Sphingol pseudoalteromonas genetically engineered bacterium as well as construction method and application thereof

A technology of sphingomonas and genetically engineered bacteria, applied in chemical instruments and methods, bacteria, bacterial peptides, etc., can solve problems such as poor temperature and pH tolerance, and improve temperature tolerance and acid-base resistance Acceptability and economical improvement effect

Active Publication Date: 2018-08-28
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Purpose of the invention: in order to solve the problem that the existing production microorganism polysaccharide Sphingomonas is poor in temperature and pH tolerance, the invention provides a kind of Sphingomonas genetically engineered bacterium, and the further purpose of the present invention is to provide The construction method of Sphingomonas genetically engineered bacteria, the further object of the present invention is to provide the application of Sphingomonas genetically engineered bacteria in the fermentation production of polysaccharides

Method used

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  • Sphingol pseudoalteromonas genetically engineered bacterium as well as construction method and application thereof
  • Sphingol pseudoalteromonas genetically engineered bacterium as well as construction method and application thereof
  • Sphingol pseudoalteromonas genetically engineered bacterium as well as construction method and application thereof

Examples

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Effect test

Embodiment 1

[0033] Example 1 Containing the Recombinant Sphingomonas Construction of Reversible Tengchong Thermotrophy GroES Gene

[0034] Step 1. Stress-resistant gene amplification

[0035] Using the Tengchong thermotrophic bacterium (CGMCC5161) genome as a template, use the software CE Design V1.0 primer design software to design synthetic primers (see Table 1 for primer sequences), and clone the stress-resistant GroES gene. The nucleotide sequence of the GroES gene is shown in SEQID NO : 1. PCR conditions for GroES gene: 1) Denaturation at 94°C for 5 min; 2) Cycle 30 times according to the following parameters: denaturation at 94°C for 1 min, annealing at 60°C for 60 s, extension at 72°C for 1 min; 3) final extension at 72°C for 10 min. After the completion of the PCR reaction, 2 μL of the product was taken, and then analyzed by electrophoresis in an agarose gel with a concentration of 1 g / 100 mL. After confirming the correct size of the fragment by imaging with a gel imaging system, ...

Embodiment 2

[0055] Embodiment 2 Construction of recombinant Sphingomonas containing anti-stress GroEL gene

[0056] The construction method is the same as in Example 1, except that the stress-resistant gene is the GroEL gene, derived from Thermus thermophilus (CGMCC15059), the nucleotide sequence is shown in SEQ ID NO: 2, the primer sequence list is shown in Table 1, and S .sp.GroEL

Embodiment 3

[0057] Embodiment 3 Construction of recombinant Sphingomonas containing stress-resistant IrrE gene

[0058] The construction method is the same as in Example 1, except that the stress-resistant gene is the IrrE gene, derived from Deinococcus radiodurans (CGMCC633), the nucleotide sequence is shown in SEQ ID NO: 5, the primer sequence list is shown in Table 1, and S. sp. IrrE.

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Abstract

The invention discloses a sphingol pseudoalteromonas genetically engineered bacterium and relates to sphingol pseudoalteromonas which stress resistance gene is guided into. The stress resistance geneis from extreme microorganisms. The invention further discloses a method for constructing the sphingol pseudoalteromonas genetically engineered bacterium through a three-parent combining method. The invention further discloses application of the sphingol pseudoalteromonas genetically engineered bacterium in fermentation to prepare welan gum or gellan gum. The sphingol pseudoalteromonas geneticallyengineered bacterium disclosed by the invention obviously improves temperature tolerance and acid-base tolerance of the sphingol pseudoalteromonas, a temperature tolerance range is 30 to 42 DEG C, and a pH tolerance range is 4.0 to 10.0. When the sphingol pseudoalteromonas genetically engineered bacterium is used in fermentation to prepare the welan gum, a microbial polysaccharide yield can reach20 to 30g / L under the condition that a pH is not regulated by an external source; when the sphingol pseudoalteromonas genetically engineered bacterium is used in fermentation to prepare the gellan gum, a microbial polysaccharide yield can reach 15 to 22g / L under the condition that a pH is not regulated by an external source; a production efficiency of microbial polysaccharides of the gellan gum,the welan gum and the like is enhanced.

Description

technical field [0001] The invention relates to a genetic engineering bacterium and its construction method and application, in particular to a sphingomonas genetic engineering bacterium and its construction method and application. Background technique [0002] Microbial polysaccharides are a typical type of microbial fermentation products, which are essential to human life and industrial production. As early as 1969, xanthan gum was approved by the FDA as an important food additive; in 1998, gellan gum was reported as the most important drug for the treatment of eye diseases; in 1992, welan gum was reported as an irreplaceable driving agent for deep sea oil production. The United States Kelco company is the world's largest supplier of microbial polysaccharides, and has developed xanthan gum and new microbial polysaccharides, namely gellan gum polysaccharides (such as gellan gum, welan gum, rhamnose gum, S88, S7) and other microbial polysaccharides. Studies have found that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/70C07K14/195C12P19/04
CPCC07K14/195C12N15/70C12N15/74C12P19/04
Inventor 李莎徐虹刘晓柳朱萍冯小海许宗奇
Owner NANJING UNIV OF TECH
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