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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

Optimization of oxygen supply in 5-L fermentor. BSF9 was cultured in LBR medium with glucose at 37 °C and 300 rpm in a 5-L fermenter at different aeration rates. 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. Agitation speed was 300 rpm. Batch fermentation with aeration rates of a 1 vvm; b 0.5 vvm; c 0.1 vvm; d glucose consumption, meso-2,3-BD production, AC, meso-2,3-BD yield, and AC + BD total yield of BSF9 during oxygen-supply optimization in batch fermentation process
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Fig4: Optimization of oxygen supply in 5-L fermentor. BSF9 was cultured in LBR medium with glucose at 37 °C and 300 rpm in a 5-L fermenter at different aeration rates. 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. Agitation speed was 300 rpm. Batch fermentation with aeration rates of a 1 vvm; b 0.5 vvm; c 0.1 vvm; d glucose consumption, meso-2,3-BD production, AC, meso-2,3-BD yield, and AC + BD total yield of BSF9 during oxygen-supply optimization in batch fermentation process

Mentions: fermentation condition, batch fermentation was conducted with different DO levels. As shown in Fig. 4a, b, and c, the aeration rate strongly influenced extracellular metabolite concentrations during 2,3-BD fermentation. It was indicated that the meso-2,3-BD yield increased from 0.164 to 0.489 g/g glucose as the aeration rate decreased from 1–0.02 VVM (Fig. 4d). Bacillus subtilis was considered to be an aerobic bacterium, and under aerobic conditions, it could secrete industrial enzymes [24], riboflavin [46, 47], AC [33, 48], etc. However, as for chiral meso-2,3-BD production using B. subtilis, limited oxygen condition was preferred for the transaction from AC to BD. As a result, the aeration rate as low as 0.02 VVM was used subsequently to achieve high meso-2,3-BD yield and production.Fig. 4


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)

Optimization of oxygen supply in 5-L fermentor. BSF9 was cultured in LBR medium with glucose at 37 °C and 300 rpm in a 5-L fermenter at different aeration rates. 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. Agitation speed was 300 rpm. Batch fermentation with aeration rates of a 1 vvm; b 0.5 vvm; c 0.1 vvm; d glucose consumption, meso-2,3-BD production, AC, meso-2,3-BD yield, and AC + BD total yield of BSF9 during oxygen-supply optimization in batch fermentation process
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Optimization of oxygen supply in 5-L fermentor. BSF9 was cultured in LBR medium with glucose at 37 °C and 300 rpm in a 5-L fermenter at different aeration rates. 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. Agitation speed was 300 rpm. Batch fermentation with aeration rates of a 1 vvm; b 0.5 vvm; c 0.1 vvm; d glucose consumption, meso-2,3-BD production, AC, meso-2,3-BD yield, and AC + BD total yield of BSF9 during oxygen-supply optimization in batch fermentation process
Mentions: fermentation condition, batch fermentation was conducted with different DO levels. As shown in Fig. 4a, b, and c, the aeration rate strongly influenced extracellular metabolite concentrations during 2,3-BD fermentation. It was indicated that the meso-2,3-BD yield increased from 0.164 to 0.489 g/g glucose as the aeration rate decreased from 1–0.02 VVM (Fig. 4d). Bacillus subtilis was considered to be an aerobic bacterium, and under aerobic conditions, it could secrete industrial enzymes [24], riboflavin [46, 47], AC [33, 48], etc. However, as for chiral meso-2,3-BD production using B. subtilis, limited oxygen condition was preferred for the transaction from AC to BD. As a result, the aeration rate as low as 0.02 VVM was used subsequently to achieve high meso-2,3-BD yield and production.Fig. 4

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