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Recombined engineering bacterium for producing UDPG (uridine diphosphate glucose) and application thereof

A technology of recombinant engineering bacteria and engineering bacteria, applied in bacteria, microorganism-based methods, microorganisms, etc., can solve the problems of massive accumulation, affecting the yield of Jinggangmycin A, increasing the cost of Jinggangmycin A, etc., and achieving a fast conversion rate. Effect

Inactive Publication Date: 2015-05-06
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been people through different mutagenesis methods and different genetic engineering techniques to transform the fermentation production strain and achieve a relatively ideal yield, but when Jinggangmycin A achieves high yield, there are a large number of Jinggang Hydroxylamine A by-products. accumulation phenomenon
The existence of the fermentation product Jinggang hydroxylamine A not only increases the cost of Jinggangmycin A separation, but also affects the yield of Jinggangmycin A

Method used

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  • Recombined engineering bacterium for producing UDPG (uridine diphosphate glucose) and application thereof
  • Recombined engineering bacterium for producing UDPG (uridine diphosphate glucose) and application thereof
  • Recombined engineering bacterium for producing UDPG (uridine diphosphate glucose) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction method of Escherichia coli pETDuet-ValG BL21 (DE3) engineering bacteria

[0031] The Streptomyces hygroscopicus genome was obtained by a small extraction method, and then the target gene ValG was introduced into the pETDuet-1 plasmid. The specific method is as follows:

[0032] 1. Mini-extraction of Streptomyces hygroscopicus genome

[0033] Streptomyces hygroscopicus subsp. jinggangensis was inoculated in Bennett's medium, cultured at 28°C and 180rpm for 18h; centrifuged at 12000rpm for 5min, the streptomyces mycelium was collected and washed with 10.3% sucrose aqueous solution for 2 times; with 500 μL, 2mg / mL lysozyme buffer (lysozyme buffer (10mmol / L) composition: 0.3mol / L sucrose, 25mmol / L Tris-HCl (pH8.0), 25mmol / L EDTA (pH8.0 ).) Re-suspend the mycelium, in a water bath at 37°C for 30 minutes until the solution is translucent; add 250 μL of 2% SDS (mass concentration, the solvent is double distilled water), mix and oscillate evenly, and bat...

Embodiment 2

[0041] Example 2 Construction method of Escherichia coli pETDuet-ValG-GalU BL21 (DE3) engineering bacteria

[0042] The E.coli JM109 genome was obtained by the CTAB method, the GalU target gene was amplified, and then the GalU target gene was introduced into the pETDuet-ValG plasmid. The specific method is as follows:

[0043] 1. Mini-extraction of Escherichia coli JM109 genome

[0044] Inoculate E.coli JM109 in LB liquid medium, 37°C, 180rpm, and culture overnight; add the culture solution with a volume concentration of 1% inoculum into fresh LB liquid medium, 37°C, 180rpm, and cultivate for 3h; take 5mL of bacteria solution, centrifuged at 5000rpm for 10min, and collected the bacteria; added 567μL TE buffer solution (pH value 8.0), added 30μL mass concentration 100g / L SDS (solvent double distilled water) and 3μL 20mg / mL proteinase K (solvent double distilled water) mixed Put in a constant temperature water bath at 37°C for 1 hour; add 100 μL 5mol / L NaCl aqueous solution and...

Embodiment 3

[0056] Example 3 Construction method of Escherichia coli pETDuet-ValG-GalU-Pgm BL21 (DE3) engineering bacteria

[0057] The E.coli JM109 genome was obtained by the CTAB method, the Pgm target gene was obtained by PCR expansion, and then the Pgm target gene was introduced into the pETDuet-ValG-GalU plasmid. The specific method is as follows:

[0058] 1. Amplification of Pgm target gene

[0059] Primers were designed according to the genome sequence of E.coli JM109:

[0060] Pgm-F:5'-TGCA CTGCAGAACGTTGCAGACAAAGGAC-3' (Pst I),

[0061] Pgm-R:5'-ATTT GCGGCCGC TTACGCGTTTTTCAGAAC-3' (Not 1). The Pgm gene was amplified by PCR using the E.coli JM109 genome as the amplification template and Pgm-F / Pgm-R as primers. PCR system: 10× buffer 5.0 μL, d NTPs 4.0 μL, Pgm-F 1.0 μL, Pgm-R 1.0 μL, E.coli JM109 genome template 3.0 μL, TaKaRa Ex Taq HS 0.25

[0062] μL, RNase Free dH 2 O supplemented to 35.75 μL. PCR program: pre-denaturation at 98°C for 2min; denaturation at 98°C for 10s...

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Abstract

The invention discloses a recombined engineering bacterium for producing UDPG (uridine diphosphate glucose) and application thereof to preparation of validamycin A. The recombined engineering bacterium is formed by transferring the ValG gene in streptomyces hygroscopicus and the GalU gene and the Pgm gene in escherichia coli JM109 into a host bacterium, wherein the ValG gene has a nucleotide sequence as shown in SEQ ID NO:1, the GalU gene has a nucleotide sequence as shown in SEQ ID NO:2, and the Pgm gene has a nucleotide sequence as shown in SEQ ID NO:3. The recombined engineering bacterium can be used for transforming validoxylamine A into validamycin A under the whole cell catalysis, has a high transformation speed in the same time and can achieve a transformation rate of 75%.

Description

(1) Technical field [0001] The invention relates to a recombinant engineering bacterium producing UDPG and its application in preparing Jinggangmycin A. (2) Background technology [0002] Scholars such as Shen Yinchu discovered an actinomycete producing active substances resistant to sheath blight, and successfully developed Jinggangmycin products (Jinggangmycin A as the main active ingredient), which replaced organic arsenic in a more environmentally friendly and efficient way Preparation, so realizing the high yield of Jinggangmycin A fermentation production has become the primary problem. Although there have been people through different mutagenesis methods and different genetic engineering techniques to transform the fermentation production strains and achieve a relatively ideal yield, but when Jinggangmycin A achieves high yield, there are a large number of Jinggang Hydroxylamine A by-products. accumulation phenomenon. The existence of the fermentation product Jinggan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/46C12R1/19
Inventor 陈小龙范永仙连振静罗星荣沈寅初
Owner ZHEJIANG UNIV OF TECH
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