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Strain and construction method thereof, and application thereof in fermentative production of high temperature resistant xanthan

A construction method and strain technology, applied in the field of fermentation and production of high-temperature-resistant xanthan gum, strains and its construction, can solve the problems of high negative mutation ratio, cumbersome processing steps, heavy screening workload, etc., and achieve the effect of reducing production costs

Active Publication Date: 2019-02-26
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although mutagenic strains can be economical mutants, the screening workload is large, the proportion of negative mutations is high, and the research cost is also greatly increased; although adding a cross-linking agent can improve the high temperature resistance of xanthan gum to a certain extent , but the processing steps are cumbersome, the cross-linking agent also needs a certain cost, and the later investment is high. For large-scale production, the production cost of xanthan gum is greatly increased

Method used

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  • Strain and construction method thereof, and application thereof in fermentative production of high temperature resistant xanthan
  • Strain and construction method thereof, and application thereof in fermentative production of high temperature resistant xanthan
  • Strain and construction method thereof, and application thereof in fermentative production of high temperature resistant xanthan

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Experimental program
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Effect test

Embodiment 1

[0094] Embodiment 1: Construction of the knockout plasmid pLO3-gumL:

[0095] Use the extraction kit to extract the genome of XANTHOMONAS CAMPESTRIS WT (strain number NRRL B-1459), the upstream and downstream homology arms of gumL use the genome of XANTHOMONAS CAMPESTRIS WT (strain number NRRL B-1459) as a template, and use primers guml-SF / gumL-SR and gumL-XF / gumL-XR and PrimeSTAR DNA polymerase (Takara Bio, Tokyo, Japan) amplification. The upstream and downstream DNA fragments were connected by overlap PCR, the product was detected by electrophoresis, and the target gene band was purified and recovered by a gel extraction kit to obtain a recombinant fragment. The recombinant fragment and pLO3 plasmid were digested with restriction enzymes SacI and XbaI at the same time, 90min at 37°C, and the digested fragment was purified and recovered by PCR using a kit, and the recovered products of the two were digested with T4 DNA ligase at 16 The recombinant plasmid pLO3-gumL was obta...

Embodiment 2

[0096] Embodiment 2: Construction of expression plasmid pBBR-gumFG:

[0097] Use an extraction kit to extract the genome of XANTHOMONAS CAMPESTRIS WT (strain number NRRL B-1459), use the XC genome as a template, use primers gumFG-F / gumFG-R and PrimeSTAR DNA polymerase to amplify the gumF and gumG genes, and pass the product through Electrophoresis detection, and the target gene band was purified and recovered by a gel recovery kit to obtain the target gene gumFG. The target fragment and the pBBRMCS plasmid were digested with restriction enzymes KpnI and Sma1 at the same time, 90min at 37°C, and the digested fragment was purified and recovered by PCR using a kit, and the recovered products of the two were digested with T4 DNA ligase at 16 The recombinant plasmid pBBR-gumFG was obtained overnight at ℃, and the recombinant plasmid was transferred into E.coli S17 competent cells for amplification of the recombinant plasmid, and single colonies with correct sequencing were picked a...

Embodiment 3

[0098] Embodiment 3: Construction of expression plasmid pMM-gumBC:

[0099] Use an extraction kit to extract the genome of XANTHOMONAS CAMPESTRIS WT (strain number NRRL B-1459), use the XC genome as a template, use primers gumBC-F / gumBC-R and PrimeSTAR DNA polymerase to amplify gumB and gumC genes, and pass the product through Electrophoresis detection, and the target gene bands were purified and recovered by the gel recovery kit to obtain the target genes gumB and gumC. The target fragment and pMMB67eH plasmid were simultaneously digested with restriction enzymes KpnI and XbaI, 37°C, 90min, and the digested fragments were purified and recovered by PCR using a kit, and the recovered products of the two were digested with T4DNA ligase at 16°C After connecting overnight, the recombinant plasmid pMM-gumBC was obtained, and the recombinant plasmid was transferred into E.coli S17 competent cells for amplification of the recombinant plasmid, and a single colony with correct sequenci...

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Abstract

The invention relates to the field of microbial genetic engineering, in particular to a strain and a construction method thereof, and application thereof in fermentative production of high temperatureresistant xanthan. The genome sequence of xanthomonas campestris is changed by using a genetic engineering technology to construct a high temperature resistant xanthan engineering strain, and the xanthan synthesized from the strain can withstand high temperature without any post-fermentation treatment, so that the production cost is greatly reduced.

Description

technical field [0001] The invention relates to the field of microbial genetic engineering, in particular to a bacterial strain and its construction method, and its application for fermenting and producing high-temperature-resistant xanthan gum. Background technique [0002] Xanthan gum (Xanthan) is a water-soluble polysaccharide polymer produced by Xanthomonas. It is widely used in more than 20 industries such as food, petroleum, geology and minerals, medicine, environmental protection, textile, ceramics, glass-lined, paint, printing and dyeing, and cosmetics for hundreds of purposes. [0003] my country is the main production area of ​​xanthan gum. In recent years, the global demand for xanthan gum has been increasing year by year, and the output of xanthan gum in my country has also been showing an increasing trend. Global sales of xanthan gum rose from 84,000 tons in 2011 to 182,000 tons in 2015. Food processing and oil drilling and mining will still be the most import...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/90C12P19/06C12R1/64
CPCC12N1/20C12N9/1025C12N15/902C12P19/06
Inventor 胡炎华马挺戴晓慧常利斌于宏艳
Owner MEIHUA BIOTECH LANGFANG CO LTD
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