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Production of novel indigo dye Corynebacterium glutamicum and its construction method and application

A technology of Corynebacterium glutamicum and indigo, applied in the biological field, can solve the problems of E.

Active Publication Date: 2022-07-22
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is worth noting that Escherichia coli is limited in the production of food and other related substances. At the same time, Escherichia coli is not an efficient production strain of glutamine, the biosynthetic raw material of Indigoidine, which makes the improvement of Indigoidine's later production encounter a bottleneck

Method used

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  • Production of novel indigo dye Corynebacterium glutamicum and its construction method and application
  • Production of novel indigo dye Corynebacterium glutamicum and its construction method and application
  • Production of novel indigo dye Corynebacterium glutamicum and its construction method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Construction of recombinant Corynebacterium glutamicum C.glutamicum ATCC 13032-(BpsA-SFP)

[0031] Using the genome of S. lavendulae as the template, the bpsA gene was amplified with the upstream and downstream primers of bpsA-F and bpsA-R, and the genome of B. subtilis was used as the template, sfp-F and sfp -R upstream and downstream primers amplify the sfp gene, using the pEC-XK99E plasmid as a template, the vector fragment pECbone is amplified with two pairs of upstream and downstream primers, pECbone1-F and pECbone1-R and pECbone2-F and pECbone2-R, respectively. By introducing a homologous recombination sequence of 20bp at the 5' end of the primer, the amplification products must have completely identical sequences (15bp-20bp) capable of mutual homologous recombination. After mixing in a certain proportion, in Exnase TM Under the catalysis of E.coli, it only takes 30 minutes to transform into E.coli JM109. The recombinant expression plasmid pEC-XK99E-(Bp...

Embodiment 2

[0035] Example 2 Extraction and analysis of indigo dye

[0036] After collecting the cells, add 10 mg / mL dimethyl sulfoxide (DMSO), break the cells by ultrasonication, centrifuge at 6000 rpm for 15 min, place the supernatant in a new 50 mL centrifuge tube and centrifuge again until the centrifuge tube The sterile body was centrifuged down. Filter the supernatant through a 0.2 μM microporous membrane, add 5 times the volume of ultrapure water with DMSO to the filtered supernatant, and centrifuge the mixture of ultrapure water and bacterial liquid at 23°C at 12,000 rpm for 1 h, and discard. The supernatant was washed with ultrapure water, and then the bacteria dissolved in ultrapure water were evaporated to dryness with a rotary evaporator. Weigh 0.02 g of evaporated indigoidine powder, dissolve it with deuterated dimethyl sulfoxide (DMSO-d6), and centrifuge it with a 1.5 mL centrifuge tube at 23°C and 12,000 rpm for 15 min. ( 1 H-NMR) detection and analysis of the chemical s...

Embodiment 3

[0037] The preparation of embodiment 3 indigo dye standard curve

[0038] Dissolve 1 mg of purified indigoidine in 1 mL of dimethyl sulfoxide (DMSO) to prepare an indigoidine solution with a concentration of 1 mg / mL. This standard solution was serially diluted to different concentrations: 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.08 mg / mL, 0.10 mg / mL. Use a spectrophotometer to detect the absorbance of the above five standard solutions with different concentrations at a wavelength of 600 nm. Taking the concentration of indigoidine as the abscissa and the absorbance as the ordinate, the standard curve of indigoidine was obtained by linear fitting. Since both the cells and the supernatant after fermentation contained indigoidine, the concentration curves of indigoidine in the cells and supernatants were drawn respectively. Standard curve R of indigoidine standard in bacteria 2 =0.996, the standard curve R of the indigoidine standard in the supernatant 2 =0.992, the linearity of th...

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Abstract

The invention relates to a method for constructing recombinant Corynebacterium glutamicum to produce novel indigo dye, belonging to the field of biological engineering. The recombinant C. glutamicum ATCC was constructed by cloning and recombining the indigo synthase gene (bpsA) and the 4'-phosphopanthyltransferase gene (sfp) and co-expressing them into Corynebacterium glutamicum. 13032-(BpsA-SFP), the recombinant Corynebacterium glutamicum can use L-glutamine as a substrate to synthesize indigo dye. In the shake flask fermentation culture LBG liquid medium, the temperature is 30°C, and the fermentation culture is carried out until the bacterial growth OD600 reaches 0.6-0.8, 0.8mM IPTG and L-glutamine 11.68g / L are added, and the culture is continued for 48h at 18°C. The highest yield of indigoidine was 1.75g / L. The invention adopts the strategy of bioengineering, and the recombinant Corynebacterium glutamicum is constructed to synthesize the target product indigoidine, the indigoidine, and provides a new idea for the high production of indigoidine by biological method.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the production of indigo recombinant Corynebacterium glutamicum and a construction method and application thereof. Background technique [0002] Dyestuff is one of the essential and important substances in human industrial production and life. my country is the world's largest producer of dyestuffs, accounting for more than 70% of the world's annual production. According to statistics from the National Bureau of Statistics, the sales of dyes in my country reached 83 billion yuan in 2015, and it is expected to exceed 100 billion yuan in 2018. Most of the industrial dyes are organically synthesized, and the ingredients contain hydrogen sulfide, alkaline substances and acidic substances. High-salt wastewater and waste acid produced by traditional dye production industries are one of the important sources of environmental pollution. With the intensification of environmental protection...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/77C12P17/16C12R1/15
Inventor 王猛徐捷刘扬程海娇
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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