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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, biomass, and productivity using BSF9 at different fermentation temperatures. BSF9 was cultivated in a mixture of 100 mL M9 and 10 g/L glucose in a 250-mL flask kept agitated at a speed of 100 rpm
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Fig6: Meso-2,3-BD production, biomass, and productivity using BSF9 at different fermentation temperatures. BSF9 was cultivated in a mixture of 100 mL M9 and 10 g/L glucose in a 250-mL flask kept agitated at a speed of 100 rpm

Mentions: Compared to most of 2,3-BD producers which mainly functioned below 37 °C (e.g., S. cerevisiae at 30 °C [20, 21]), B. subtilis could grow well and had the potential to produce 2,3-BD efficiently at a higher temperature. Considering this possibility, we tested the 2,3-BD production ability of BSF9 at 37, 42, 46, and 50 °C, respectively. As shown in Fig. 6, in the minimal medium, the titer of meso-2,3-BD was almost unchanged at 37, 42, and 46 °C, while the meso-2,3-BD productivity increased when the cultivation temperature was increased from 37 to 46 °C. However, the titer and productivity at 50 °C decreased by 28.6 and 36.3 % compared to those at 37 °C, respectively, which can be attributed to the much lower biomass and about 0.8 g/L acetate produced at this temperature. This problem could be got rid of with the addition of a small amount of organic nitrogen sources (e.g., 5 g/L corn steep liquor powder or yeast power) to obtain a satisfactory meso-2,3-BD production and yield. (data not shown).Fig. 6


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, biomass, and productivity using BSF9 at different fermentation temperatures. BSF9 was cultivated in a mixture of 100 mL M9 and 10 g/L glucose in a 250-mL flask kept agitated at a speed of 100 rpm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Meso-2,3-BD production, biomass, and productivity using BSF9 at different fermentation temperatures. BSF9 was cultivated in a mixture of 100 mL M9 and 10 g/L glucose in a 250-mL flask kept agitated at a speed of 100 rpm
Mentions: Compared to most of 2,3-BD producers which mainly functioned below 37 °C (e.g., S. cerevisiae at 30 °C [20, 21]), B. subtilis could grow well and had the potential to produce 2,3-BD efficiently at a higher temperature. Considering this possibility, we tested the 2,3-BD production ability of BSF9 at 37, 42, 46, and 50 °C, respectively. As shown in Fig. 6, in the minimal medium, the titer of meso-2,3-BD was almost unchanged at 37, 42, and 46 °C, while the meso-2,3-BD productivity increased when the cultivation temperature was increased from 37 to 46 °C. However, the titer and productivity at 50 °C decreased by 28.6 and 36.3 % compared to those at 37 °C, respectively, which can be attributed to the much lower biomass and about 0.8 g/L acetate produced at this temperature. This problem could be got rid of with the addition of a small amount of organic nitrogen sources (e.g., 5 g/L corn steep liquor powder or yeast power) to obtain a satisfactory meso-2,3-BD production and yield. (data not shown).Fig. 6

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