To our knowledge, this is the first report indicating that metabolic engineered B. subtilis could produce chiral meso-2,3-BD with high purity under limited oxygen conditions. These results further demonstrated that B. subtilis as a class I microorganism is a competitive industrial-level meso-2,3-BD producer.

No MeSH data available.


Related in: MedlinePlus