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Method for preparing genetically engineered bacteria for efficiently compounding pantothenic acid and application thereof

A technology of genetically engineered bacteria and pantothenic acid, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of low enzyme activity, substrate toxicity, high production cost, etc., achieve high yield, short fermentation time, highly active effect

Active Publication Date: 2017-05-17
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] What the present invention aims to solve is efficient, environment-friendly synthesis of pantothenic acid, due to the existence of substrate toxicity in chemical synthesis of pantothenic acid, high production cost in splitting, small production volume, and poor optical purity of products; and microbial synthesis of pantothenic acid is mainly Escherichia coli or Bacillus subtilis expresses itself or the same kind of pantothenic acid synthase to synthesize pantothenic acid, and the enzyme activity is the decisive link of the synthesis efficiency, and the current enzyme activity is relatively low

Method used

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  • Method for preparing genetically engineered bacteria for efficiently compounding pantothenic acid and application thereof
  • Method for preparing genetically engineered bacteria for efficiently compounding pantothenic acid and application thereof
  • Method for preparing genetically engineered bacteria for efficiently compounding pantothenic acid and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Construction of Genetically Engineered Bacteria

[0019] Respectively selected from Escherichia coli (Escherichia coli BL21 (DE3)), Corynebacterium glutamicum ATCC 13032, Bacillus subtilis (Bacillus subtilissubsp.subtilis str.168), Bacillus cereus (Bacillus cereus E33L), Enterobacter cloacae EcWSU1, Bacillus thuringiensis BMB171, and Escherichia coli high-efficiency expression system BL21(DE3) / PET30 were used to construct genetically engineered bacteria.

[0020] Using Escherichia coli (E.coliBL21(DE3)) panc gene (Sequence Listing 1) as a template, the fragment panC was obtained by PCR amplification with primers E-panc-for and E-panc-rev respectively E , the PCR product panC E The fragment was recovered by 1% agarose electrophoresis, and after double digestion with restriction endonucleases BamH 1 and Xho 1, it was connected to the vector pET30 after the same digestion to obtain the recombinant plasmid pET30-panC E , transform E.coli DH5α competent cells, colony PCR s...

Embodiment 2

[0030] Enzyme Activity Detection of Genetic Engineering Strains

[0031] Pick up E.coli BL21(DE3) / pET30-panC from the plate E , E.coli BL21(DE3) / pET30-panC C , E.coli BL21(DE3) / pET30-panC b , E.coli BL21(DE3) / pET30-panC bc , E.coli BL21(DE3) / pET30-panC ec and E.coli BL21(DE3) / pET30-panC bt A single colony was inoculated in 5 mL of LB medium (containing 50 μg / ml Kan) and cultured overnight at 37°C. The above culture solution was transferred to 20 mL of LB medium (containing 50 μg / ml Kan) at a ratio of 1:100, cultured at 37°C, and when the OD reached 0.4-0.6, 0.2mM IPTG was added, and cultured at 30°C for 16h. Collect 10OD bacteria, use 1ml buffer (100mM Hepes, 20mM MgCl 2 .6H 2 O, 1 mM EDTA, pH 8.0) to suspend, then sonicate the bacterial cells, centrifuge to get the supernatant and dilute 10 times. Take 5 μL of the diluted liquid and add 1.1 mL of reaction buffer (25 mM β-alanine, 25 mM DL-pantoic acid, 4.5 mM ATP, 10 mM MgCl 2 , 15mM KCl). React at 37°C for 20 minut...

Embodiment 3

[0038] high density fermentation

[0039] (1) Pick the engineering bacteria E.coli BL21(DE3) / PET30-panC containing the pantothenate synthase of Corynebacterium glutamicum from the plate C Single colonies were inoculated in 10 mL of LB liquid medium ((yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L)), and cultivated overnight at 37 °C;

[0040] (2) Take 3mL and transfer to 100mL high-density fermentation medium (glucose 20g / L, (NH 4 ) 2 SO 4 9g / L, Na 2 CO 3 2g / L, KH 2 PO 4 6.67g / L, (NH 4 ) 2 HPO 4 4g / L, MgSO 4 ·7H 2 O 0.8g / L, citric acid 0.8g / L, NaHCO 3 2g / L, 5mL ion mother solution, pH 7.0), 50μg / ml kanamycin (Kan), cultured at 37°C for 12h;

[0041] (3) Take 60ml of the 100ml medium in the second step, and transfer it to a 1L fermenter containing 600mL of fermentation medium at a ratio of 1:10 for cultivation, control dissolved oxygen at 15%, and cultivate at 30°C; ferment for 8h Add feeding material (glucose, 650g / L); Add IPTG (final concentratio...

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Abstract

The invention relates to a method for acquiring genetically engineered bacteria capable of efficiently producing pantothenic acid by introducing the pantothenic acid synthetase genes from different sources into escherichia coli. An efficient escherichia coli protein expression system is adopted for heterologously expressing the pantothenic acid synthetase from different sources, thereby acquiring high-activity pantothenic acid synthetase strain. The strain can efficiently express the pantothenic acid synthetase and convert substrate pantoic acid and beta-alanine into pantothenic acid; the enzyme activity reaches up to 33.52U / mL; the engineering bacteria are fermented for 38h; the generated pantothenic acid is 101.2g / L. The strain has the characteristics of high activity, short fermentation time, high yield, and the like.

Description

technical field [0001] The invention relates to the field of biological fermentation, in particular to genetically engineered bacteria for producing pantothenic acid. Background technique [0002] D-pantothenic acid (D-pantothenic acid), also known as vitamin B5, is a kind of water-soluble vitamin B family and an important precursor of CoA and acyl carrier protein ACP. According to the KEGG database, CoA (Kanechisa M, 2006) participates in There are more than 400 kinds of enzymatic reactions involved in fatty acid metabolism, cell signaling, tricarboxylic acid cycle and other central metabolic reactions (GaneshSamala, 2015). Natural pantothenic acid has dextrorotation, that is, D-pantothenic acid, which is an important food additive and feed additive, as well as an important vitamin drug. It is clinically used to treat vitamin B deficiency, peripheral neuritis, postoperative ileus, streptomycin poisoning and rheumatoid diseases. [0003] The commercial form of pantothenic ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/00C12N15/52C12P13/02C12R1/19
CPCC12N9/93C12P13/02C12Y603/02001
Inventor 蔡真张君丽奇古王瑞研李寅
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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