High-inosine-yield strain breeding method

A strain and inosine technology, applied in the field of biomedicine, can solve the cumbersome and difficult problems of improvement, and achieve the effects of simple and feasible method, improved inosine production ability, and stable genetic expression

Inactive Publication Date: 2017-01-04
上海瑞丰农业科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is often very cumbersome and difficult to improve the cellular characteristics conferred by multiple or dozens of genes scattered in different positions of the genome through traditional DNA recombination technology

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Inoculate the myocardin producing strain (No. CICC21929) purchased from China Industrial Microbial Culture Collection Center into the seed culture solution (100ml containing 2.2g of glucose, 1.4g of yeast extract, 1mL of corn steep liquor, 0.8g of urea, 1.0g of peptone, NaCl 0.25g, Ade 2.5mg, pH 7.0), cultured at 37°C for 24 hours, centrifuged, washed with normal saline for 3 times and resuspended, and configured by plate colony counting method to about 10 8 cells / mL of bacterial suspension. Take 1 mL of bacterial liquid and mix equal volumes of different antibiotic solutions, irradiate with 20W ultraviolet lamp at 30cm for 60s, treat at 37°C and 180rpm for 60min, and immediately dilute with normal saline to terminate the reaction.

[0037] (2) Take 70 μL of the diluted Bacillus subtilis solution and spread it on the plate of LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0) containing sulfaguanidine and antibiotics, 37 Cultivate at ℃ for 24h, t...

Embodiment 2

[0041] (1) Take one loop of activated gentamicin, rifampicin or streptomycin-resistant strains on the plate and insert it into a Erlenmeyer flask containing 25mL of LB liquid medium, shake and culture at 200rpm at 37°C for 20h, and take 1mL The bacterial solution was transferred to another same LB liquid medium, and cultured to the middle and late logarithm under the same conditions.

[0042] (2) Take 10 mL of bacterial liquid in a 50 mL centrifuge tube, centrifuge at 8000 rpm for 5 min, discard the supernatant, and collect the bacterial cells. Add 1mL SMMP solution: 2×SMM (sucrose 0.5M, maleic acid 0.02M, MgCl 2 ·7H 2 O 0.02M, pH 7.0)+4×PAB (beef extract 1.5g / L, peptone 5g / L, yeast extract 1.5g / L, NaCl 3.5g / L, glucose 1g / L, KH 2 PO41.32g / L, K 2 HPO 4 3.68g / L). Suspend the bacteria, add lysozyme (final concentration: 0.2-1mg / mL), mix well, place in a 37°C water bath, and take samples for microscopic examination at intervals of 15 minutes to observe the formation of protop...

Embodiment 3

[0047] Example 3 High-yield inosine strain ability detection

[0048] (1) The paper chromatography method was used to analyze the ability of the primary screened strain to produce inosine through fermentation. Cut No. 3 filter paper with a length of 30 cm and a width of 20 cm, draw a straight line with a pencil at a distance of 2 cm from the edge of the filter paper, and set a sample point every 2 cm; use a pipette gun to draw 1 mL of fermentation broth into a 1.5 mL small centrifuge tube, and centrifuge at 12000 rpm for 10 min. Take the supernatant. Use a micro-sampler to place the inosine standard or the processed sample to be tested on the corresponding spot on the filter paper, and let it dry naturally or with a hair dryer. Spotting is 5-10 μL each time, and a total of 20 μL is spotted, and the spot diameter is controlled within 4 mm; roll the spotted filter paper into a cylinder, sew it, and place it on a layer filled with a developing agent about 5 mm deep In the analy...

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PUM

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Abstract

The invention discloses a high-inosine-yield strain breeding method which comprises the following steps: irradiating the initial strains under ultraviolet light, screening out an antibiotic/sulfaguanidine-resistant double-antimicrobial strain by a gradient plate process, and carrying out shake flask fermentation on the double-antimicrobial strain to obtain the strain with higher inosine yield; and fusing two or more different antibiotic-resistant strain bioplasms by genome rearrangement, screening out the multi-antibiotic-resistant strain, and carrying out shake flask fermentation to screen out the high-inosine-yield strain. The method greatly enhances the Bacillus subtilis inosine yield, and maintains favorable characteristics of the strain.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a method for breeding high-yield inosine-producing strains, in particular to a method for organically combining ribosome resistance screening and genome rearrangement technology to screen high-yielding inosine-producing strains. Background technique [0002] Inosine is a multi-purpose purine nucleoside, which is widely used in the synthesis of food freshness enhancer IMP, and is not easily destroyed in conventional storage and food baking and cooking. With the improvement of people's living standards, consumers have higher and higher requirements for food flavors. Nucleotide flavor enhancers are being used to produce various functional foods, including cold-resistant foods and diet foods. In addition, purine nucleosides can affect the activity of the nervous system, produce cardiovascular effects, and have important physiological functions such as sedation, antispasmodic, di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N13/00C12N15/01C12N15/03C12R1/125
Inventor 彭日荷姚泉洪王荣谈田永生王丽娟丁卫星严培兰王波孙斌
Owner 上海瑞丰农业科技有限公司
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