Xanthomonadins synthesis related gene and application thereof in constructing colorless xanthan gum genetically engineered bacteria

A technology of genetic engineering and xanthan gum, which is applied in the construction of colorless xanthan gum-producing engineering bacteria, can solve the problems of cumbersome operation, large solvent consumption, and high production cost, so as to save production cost and improve product quality , The effect of simplifying the production process

Active Publication Date: 2019-07-16
GUANGDONG FOOD & DRUG VOCATIONAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the production process of xanthan gum, due to the requirements of the quality standards of xanthan gum products, it is necessary to remove the cytochrome produced in the fermentation process, and phytoflavin is the main component of the cytochrome of the bacterial cell. At present, solvents are usually used in the industry The removal of cytochrome by chemical reaction has the problems of large solvent consumption, cumbersome operation, and high production cost. Therefore, the step of removing cytochrome has become a major obstacle restricting the improvement of xanthan gum quality and saving production costs.

Method used

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  • Xanthomonadins synthesis related gene and application thereof in constructing colorless xanthan gum genetically engineered bacteria
  • Xanthomonadins synthesis related gene and application thereof in constructing colorless xanthan gum genetically engineered bacteria
  • Xanthomonadins synthesis related gene and application thereof in constructing colorless xanthan gum genetically engineered bacteria

Examples

Experimental program
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Embodiment 1

[0036] Embodiment 1: Cloning of XC_4092 gene

[0037] This embodiment discloses the cloning method of XC_4092 gene, comprising the following steps:

[0038] S1. According to the DNA sequence of the XC_4092 gene, design a pair of amplification primers: Up primer and Down primer, the sequence of the Up primer is shown in SEQ ID NO.9, and the sequence of the Down primer is shown in SEQ ID NO.10;

[0039] S2, to Xcc The total DNA of 8004 was used as a template, PCR amplification was carried out with Up primer and Down primer, and the XC_4092 gene fragment of about 740 bp was obtained;

[0040] S3. Ligate the XC_4092 gene fragment obtained in step S2 into the pMD19-T vector, and ligate at 16°C for 2 hours to obtain plasmid pYYH-1;

[0041]S4. Sequencing the plasmid pYYH-1 in step S3 to prove that the inserted gene fragment is correct.

[0042] What needs to be explained about the above steps is that the PCR amplification program in step S2 is: ①pre-denaturation at 95°C for 10 m...

Embodiment 2

[0048] Example 2: Construction of Gene Knockout Recombinant Plasmid

[0049] This example discloses a method for constructing a gene knockout recombinant plasmid, aiming to use two homologous recombination gene knockout methods to delete Xcc Gene XC_4092 in the 8004 genome, thereby achieving the purpose of constructing the engineering bacteria producing colorless xanthan gum. The construction method of the XC_4092 gene knockout recombinant plasmid is as follows:

[0050] S1. Design PCR primer pairs P1-F and P2-R for the upstream fragment of the XC_4092 gene (about 520bp), and PCR primer pairs P3-F and P4-R for the downstream fragment of the XC_4092 gene (about 570bp);

[0051] S2, to Xcc The total DNA of 8004 was used as a template, and the upstream fragment of the XC_4092 gene and the downstream fragment of the XC_4092 gene were respectively amplified by PCR;

[0052] S3. Using the fusion PCR method, using the upstream and downstream fragments of the XC_4092 gene obtaine...

Embodiment 3

[0065] Embodiment 3: produce the construction of colorless xanthan gum engineering bacteria

[0066] This embodiment discloses a method for constructing a colorless xanthan gum-producing engineering bacterium, that is, constructing a colorless xanthan gum-producing engineering bacterium through two homologous recombination gene knockout methods, including the following steps:

[0067] S1, using CaCl 2 Prepare competent Escherichia coli S17-1 by induction method, introduce the recombinant plasmid pYYH-2 in Example 2 into competent Escherichia coli S17-1, and obtain transformants E. coli S17-1 / pYYH-2;

[0068] S2, the transformant obtained in step S1 E. coli S17-1 / pYYH-2 was introduced by conjugation transfer Xcc 8004, and then cultured on resistant plates, so that the XC_4092 gene deletion fragment on pYYH-2 was compatible with Xcc The XC_4092 gene fragment on the 8004 genome undergoes homologous recombination exchange, and the single colony grown on the resistance plat...

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Abstract

The present invention discloses a xanthomonadins synthesis related gene XC_4092. A gene sequence is as shown in SEQ ID NO.1. An encoded product has a 3-ketoacyl-ACP reductase activity and can catalyzea reduction reaction in xanthomonadins synthesis. The present invention further discloses an application of the gene XC_4092 in production of the colorless xanthan gum, xanthomonas campestris pathogenic strains of wild strains Xcc8004 are used as starting strains, the gene XC_4092 in a Xcc8004 genome is inactivated to complete gene modification to construct the colorless xanthan gum genetically engineered bacteria. Compared with the Xcc8004, the genetically engineered bacteria cannot synthesize xanthomonadins during fermentation processes, effectively improve appearance color of xanthan gum,and thus greatly improve quality of the xanthan gum product.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a phytoxanthin synthesis gene of Xanthomonas campestris pathogenic species, and the application of the phytoxanthin synthesis gene in constructing an engineering bacterium producing colorless xanthan gum. Background technique [0002] Xanthan gum is a pathogenic species of Xanthomonas campestris Xanthomonas campestris pv. campestris , Xcc The exopolysaccharide polymer produced by ) is the most widely used microbial polysaccharide that has been developed. It has good viscosity-increasing and thixotropic properties, high stability to heat, acid, and alkali, and can be compatible with various solvents. Allow. At present, xanthan gum is used as a suspending agent, thickener, emulsifier, stabilizer, etc. in the fields of medicine, food, beverage, cosmetics, detergent, and oil exploration. [0003] Phytoflavin is an important metabolite of Xanthomonas, which i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/04C12N1/21C12N15/74C12P19/06
CPCC12N9/0006C12N15/74C12Y101/011C12P19/06
Inventor 余永红王海洪马建荣
Owner GUANGDONG FOOD & DRUG VOCATIONAL COLLEGE
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