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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

Changes in the levels of intracellular NADH/NAD+ ratios in different strains. Bacteria were cultivated using a mixture of 100 mL M9 and 10 g/L glucose in a 250-mL flask kept agitated at a speed of 100 rpm and 37 °C. The intracellular NADH and NAD+ were extracted after 30 h. Data show average values and standard deviations of triplicate experiments
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Fig2: Changes in the levels of intracellular NADH/NAD+ ratios in different strains. Bacteria were cultivated using a mixture of 100 mL M9 and 10 g/L glucose in a 250-mL flask kept agitated at a speed of 100 rpm and 37 °C. The intracellular NADH and NAD+ were extracted after 30 h. Data show average values and standard deviations of triplicate experiments

Mentions: As shown in Table 1, a small amount of acetate had been detected in the media of strains BSF2 when glucose was depleted, while lactate and succinate accumulated with maximum concentrations of 0.36 and 0.09 g/L, respectively. To eliminate the accumulation of byproducts, acetate and lactate, pta (coding phosphate acetyltransferase) and ldh (coding l-lactate dehydrogenase) were deleted, respectively. Inactivation of pta had almost no effect on acetate reduction of BSF4 (0.14 ± 0.01 g/L) compared with BSF3 (bdhA deleted in BSF2 for abolishing of d-(−)-2,3-BD) (0.17 ± 0.01 g/L), indicating there might be other major acetate or acetyl-phosphate synthetic pathways in B. subtilis [41]. However, the biomass decreased from 0.898 g/L DCW for BSF3 to 0.828 g/L DCW for BSF4, while the glucose consumption rate slightly decreased from 0.233 g/(L h) of BSF3 to 0.222 g/(L h) of BSF4. The disruption of ldh abolished lactate accumulation of BSF5; however, it drastically increased the formations of acetate and succinate to 0.71 and 0.29 g/L, respectively. The increasing concentrations of acetate and succinate might presumably have resulted from an unknown regulatory mechanism that affected the other acetate synthesis pathway of ldh-deleted mutant BSF5. In our previous study, we observed that bdhA deletion resulted in a lower biomass (0.960 g/L for BSF1 versus 0.898 g/L for BSF3) and a slower glucose consumption rate [0.270 g/(L h) for BSF1 versus 0.233 g/(L h) for BSF3]. Here, we found that the knockout of ldh in bdhA-deleted mutant BSF4 further decreased the biomass formation (from 0.828 to 0.814 g/L) and glucose consumption rate [from 0.222 to 0.208 g/(L h)]. It was also reported that deletion of ldh in B. subtilis resulted in drastic growth reduction under anaerobic conditions [42]. All these results might be ascribed to the fact that blocking the biosynthesis pathways of reducing metabolites caused the imbalance of reducing power to some extent, which was demonstrated by the intracellular NADH/NAD+ analysis (Fig. 2). The NADH/NAD+ level increased from 0.61 for BSF1 to 0.71 for BSF4, and further increased to 0.83 for BSF5. For all the strains containing ldh, lactate was accumulated to its highest concentration when glucose was depleted and was quickly reused when glucose was exhausted, indicating that lactate might be a direct carbon source for B. subtilis after the glucose was depleted in minimal medium. Although deletion of ldh did not increase metabolic flux in 2,3-BD pathway, it could increase the NADH/NAD+ ratio for further conversion of AC to 2,3-BD, as the NADH availability was the key factor for 2,3-BD production [25].Fig. 2


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)

Changes in the levels of intracellular NADH/NAD+ ratios in different strains. Bacteria were cultivated using a mixture of 100 mL M9 and 10 g/L glucose in a 250-mL flask kept agitated at a speed of 100 rpm and 37 °C. The intracellular NADH and NAD+ were extracted after 30 h. Data show average values and standard deviations of triplicate experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Changes in the levels of intracellular NADH/NAD+ ratios in different strains. Bacteria were cultivated using a mixture of 100 mL M9 and 10 g/L glucose in a 250-mL flask kept agitated at a speed of 100 rpm and 37 °C. The intracellular NADH and NAD+ were extracted after 30 h. Data show average values and standard deviations of triplicate experiments
Mentions: As shown in Table 1, a small amount of acetate had been detected in the media of strains BSF2 when glucose was depleted, while lactate and succinate accumulated with maximum concentrations of 0.36 and 0.09 g/L, respectively. To eliminate the accumulation of byproducts, acetate and lactate, pta (coding phosphate acetyltransferase) and ldh (coding l-lactate dehydrogenase) were deleted, respectively. Inactivation of pta had almost no effect on acetate reduction of BSF4 (0.14 ± 0.01 g/L) compared with BSF3 (bdhA deleted in BSF2 for abolishing of d-(−)-2,3-BD) (0.17 ± 0.01 g/L), indicating there might be other major acetate or acetyl-phosphate synthetic pathways in B. subtilis [41]. However, the biomass decreased from 0.898 g/L DCW for BSF3 to 0.828 g/L DCW for BSF4, while the glucose consumption rate slightly decreased from 0.233 g/(L h) of BSF3 to 0.222 g/(L h) of BSF4. The disruption of ldh abolished lactate accumulation of BSF5; however, it drastically increased the formations of acetate and succinate to 0.71 and 0.29 g/L, respectively. The increasing concentrations of acetate and succinate might presumably have resulted from an unknown regulatory mechanism that affected the other acetate synthesis pathway of ldh-deleted mutant BSF5. In our previous study, we observed that bdhA deletion resulted in a lower biomass (0.960 g/L for BSF1 versus 0.898 g/L for BSF3) and a slower glucose consumption rate [0.270 g/(L h) for BSF1 versus 0.233 g/(L h) for BSF3]. Here, we found that the knockout of ldh in bdhA-deleted mutant BSF4 further decreased the biomass formation (from 0.828 to 0.814 g/L) and glucose consumption rate [from 0.222 to 0.208 g/(L h)]. It was also reported that deletion of ldh in B. subtilis resulted in drastic growth reduction under anaerobic conditions [42]. All these results might be ascribed to the fact that blocking the biosynthesis pathways of reducing metabolites caused the imbalance of reducing power to some extent, which was demonstrated by the intracellular NADH/NAD+ analysis (Fig. 2). The NADH/NAD+ level increased from 0.61 for BSF1 to 0.71 for BSF4, and further increased to 0.83 for BSF5. For all the strains containing ldh, lactate was accumulated to its highest concentration when glucose was depleted and was quickly reused when glucose was exhausted, indicating that lactate might be a direct carbon source for B. subtilis after the glucose was depleted in minimal medium. Although deletion of ldh did not increase metabolic flux in 2,3-BD pathway, it could increase the NADH/NAD+ ratio for further conversion of AC to 2,3-BD, as the NADH availability was the key factor for 2,3-BD production [25].Fig. 2

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