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A Zymomonas mobilis resistant to furan formaldehyde and its preparation method and application

A technology of Zymomonas and furanformaldehyde, which is applied in the field of bioengineering, can solve problems such as the improvement of furanformaldehyde tolerance, and achieve the effects of less stringent process requirements, easy operation, and great application prospects

Active Publication Date: 2018-03-16
BIOGAS SCI RES INST MIN OF AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the existing research, there is no report on the improvement of furfuralaldehyde tolerance of Zymomonas mobilis. The main reason is that it is not yet known what kind of genetic modification of Zymomonas mobilis can obtain furfuralaldehyde-resistant mutations. body

Method used

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  • A Zymomonas mobilis resistant to furan formaldehyde and its preparation method and application
  • A Zymomonas mobilis resistant to furan formaldehyde and its preparation method and application
  • A Zymomonas mobilis resistant to furan formaldehyde and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Construction of recombinant plasmids

[0031] Using the GeneMorph Ⅱ Random Mutagenesis Kit to perform error-prone polymerase chain reaction on the PCR product of RpoD, the mutant RpoD gene with point mutations of K219E, V522G and Q649L was screened out. The reaction product was purified using E.Z.N.A®Gel Extraction Kit, the concentration of low-melting point agarose gel was 1%, and the Bam H I and Xba I treatment, and then ligated to the restriction site corresponding to the plasmid pBBR1MCS-tet to obtain the recombinant plasmid, said pBBR1MCS-tet contains the promoter and terminator of pyruvate decarboxylase.

[0032] Construction of control plasmid

[0033] After purifying the PCR product of RpoD using E.Z.N.A®Gel Extraction Kit, use Bam H I and Xba I treatment, and then ligated to the restriction site corresponding to the plasmid pBBR1MCS-tet to obtain the control plasmid, said pBBR1MCS-tet contains the promoter and terminator of pyruvate decarboxylase.

Embodiment 2

[0035] Preparation of furan formaldehyde-resistant Zymomonas mobilis (strain of the present invention):

[0036] The recombinant plasmid obtained in Example 1 was transformed into Z.mobilis ZM4 by means of electroporation, and the bacteria transformed by the plasmid were spread on the RM agar solid plate medium containing 5 μg / ml tetracycline, and single clones were scraped. Place in RM liquid medium and culture overnight at 30°C.

[0037]According to 10% (v / v), the overnight culture liquid was added to 10ml RM liquid medium with furan formaldehyde concentration of 1g / L, 2g / L and 3g / L for cultivation, and then the obtained culture was spread on furan Cultured on a solid medium with a formaldehyde concentration of 3% and 5 μg / ml tetracycline, picked a single clone, extracted the plasmid, and sequenced the DNA.

[0038] Preparation of control strains:

[0039] The control plasmid obtained in Example 1 was transformed into Z. mobilis ZM4 by means of electroporation, and the bac...

Embodiment 3

[0041] The furfuralaldehyde-resistant Zymomonas mobilis and the control strain of Example 2 were cultured on RM liquid medium at 30° C. until the OD600 was 1.0, and then 10-fold serial dilution was performed. Each dilution was placed in RM solid medium with different concentrations of furfuraldehyde, and cultured at 30° C. for 4-5 days.

[0042] When drawing the growth curves of the bacterial strains of the present invention and the control bacterial strains, the bacteria were placed in the Bioscreen C system for cultivation. At 10% (V / V) inoculum size, overnight bacteria were respectively placed in 1ml of fresh RM liquid medium with furfuraldehyde concentration of 2g / L and 3g / L for cultivation, and the initial OD600 value was 0.15-0.2. Then the bacteria were added to the sample wells of the Bioscreen plate, 300 μl per well, and three groups of parallel samples were set up. The temperature is controlled at 30 degrees Celsius, the detection optical density is 600nm, and the ab...

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Abstract

The invention provides a strain of furfuralaldehyde-resistant Zymomonas mobilis, the furfurylaldehyde-resistant Zymomonas mobilis contains the rpoD gene after mutation transformation, and the point mutations of the RpoD protein encoded by the gene are K219E, V522G and Q649L , the furfuralaldehyde-resistant Zymomonas mobilis can tolerate furfuralaldehyde at a concentration of at least 3g / L; the taxonomic name of the furfurylformaldehyde-resistant Zymomonas mobilis is Zymomonas. Culture Collection Management Center, the preservation number is CGMCC NO.10616, and the preservation time is March 12, 2015. The invention also provides a method for preparing the bacterial strain, which includes: preparation of mutant rpoD gene, construction of recombinant plasmid, transformation of plasmid and screening by using different gradients of furanformaldehyde. The invention also discloses the application of the bacterial strain in producing ethanol.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a strain of Zymomonas mobilis resistant to furan formaldehyde and its preparation method and application. technical background [0002] Lignocellulose contains 70% carbohydrates and is an important raw material for bacteria to produce ethanol, a renewable energy fuel. However, in lignocellulose, lignin and hemicellulose are covalently bonded, and cellulose molecules are embedded in it, forming a strong natural barrier, which makes it difficult for general microorganisms to enter and degrade it. Therefore, the production of fuel ethanol from lignocellulosic raw materials requires pretreatment. Among them, dilute acid pretreatment is a more commonly used and mature method, but after this method, a large number of inhibitory products that inhibit bacterial fermentation will be produced, such as furfural, formic acid, acetic acid, etc. [0003] In order to overcome the inh...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/54C12P7/06C12R1/01
CPCC12P7/06C12N1/205C12R2001/01Y02E50/10
Inventor 谭芙蓉何明雄吴波代立春秦晗祝其丽胡启春
Owner BIOGAS SCI RES INST MIN OF AGRI
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