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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

The meso-2,3-BD biosynthetic pathway in B. subtilis. Enzymes overexpressed or introduced are underlined and depicted in green, and those interrupted are shown in red with a cross. Expanded names of enzymes and their coding genes: ALS acetolactate synthase, alsS; ALD acetolactate decarboxylase, alsD; ACK acetate kinase, ack; ALDH acetaldehyde dehydrogenase, adh; ADH alcohol dehydrogenase, ack; LDH lactate dehydrogenase, ldh; PDH pyruvate dehydrogenase, pdhABCD; PTA phosphotransacetylase, pta; PYC pyruvate carboxylase; E1, d-(−)-butanediol dehydrogenase (EC 1.1.1.4), bdhA; E2, L-(+)-butanediol dehydrogenase (EC 1.1.1.76), budC
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Fig1: The meso-2,3-BD biosynthetic pathway in B. subtilis. Enzymes overexpressed or introduced are underlined and depicted in green, and those interrupted are shown in red with a cross. Expanded names of enzymes and their coding genes: ALS acetolactate synthase, alsS; ALD acetolactate decarboxylase, alsD; ACK acetate kinase, ack; ALDH acetaldehyde dehydrogenase, adh; ADH alcohol dehydrogenase, ack; LDH lactate dehydrogenase, ldh; PDH pyruvate dehydrogenase, pdhABCD; PTA phosphotransacetylase, pta; PYC pyruvate carboxylase; E1, d-(−)-butanediol dehydrogenase (EC 1.1.1.4), bdhA; E2, L-(+)-butanediol dehydrogenase (EC 1.1.1.76), budC

Mentions: In this work, a series of mutant strains were constructed to improve the yield and production of meso-2,3-BD step by step, including disruption of all the target genes (acoA, bdhA, pta, and ldh), introduction of meso-2,3-butadediol coding gene budC from K. pneumoniae CICC10011, and overexpressions of alsS and alsD in 2,3-BD synthetic pathway (Fig. 1) by means of a modified marker-less genetic manipulation system [38, 39]. In addition, deletion of ldh and overexpression of udhA were employed to improve the availability of NADH for AC reductase (AR). Batch cultures under microaerobic conditions were employed to investigate the performances of these genetically modified B. subtilis mutants. Finally, strain BSF9 produced 103.7 g/L meso-2,3-BD (purity >99 %) with a yield of 0.487 g/g glucose in fed-batch experiment.Fig. 1


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)

The meso-2,3-BD biosynthetic pathway in B. subtilis. Enzymes overexpressed or introduced are underlined and depicted in green, and those interrupted are shown in red with a cross. Expanded names of enzymes and their coding genes: ALS acetolactate synthase, alsS; ALD acetolactate decarboxylase, alsD; ACK acetate kinase, ack; ALDH acetaldehyde dehydrogenase, adh; ADH alcohol dehydrogenase, ack; LDH lactate dehydrogenase, ldh; PDH pyruvate dehydrogenase, pdhABCD; PTA phosphotransacetylase, pta; PYC pyruvate carboxylase; E1, d-(−)-butanediol dehydrogenase (EC 1.1.1.4), bdhA; E2, L-(+)-butanediol dehydrogenase (EC 1.1.1.76), budC
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: The meso-2,3-BD biosynthetic pathway in B. subtilis. Enzymes overexpressed or introduced are underlined and depicted in green, and those interrupted are shown in red with a cross. Expanded names of enzymes and their coding genes: ALS acetolactate synthase, alsS; ALD acetolactate decarboxylase, alsD; ACK acetate kinase, ack; ALDH acetaldehyde dehydrogenase, adh; ADH alcohol dehydrogenase, ack; LDH lactate dehydrogenase, ldh; PDH pyruvate dehydrogenase, pdhABCD; PTA phosphotransacetylase, pta; PYC pyruvate carboxylase; E1, d-(−)-butanediol dehydrogenase (EC 1.1.1.4), bdhA; E2, L-(+)-butanediol dehydrogenase (EC 1.1.1.76), budC
Mentions: In this work, a series of mutant strains were constructed to improve the yield and production of meso-2,3-BD step by step, including disruption of all the target genes (acoA, bdhA, pta, and ldh), introduction of meso-2,3-butadediol coding gene budC from K. pneumoniae CICC10011, and overexpressions of alsS and alsD in 2,3-BD synthetic pathway (Fig. 1) by means of a modified marker-less genetic manipulation system [38, 39]. In addition, deletion of ldh and overexpression of udhA were employed to improve the availability of NADH for AC reductase (AR). Batch cultures under microaerobic conditions were employed to investigate the performances of these genetically modified B. subtilis mutants. Finally, strain BSF9 produced 103.7 g/L meso-2,3-BD (purity >99 %) with a yield of 0.487 g/g glucose in fed-batch experiment.Fig. 1

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