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Construction method of glucose-tolerant high-secretion genetic engineering recipient bacterium

A technology of genetic engineering and construction method, applied in the field of genetic modification and construction of glucose-tolerant and high-secretion genetically engineered receptor bacteria, can solve the problems of genetic and unstable endotoxin contained in the engineered receptor bacteria, and achieve large yield and increased secretion. Effect

Pending Publication Date: 2021-03-05
TIANJIN UNIVERSITY OF TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problems that the currently used engineering receptor bacteria contain endotoxin or genetic instability, and various engineering receptor bacteria are limited by the CCR effect in industrial applications, and improve industrial productivity, and provide a sugar-resistant high-secretion Construction method of type genetic engineering recipient bacterium

Method used

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  • Construction method of glucose-tolerant high-secretion genetic engineering recipient bacterium
  • Construction method of glucose-tolerant high-secretion genetic engineering recipient bacterium
  • Construction method of glucose-tolerant high-secretion genetic engineering recipient bacterium

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Experimental program
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Effect test

Embodiment 1

[0023] 1. Construction of recombinant plasmids

[0024] 1. Design of gRNA

[0025] Determine the target gene that has a CCR effect and contains the cis element of crebox in the sugar metabolism pathway, specifically select the following seven target genes:

[0026] ackA UP12_RS13195 acetate kinase

[0027] ycgE UP12_RS01730 aminotransferase

[0028] ptsH UP12_RS06700 phosphocarrierprotein HPr

[0029] budA UP12_RS16915 acetolactate decarboxylase

[0030] acsA UP12_RS13395 acetyl-CoA synthetase

[0031] xylA UP12_RS09365 xylose isomerase

[0032] sucC UP12_RS07835 succinate-CoAligase[ADP-forming]subunitbeta

[0033] Determine the crebox sequence and specific location in the target gene.

[0034] After positioning with gRNA, the Cas9 protein cuts about 3bp at the 5' end of the binding site. Select the cre box and the subsequent 20bp sequence and select PAM as NNG to design the target fragment. Each gene corresponds to multiple Group possible target fragments, summarize t...

Embodiment 2

[0093] Glucose fermentation medium (GYN): 2g yeast powder, 0.05g MgSO4·7H2O, add 1% volume of urea mother solution (20% 0.05Mpa sterilization for 20min), add glucose according to different concentrations. Add deionized water to 100mL, pH 7.2-7.5, sterilize at 0.08Mpa for 20min.

[0094]Pick the recombinant Bacillus pumilus LG3145 and culture it in 4 μg / ml liquid medium containing chloramphenicol for 12 hours, then take 200 μL and add it to 100 ml glucose fermentation medium at 150 rpm, shake and culture at 37°C for 24 hours to observe the growth status. And use the ultraviolet spectrophotometer to take samples every two hours to measure the bacterial concentration. Bacillus pumilus LG3145 grows much better than wild bacteria in the case of high sugar.

[0095] When the recombinant Bacillus pumilus LG3145 was cultured in different concentrations of glucose fermentation medium, the sugar tolerance of Bacillus pumilus LG3145 was significantly improved, and the sugar tolerance cou...

Embodiment 3

[0101] Determination of Extracellular Protein Activity of Recombinant Bacillus pumilus

[0102] 1. Preparation of double-layer skimmed milk powder solid medium: lower layer: 2.5g of agar was dissolved in 100ml of deionized water, autoclaved at 121°C for 30 minutes, and the solution was spread on the bottom of the petri dish and allowed to cool and solidify. Upper layer: 2g of skimmed milk powder, 2.5g of agar dissolved in 100ml of deionized water, autoclaved at 105°C for 20 minutes to obtain 2% milk medium, spread the solution on the solidified lower layer of agar, let cool and solidify.

[0103] 2. Pick a single colony from the plate, inoculate it into a test tube containing 5mL LB medium, culture overnight at 37°C, 120-200r / min, transfer 1mL of the bacterial solution to a 250mL Erlenmeyer flask inoculated with 100mL LB medium , 37°C, 120-200r / min culture. Take 1ml every two hours, centrifuge at 12000r for 5min, get the supernatant, take 10ul supernatant and spot on the plat...

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Abstract

The invention relates to a construction method of a glucose-tolerant high-secretion genetic engineering recipient bacterium. The method comprises the following steps: a, selecting target genes inhibited by CCR, such as ackA, ycgE, ptsH, budA, acsA, xylA and sucC, and determining a site and base sequence of cre box of each target gene; b, designing a group of gRNAs with the similarity of 30-100% with cre box consensus sequence target fragments, and ensuring that editing of a plurality of target cre box fragments can be realized; and c, constructing integrated plasmid containing a CRISPR / Cas9 locus and the gRNAs, and transferring the integrated plasmid into bacillus to realize editing of a plurality of cre box sites of a genome. By adopting the gene modification method provided by the invention, the CCR repression effect of a plurality of genes can be removed at the same time, so that secondary metabolites of bacteria are increased, and the high-secretion genetic engineering recipient bacterium can be obtained. The CCR effect is removed, so that the glucose tolerance of the engineering bacterium is improved, and the engineering bacterium has relatively strong cell secretion capability and can be used as recipient cells for microbial fermentation.

Description

technical field [0001] The invention relates to the fields of molecular biology and genetic engineering, in particular to a genetic modification and construction method of a sugar-tolerant high-secretion genetic engineering recipient bacterium. Background technique [0002] The common engineering recipient bacteria used in the industry are generally Escherichia coli and Bacillus subtilis, etc., using gene recombination technology to introduce a specific protein gene into the recipient bacteria, so as to obtain the required protein, and then obtain it for treatment by separation and purification protein drugs, etc. Among them, Escherichia coli has the advantages of clear genetic background, complete receptor system, rapid growth and simple cultivation, etc., but the products of Escherichia coli are complicated, difficult to separate and have various endotoxins, which limit its industrial application. Bacillus subtilis also has the advantages of fast growth, simple culture, c...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N15/55C12R1/07
CPCC12N15/75C12N9/22
Inventor 应明王迎香曹林峰毕美营陈美廷
Owner TIANJIN UNIVERSITY OF TECHNOLOGY
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