Sphingomonas paucimobilis gene knockout mutant strain and construction method thereof

A sphingomonas and gene knockout technology, applied in the field of genetic engineering, can solve the problems of affecting the production of glue, inability to use, and unstable glue production, so as to improve the purity and transparency, reduce the accumulation of intermediate metabolites, and inhibit The effect of PHB

Inactive Publication Date: 2017-11-24
上海北连生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it was found in the experiment that inhibiting the generation of PHB led to a large accumulation of the intermediate product beta-hydroxybutyrate coenzyme A in the previous step, and the large accumulation of organic acids seriously affected the yield of the gum
Although the spontaneous mutant strains recovering the gel production ability were screened in the fermentation process, the gel production was unstable, which made it impossible to use in actual production

Method used

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  • Sphingomonas paucimobilis gene knockout mutant strain and construction method thereof
  • Sphingomonas paucimobilis gene knockout mutant strain and construction method thereof
  • Sphingomonas paucimobilis gene knockout mutant strain and construction method thereof

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Experimental program
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Embodiment 1

[0036] This embodiment is the construction of Sphingomonas paucimobilis gene knockout mutant strain, specifically:

[0037] 1) Amplification of the acetoacetyl-CoA reductase gene (phaB) fragment

[0038] In order to determine the highly conserved region, the amino acid sequence of the acetoacetyl-CoA reductase of bacteria related to Sphingomonas paucimobilis was searched from the NCBI (national center for biotechnology information) gene bank, and multiple sequence alignment analysis was performed using Clustal W . as attached figure 1 As shown, in the conserved region, the amino acid sequences of two regions with low degeneracy were selected as VAIVTGG and INGGQHM (indicated by the arrows) to design degenerate primers, and PCR was performed using the genomic DNA of Sphingomonas paucimobilis as a template A 708bp gene fragment (SEQ ID NO.3) was amplified. The primers used were: GTNGCNATNGTNACNGGNGG (SEQ ID NO. 1) and CATRTGYTGNCCNCCRTTDAT (SEQ ID NO. 2). The amplification c...

Embodiment 2

[0046] Shake Flask Fermentation Test of phaB Deletion Mutant Strain

[0047] The two phaB deletion strains in Example 1 were subjected to a shake flask fermentation experiment in a fermentation medium containing suitable organic and inorganic nitrogen sources, corn syrup and inorganic salts, and the fermentation broth was recovered by isopropanol precipitation and dried to obtain the total precipitate TPM (Total Precipitable Material, TPM). As a result, it was found that the fermented liquid was very dilute, measured at 30 rpm using a Brookfield viscometer, and the viscosity was only 56 cp, and the isopropanol precipitation hardly obtained precipitate, and the fermented liquid had thick sour gas. The analysis of the composition of the fermentation broth showed that a large amount of organic acids were produced during the fermentation process, because when the synthesis pathway of PHB was hindered, the metabolic pathway was changed to the synthesis of organic acids. The large ...

Embodiment 3

[0057] Comparison of strength and transparency of gellan gum produced by phaB deletion mutant strain and wild strain

[0058] Gellan gum will form a gel after heating and cooling, and it is widely used in the food field due to its unique texture and rheological properties. As the main indicator of texture, the strength of gelatin is also one of the important evaluation parameters of gellan gum performance. Therefore, we compared the strength of gellan gum produced by the fermentation of phaB deletion mutant strains and wild strains. The breaking tension generated at the surface.

[0059]The gelatin gum produced by the fermentation of phaB deletion mutant strains PDGG-1 and PDGG-2 and wild strain WT was dried and pulverized, passed through an 80 mesh sieve, and prepared into gel as follows. Add 1mL of 0.3M CaCl2.2H2O solution to 147.5ML deionized water in a beaker. Place the mixture on a magnetic stirrer at a speed of 700rpm and slowly add 1.5g of gellan gum while stirring, c...

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Abstract

The invention provides a sphingomonas paucimobilis gene knockout mutant strain and a construction method thereof. The strain is collected in China Center for Type Culture Collection and has the collection number of CCTCC No. 2016610. A PHB deletion sphingomonas paucimobilis gene knockout mutant strain is constructed, and genes of acetoacetyl coenzyme A reductase in a PHB synthetic route of sphingomonas paucimobilis are knocked out by utilizing homologous recombination so as to obtain the gene deletion mutant strain, so that production of the PHB is inhibited, and the purity and transparency of colloid are improved; and meanwhile, the colloid productive rate and colloid production stability are maintained, so that the possibility of removing transparent high acyl gellan gum of the PHB in large-scale production is greatly improved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a gene knockout mutant strain of Sphingomonas paucikinis and a construction method thereof. Background technique [0002] Gellan gum is an exopolysaccharide produced by Sphingomonas elodea. In 1988, Japan first approved the application of gellan gum in food. Subsequently, countries such as the U.S. and Europe also approve its use in food as a gelling agent, a stabilizing agent, a suspending agent and a thickening agent. Due to its superior gel ability, gel stability, excellent suspension, dispersibility and good flavor release, it has attracted more and more attention from many industries such as food, pharmaceuticals and cosmetics in recent years. [0003] The basic structure of gellan gum molecule is a main chain, which is composed of repeating tetrasaccharide units. The monosaccharides involved in the formation are glucose, glucuronic acid and rhamnose, and the m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12P19/04
CPCC12N9/0006C12N15/74C12P19/04C12Y101/01036
Inventor 余丰年王晋源
Owner 上海北连生物科技有限公司
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