A method for improving the stress resistance of Clostridium beijerinckii to 4-hydroxycinnamic acid

A technology of hydroxycinnamic acid and Clostridium beijerinckii is applied in the field of genetic engineering to achieve the effects of high butanol yield, improved stress resistance and high efficiency stress resistance

Active Publication Date: 2019-08-09
GUANGDONG PROVINCIAL BIOENGINEERING INST (GUANGZHOU SUGARCANE IND RES INST)
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specific mechanism of the inhibition of 4-hydroxycinnamic acid on the bacteria has not yet been clarified, and more and more attention has been paid to improving the stress resistance of solventogenic bacteria and increasing the yield of butanol through genetic engineering.

Method used

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  • A method for improving the stress resistance of Clostridium beijerinckii to 4-hydroxycinnamic acid

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Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1: the construction method of Clostridium beijerinckii Cbei_2996 gene inactivation mutant

[0047] (1) Design introns:

[0048] According to the Cbei_2996 gene sequence of Clostridium beijerinckii included in the NCBI database (as shown in SEQ ID No: 1), that is, the base sequence encoding coenzyme Q oxidoreductase subunit A ( nuo A), with the help of software to design a suitable insertion gene site (http: / / www.clostron.com), through the previous experimental screening and verification, select the insertion between the 62nd and 63rd bases of the Cbei_2996 gene sequence, and generate Intron sequence, the synthetic intron is S-62, and its sequence is shown in SEQ ID NO:2.

[0049] (2) Construction of Cbei_2996 insertion inactivation vector:

[0050] Use XhoI and BsrGI to double-enzyme-cut the vector pWJ (provided by Yang Sheng, Shanghai Academy of Biological Sciences, its sequence is shown in SEQ ID NO: 5, and the plasmid map of pWJ is shown in figure 1 sho...

Embodiment 2

[0061] Embodiment 2: the passage stability of the recombinant bacterium of Cbei_2996 gene inactivation

[0062] The culture medium formula described in the present embodiment:

[0063] Plate medium: yeast powder 3 g / L, peptone 5 g / L, soluble starch 10 g / L, ammonium acetate 2 g / L, sodium chloride 3 g / L, magnesium sulfate heptahydrate 3 g / L, diphosphate Potassium hydrogen 1 g / L, dipotassium hydrogen phosphate 1 g / L, ferrous sulfate heptahydrate 0.1 g / L, agar 15 g / L, the rest is water, pH 6.

[0064] Seed medium: yeast powder 3 g / L, peptone 5 g / L, soluble starch 10 g / L, ammonium acetate 2 g / L, sodium chloride 3 g / L, magnesium sulfate heptahydrate 3 g / L, diphosphate Potassium hydrogen 1 g / L, dipotassium hydrogen phosphate 1 g / L, ferrous sulfate heptahydrate 0.1 g / L, the rest is water, pH 6.

[0065] Fermentation medium: glucose 30 g / L, ammonium acetate 2.2 g / L, potassium dihydrogen phosphate 0.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, sodium chloride 0.01 g / L, magnesium sul...

Embodiment 3

[0071] Embodiment 3: Recombinant bacteria and departure bacterial strains are investigated to the stress resistance of 4-hydroxycinnamic acid

[0072] Plate medium: yeast powder 3 g / L, peptone 5 g / L, soluble starch 10 g / L, ammonium acetate 2 g / L, sodium chloride 3 g / L, magnesium sulfate heptahydrate 3 g / L, diphosphate Potassium hydrogen 1 g / L, dipotassium hydrogen phosphate 1 g / L, ferrous sulfate heptahydrate 0.1 g / L, agar 15 g / L, the rest is water, pH 6.

[0073] Seed medium: yeast powder 3 g / L, peptone 5 g / L, soluble starch 10 g / L, ammonium acetate 2 g / L, sodium chloride 3 g / L, magnesium sulfate heptahydrate 3 g / L, diphosphate Potassium hydrogen 1 g / L, dipotassium hydrogen phosphate 1 g / L, ferrous sulfate heptahydrate 0.1 g / L, the rest is water, pH 6.

[0074] Fermentation medium: glucose 30 g / L, ammonium acetate 2.2 g / L, potassium dihydrogen phosphate 0.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, sodium chloride 0.01 g / L, magnesium sulfate heptahydrate 0.2 g / L L, ferro...

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Abstract

The invention discloses a method for improving stress resistance of clostridium beijerinckii to 4-hydroxycinnamic acid. The method comprises the steps that genes of a coenzyme Q oxidation-reduction enzyme subunit A with encoding relying on NADH in the clostridium beijerinckii are inactivated to be not normally expressed in the clostridium beijerinckii, and the nucleotide sequence is shown in SEQ ID NO:1. The genes of the coenzyme Q oxidation-reduction enzyme subunit A with encoding relying on the NADH in the clostridium beijerinckii are inactivated through the type-two intron gene knockout technology. Results show that the recombination strain has the good passage stability; when the concentration of 4-hydroxycinnamic acid in the culture medium is 0.5 g / L, the original strain 8052 hardly produces a solvent, the built recombinant bacteria butanol yield reaches 3.4 g / L, and it shows that the stress resistance of the clostridium beijerinckii to the 4-hydroxycinnamic acid is improved through the method.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for improving the stress resistance of Clostridium beijerinckii to 4-hydroxycinnamic acid. Background technique [0002] Petroleum depletion, energy shortage and other problems are becoming more and more serious; butanol has similar properties to petroleum energy, and is widely recognized as a second-generation bioenergy; furthermore, biological production of butanol has gradually become a research hotspot. Traditional acetone-butanol-ethanol fermentation (ABE fermentation) generally uses glucose, xylose, etc. as substrate raw materials directly, and the consumption cost is high. Researchers have to focus on cheap waste and renewable lignocellulosic raw materials (bagasse , corncobs, straw, etc.). However, when the lignocellulosic raw material is pretreated to become a carbon source available for microorganisms, a large amount of organic acids, f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P7/16C12R1/145
CPCC12N9/004C12P7/16Y02E50/10
Inventor 郭亭孙东磊王庆福斑文婷黄清铧陈瑞荣梁磊安玉兴
Owner GUANGDONG PROVINCIAL BIOENGINEERING INST (GUANGZHOU SUGARCANE IND RES INST)
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