Method for enhancing 2,3-butanediol synthesis by improving intracellular coenzyme level
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A technology of butanediol and content, applied in the field of genetic engineering, can solve problems that do not meet industrial safety production
Active Publication Date: 2015-12-16
JIANGNAN UNIV +1
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Problems solved by technology
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] Example 1: Amplification of target gene and construction of recombinant Bacillus amyloliquefaciens
[0009] The specific process of obtaining fragment nox::fdh is as follows:
[0010] Firstly, using the chromosomal DNA of strain B10-127 as a template, using primers P1 and P2, the nucleotide sequence of the gene nox was amplified by PCR technology as shown in SEQ ID NO:1, and the purified nox gene was ligated to the plasmid pMD-18T Above, the recombinant plasmid T-nox was constructed, and after double enzyme digestion verification, the recombinant plasmid was constructed successfully. Subsequently, primers P3 and P4 were used to amplify the gene fdh with the nucleotide sequence shown in SEQ ID NO: 2 by PCR, and the purified fdh gene was digested with restriction enzymes NdeI and MluI, and the same as the above two Restriction enzyme digested plasmid pMA5-HpaII was ligated to obtain pMA5-HpaII-fdh, which was amplified by PCR with primers P5 and P6 to obtain HpaII-fdh; and ins...
Embodiment 2
[0014] Example 2: Verification of fermentation performance of original bacteria and recombinant bacteria
[0015] (1) Seed cultivation
[0016] Pick a single colony from the activation plate and inoculate it in the seed culture medium. The seed culture temperature is 37℃, the shaker speed is 160r / min, and the culture time is about 12h. The composition of the seed culture medium: yeast extract 5g / L, tryptone 10g / L, NaCl10g / L.
[0017] (2) Fermentation culture
[0018] The initial fermentation culture volume is 2.5L, and the fermentation medium components used are as follows:
[0019] Fermentation medium composition: 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 it at 121°C for 30 minutes.
[0020] Fermentation and fermentation conditions: the above-mentioned cultivated seed liquid is inoculated into the fermentation medium according to the inocul...
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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] A method for enhancing the synthesis of 2,3-butanediol by increasing the level of intracellular cofactors 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 production of various chemicals, functional materials and energy materials through chemical, biological and integrated methods of bio-refining technology is increasing. More and more attention is paid at home and abroad. 2,3-Butanediol is an important chemical raw material and liquid fuel, which is widely used in chemical, food, fuel, aerospace and other fields. 2,3-Butanediol is dehydrogenated to diacetyl, which is a high-value food flavor with a certain antibacterial effect; under the action of dehydrogenase, 3-hydroxy-2-butanone (ethyl Yin) is a widely used natural food flavor; the methyl ethyl ke...
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