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Increased production of 2,3-butanediol in bacillus amyloliquefaciens by enhancing the regeneration rate of the coenzyme cycle

A recycling and butanediol technology, applied in the field of genetic engineering, can solve problems that do not conform to industrial safety production

Active Publication Date: 2015-09-30
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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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

Method used

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  • Increased production of 2,3-butanediol in bacillus amyloliquefaciens by enhancing the regeneration rate of the coenzyme cycle
  • Increased production of 2,3-butanediol in bacillus amyloliquefaciens by enhancing the regeneration rate of the coenzyme cycle
  • Increased production of 2,3-butanediol in bacillus amyloliquefaciens by enhancing the regeneration rate of the coenzyme cycle

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Experimental program
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Effect test

Embodiment 1

[0011] Embodiment 1: Amplification of target gene and construction of recombinant Bacillus amyloliquefaciens

[0012] The schematic diagram of the construction of plasmid pMA5-HpaII-acr-HapII-gapdh is as follows figure 1 As shown, the specific process is as follows:

[0013] First, using the chromosomal DNA of the bacterial strain B10-127 as a template, using primers P1 and P2, a 901bp nucleotide sequence of the acr gene shown in SEQ ID NO: 1 was amplified by PCR technology, and the purified acr After the gene was digested by restriction endonucleases NdeI and BamHI, it was ligated with the plasmid pMA5-HapII which was also digested by the above two restriction endonucleases (the sequence of pMA5-HapII is shown in SED ID NO: 3), and the recombinant plasmid pMA5 was constructed -HapII-acr, verified by double enzyme digestion, indicating that the recombinant plasmid was constructed successfully. Subsequently, using primers P3 and P4 to obtain the gene gapdh whose nucleotide se...

Embodiment 3

[0020] Embodiment 3: Fermentation performance verification of bacteria original bacteria and recombinant bacteria

[0021] (1) Seed cultivation

[0022] Pick a single colony from the activated plate and inoculate it into 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.

[0023] (2) Fermentation culture

[0024] The initial fermentation culture volume is 2.5L, and the components of the fermentation medium used are as follows:

[0025] 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 at 121°C for 30min.

[0026] Fermentation Fermentation conditions: inoculate the above-mentioned cultivated seed liquid into the fermentation medium according to the i...

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Abstract

The key enzyme genes, namely 3-phosphoglyceraldehyde dehydrogenase (gapdh) and 3-hydroxyl-2- butanone reductase (acr), which participate in the coenzyme cyclic regeneration process in the synthesis route of 2,3-butylene glycol, are cloned from B. amyloliquefaciens B10-127; then the genes are inoculated to a shuttle expression plasmid pMA5-HpaII so as to successfully establish a shuttle expression plasmid containing gene gapdh and gene acr pMA5-HpaII-acr-HapII-gapdh, and the pMA5-HpaII-acr-HapII-gapdh is transferred to bacillus amyloliquefaciens B10-127 so as to obtain bacillus amyloliquefaciens, which is strengthened in gapdh and acr gene expression, namely pMA5-HpaII-acr-HapII-gapdh / B. amyloliquefaciens. The 2,3-butylene glycol output of the recombinant bacterium is raised by 14.7% compared to that of the original bacterium, the byproduct contents of 3-hydroxyl-2-butone, lactic acid and butanedioic acid are reduced by 65.6%, 43.8% and 42.4, and moreover the fermentation period is shortened. Through the processes of material supplement and fermentation, the 2,3-butylene glycol output can reach 121.3 g / L.

Description

technical field [0001] The method improves the yield of 2,3-butanediol synthesized by Bacillus amyloliquefaciens by strengthening the regeneration rate of the coenzyme cycle, and 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 generate diacetyl, which is a high-value food flavoring agent with certain antibacterial effect; under the action of dehydrogenase, the genera...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/75C12P7/18C12R1/07
Inventor 饶志明杨套伟张显徐美娟满在伟
Owner JIANGNAN UNIV
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