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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

Metabolic distribution of reducing power [H] in B. subtilis mutants. The arrows represent the delivery of the [H] in NADH, and the dashed arrows represent the creation of the [H]. The acetoin synthesis and breakdown pathway are indicated in the oval frame. Overexpressed genes are underlined. Disrupted pathway steps ar indicated by broken arrows
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Fig3: Metabolic distribution of reducing power [H] in B. subtilis mutants. The arrows represent the delivery of the [H] in NADH, and the dashed arrows represent the creation of the [H]. The acetoin synthesis and breakdown pathway are indicated in the oval frame. Overexpressed genes are underlined. Disrupted pathway steps ar indicated by broken arrows

Mentions: The overall strategy used to increase the pool of NADH available and reduce its consumption (Fig. 3) was as follows. The udhA gene was overexpressed in BSF25 and BSF9, transforming NADPH [produced by the pentose phosphate pathway (PPP)] to NADH supply. To reduce the consumption of NADH in lactate synthetic pathway, ldh was deleted to block the pathway, and the deletion had increased 2,3-BD yield and titer of BSF9 (Δldh) compared with BSF25 in the M9 medium. Afterward, high volume and low agitation speed were adopted to reduce the DO level. Therefore, the wastage of [H] in NADH produced by Embden Meyerhof Parnas pathway (EMP), tricarboxylic acid cycle (TCA) and from the reoxidation of excess NADPH, could be considerably reduced in the respiratory chain. Furthermore, rich medium with more reducing substrate could allow higher NADH availability and cell density, and hence the LBR medium was available to be used subsequently, thus leading to the improved 2,3-BD production in this study.Fig. 3


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)

Metabolic distribution of reducing power [H] in B. subtilis mutants. The arrows represent the delivery of the [H] in NADH, and the dashed arrows represent the creation of the [H]. The acetoin synthesis and breakdown pathway are indicated in the oval frame. Overexpressed genes are underlined. Disrupted pathway steps ar indicated by broken arrows
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Metabolic distribution of reducing power [H] in B. subtilis mutants. The arrows represent the delivery of the [H] in NADH, and the dashed arrows represent the creation of the [H]. The acetoin synthesis and breakdown pathway are indicated in the oval frame. Overexpressed genes are underlined. Disrupted pathway steps ar indicated by broken arrows
Mentions: The overall strategy used to increase the pool of NADH available and reduce its consumption (Fig. 3) was as follows. The udhA gene was overexpressed in BSF25 and BSF9, transforming NADPH [produced by the pentose phosphate pathway (PPP)] to NADH supply. To reduce the consumption of NADH in lactate synthetic pathway, ldh was deleted to block the pathway, and the deletion had increased 2,3-BD yield and titer of BSF9 (Δldh) compared with BSF25 in the M9 medium. Afterward, high volume and low agitation speed were adopted to reduce the DO level. Therefore, the wastage of [H] in NADH produced by Embden Meyerhof Parnas pathway (EMP), tricarboxylic acid cycle (TCA) and from the reoxidation of excess NADPH, could be considerably reduced in the respiratory chain. Furthermore, rich medium with more reducing substrate could allow higher NADH availability and cell density, and hence the LBR medium was available to be used subsequently, thus leading to the improved 2,3-BD production in this study.Fig. 3

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