A method for enhancing the synthesis of 2,3-butanediol by increasing the level of intracellular coenzyme
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A technology of butanediol and oxidase, applied in the field of genetic engineering, can solve problems such as non-compliance with industrialized safe production and the like
Active Publication Date: 2018-05-01
JIANGNAN UNIV +1
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Most of the currently reported 2,3-butanediol producing strains are Klebsiella, Enterobacter, and Serratia, etc. These strains are potentially pathogenic and do not meet the Meet the requirements of industrial safety production
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Embodiment 1
[0008] Embodiment 1: Amplification of target gene and construction of recombinant Bacillus amyloliquefaciens
[0009] The specific process of obtaining the fragment nox::fdh is as follows:
[0010] First, using the chromosomal DNA of strain B10-127 as a template, using primers P1 and P2, amplified by PCR technology to obtain the gene nox nucleotide sequence as shown in SEQ ID NO: 1, the purified nox gene was connected to the plasmid pMD On -18T, the recombinant plasmid T-nox was constructed, and after verification by double enzyme digestion, it indicated that the recombinant plasmid was constructed successfully. Subsequently, use primers P3 and P4 to obtain nucleotide sequence by PCR amplification and be the gene fdh shown in SEQ ID NO: 2, after the fdh gene after the purification is digested by restriction endonuclease Nde I and MluI, with same process The plasmid pMA5-HpaII digested by the above two restriction endonucleases was connected to obtain pMA5-HpaII-fdh, and HpaII...
Embodiment 2
[0014] Embodiment 2: Fermentation performance verification of bacteria original bacteria and recombinant bacteria
[0015] (1) Seed cultivation
[0016] Pick a single colony from the activated plate and inoculate it in the seed medium. The seed culture temperature is 37°C, the shaker speed is 160r / min, and the culture time is about 12h. The composition of the seed medium: yeast extract 5g / L, tryptone 10g / min L, NaCl10g / L.
[0017] (2) Fermentation culture
[0018] The initial fermentation culture volume is 2.5L, and the components of the fermentation medium used are as follows:
[0019] Fermentation medium components: glucose 160g / L, corn steep liquor 5g / L, urea 3g / L, sodium citrate 6g / L, K 2 HPO 4 4g / L, MgSO 4 0.2g / L; adjust the pH of the above fermentation medium to 6.5 with 5mol / L NaOH, and sterilize at 121°C for 30min.
[0020] Fermentation Fermentation conditions: inoculate the above-mentioned cultivated seed liquid into the fermentation medium according to the inoc...
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Abstract
The last step of synthesis of 2,3-butanediol is that acetoin reductase performs catalytic reduction on acetoin, 2,3-butanediol is synthesized, the reaction requires participation of reduced coenzyme NADH (nicotinamide adenine dinucleotide hydrogen), and thus the high-low level of intracellular coenzyme NADH is an important factor affecting conversion of acetoin to 2,3-butanediol. In order to further improve the synthesis capability of strain 2,3-butanediol and reduce accumulation of byproduct acetoin, HpaII-fdh fragments are inserted into a gene nox, a fragment nox::HpaII-fdh is obtained and guided into bacillus amyloliquefaciens B10-127, and recombinant bacillus amyloliquefaciens (renamed as N delta F) with the fdh gene inserted into the gene nox on a genome is obtained in a homologous recombination principle. Fermentation performance detection of 2,3-butanediol is performed on original bacteria and recombinant bacteria N delta F. A result proves that by comparison with the original bacteria, the yield of the recombinant bacteria 2,3-butanediol is improved by 18.9%, and the yield of the byproduct acetoin is reduced by 70.8%. Accordingly, by means of the strategy, the yield of 2,3-butanediol can be improved, and accumulation of the byproduct acetoin can be reduced.
Description
technical field [0001] The invention relates to a method for enhancing the synthesis of 2,3-butanediol by increasing the level of intracellular cofactors, which belongs to the technical field of genetic engineering. Background technique [0002] With the increasing shortage of energy resources and the increasingly serious environmental problems, the development of renewable biomass resources as raw materials, the biorefining technology of producing various chemicals, functional materials and energy substances through chemical, biological and integrated methods is becoming more and more important. more and more attention at home and abroad. 2,3-Butanediol is an important chemical raw material and liquid fuel, widely used in chemical industry, food, fuel, aerospace and other fields. 2,3-butanediol is dehydrogenated to form diacetyl, which is a high-value food flavor with certain antibacterial effect; under the action of dehydrogenase, the generated 3-hydroxy-2-butanone (aceto...
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