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Method for improving ferulic acid stress resistance of clostridium beijerinckii

A clostridium beijerinckii, stress-resistant technology, applied in the field of genetic engineering, can solve problems such as reducing the amount of solvent, and achieve the effects of high butanol yield, high stress resistance, and strong stress resistance

Active Publication Date: 2015-09-09
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Richmond et al. found that syringaldehyde inhibited the expression of CoAT, blocked the conversion of acetic acid and butyric acid, and led to a decrease in the amount of solvent

Method used

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  • Method for improving ferulic acid stress resistance of clostridium beijerinckii
  • Method for improving ferulic acid stress resistance of clostridium beijerinckii
  • Method for improving ferulic acid stress resistance of clostridium beijerinckii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction method of Clostridium beijerinckii Cbei_4693 gene inactivation mutant.

[0036] 1. Design introns:

[0037] According to the Cbei_4693 gene sequence (as shown in SEQ ID No: 1) of Clostridium beijerinckii included in the NCBI database, a suitable insertion gene site is designed with the help of software ( http: / / www.clostron.com), choose to insert between the 335th and 336th bases, and generate an intron sequence, and synthesize an intron sequence S-335 whose sequence is shown in SEQ ID NO:2.

[0038] 2. Construction of Cbei_4693 insertion inactivation vector:

[0039] The vector pWJ (provided by Mr. Yang Sheng, Shanghai Academy of Biological Sciences, whose sequence is shown in SEQ ID NO: 5) and the DNA fragment S-335 were double-digested with XhoI and BsrGI, respectively. After the digested product was purified by a purification kit (Takara), it was ligated by T4 ligase (Takara) overnight. Transform the ligated recombinant plasmid into Esche...

Embodiment 2

[0050] Example 2: Passage stability of recombinant bacteria with inactivated Cbei_4693 gene.

[0051] The culture medium formula described in the present embodiment:

[0052] Plate medium: yeast powder 3g / L, peptone 5g / L, soluble starch 10g / L, ammonium acetate 2g / L, sodium chloride 3g / L, magnesium sulfate heptahydrate 3g / L, potassium dihydrogen phosphate 1g / L, Dipotassium hydrogen phosphate 1g / L, ferrous sulfate heptahydrate 0.1g / L, agar 15g / L, the rest is water, pH 6.

[0053] Seed medium: yeast powder 3g / L, peptone 5g / L, soluble starch 10g / L, ammonium acetate 2g / L, sodium chloride 3g / L, magnesium sulfate heptahydrate 3g / L, potassium dihydrogen phosphate 1g / L, Dipotassium hydrogen phosphate 1g / L, ferrous sulfate heptahydrate 0.1g / L, the rest is water, pH 6.

[0054] Fermentation medium: glucose 30g / L, ammonium acetate 2.2g / L, potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 0.5g / L, sodium chloride 0.01g / L, magnesium sulfate heptahydrate 0.2g / L , ferrou...

Embodiment 3

[0061] Example 3: Investigation of stress resistance of recombinant bacteria and starting strains to ferulic acid.

[0062] Plate medium: yeast powder 3g / L, peptone 5g / L, soluble starch 10g / L, ammonium acetate 2g / L, sodium chloride 3g / L, magnesium sulfate heptahydrate 3g / L, potassium dihydrogen phosphate 1g / L, Dipotassium hydrogen phosphate 1g / L, ferrous sulfate heptahydrate 0.1g / L, agar 15g / L, the rest is water, pH 6.

[0063] Seed medium: yeast powder 3g / L, peptone 5g / L, soluble starch 10g / L, ammonium acetate 2g / L, sodium chloride 3g / L, magnesium sulfate heptahydrate 3g / L, potassium dihydrogen phosphate 1g / L, Dipotassium hydrogen phosphate 1g / L, ferrous sulfate heptahydrate 0.1g / L, the rest is water, pH 6.

[0064] Fermentation medium: glucose 30g / L, ammonium acetate 2.2g / L, potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 0.5g / L, sodium chloride 0.01g / L, magnesium sulfate heptahydrate 0.2g / L , ferrous sulfate heptahydrate 0.01g / L, manganese sulfate mo...

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Abstract

The invention provides a method for improving the ferulic acid stress resistance of clostridium beijerinckii, and belongs to the technical field of gene engineering. According to the method, an FMN redoxin gene, coded in dependence on NADPH, in the clostridium beijerinckii is inactivated, so that the FMN redoxin gene cannot be normally expressed in the clostridium beijerinckii; the clostridium beijerinckii NCIMB 8052 is adopted; the FMN redoxin gene, coded in dependence on NADPH, in the clostridium beijerinckii is Cbei_4693 of which the nucleotide sequence is shown as SEQ ID NO: 1. The FMN redoxin gene, coded in dependence on NADPH, in the clostridium beijerinckii is inactivated according to the group II intron gene knockout technology. A recombinant strain obtained according to the method provided by the invention is relatively high in passage stability; when the concentration of ferulic acid in a culture medium is 0.5 g / L, the butanol yield of a starting strain 8052 is 0.7 g / L, and the butanol yield of the obtained recombinant strain reaches 5.6 g / L, that is, the ferulic acid stress resistance of the clostridium beijerinckii is improved according to 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 ferulic acid. Background technique [0002] With the depletion of petroleum energy, bio-butanol as a second-generation energy has become a research hotspot. The traditional acetone-butanol-ethanol fermentation (ABE fermentation) directly uses glucose, xylose, etc. as substrate raw materials, and the consumption cost is high. The use of cheap waste and renewable lignocellulosic raw materials (corncobs, bagasse, straw, etc.) to produce butanol through microbial fermentation has become one of the research focuses. However, when lignocellulosic raw materials are pretreated to become sugars available to microorganisms, a large amount of inhibitors such as organic acids, furfural, 5-hydroxymethylfurfural, and phenolic compounds are also degraded. However, in the process of removing the inh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/16
Inventor 应汉杰刘俊郭亭沈晓宁许佳慧柳东牛欢青陈勇吴菁岚朱晨杰庄伟
Owner NANJING UNIV OF TECH
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