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Method for producing cis-4-hydroxy-L-proline through recombinant escherichia coli

A production method, proline technology, applied in the fields of biochemical equipment and methods, recombinant DNA technology, botany equipment and methods, etc.

Inactive Publication Date: 2015-09-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the production of cis-4-hydroxy-L-proline by biotransformation in China

Method used

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  • Method for producing cis-4-hydroxy-L-proline through recombinant escherichia coli
  • Method for producing cis-4-hydroxy-L-proline through recombinant escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: the design of proline-cis-4-hydroxylase gene sequence

[0024] According to the codon usage frequency of Escherichia coli to optimize gene codons, eliminate low-usage codons, and use the synonymous transformation method to eliminate Hind Ⅲ, BamH I restriction sites, and modify the stop codon in the original sequence to TAAT strong terminator, in order to facilitate the connection of the proline-cis-4-hydroxylase gene to other vectors, a Hind III restriction site was added before the start codon, and an enzyme was inserted behind the terminator Cutting site BamH Ⅰ.

[0025] The secondary structure of mRNA should also be considered to ensure that the codon translation pocket consisting of the AUG start codon and a few bases after it is open, reducing the energy potential of ribosomes binding to mRNA, so that ribosomes can smoothly Translate backwards along the start codon.

[0026] The original sequence of the proline-cis-4-hydroxylase gene sequence in thi...

Embodiment 2

[0027] Embodiment 2: Construction of proline-cis-4-hydroxylase gene expression vector and recombinant Escherichia coli

[0028] On the pES vector containing the tryptophan tandem promoter (ptrp2), there are Hind III and BamH I restriction sites at the 3' end of the promoter, and the synthetic proline-cis-4-hydroxylase gene The 5' end sequence has a Hind Ⅲ restriction site, and the 3' end has a BamH Ⅰ restriction site, so both Hind Ⅲ and BamH Ⅰ are used for the pES vector and proline-cis-4-hydroxylase gene After double enzyme digestion and gel recovery, the gel recovery product was ligated with T4DNA ligase.

[0029] The ligation solution was transformed into Escherichia coli JM109 competent cells, and then the plasmids were extracted for enzyme digestion verification, and the plasmids with correct enzyme digestion results were sequenced to verify whether the gene sequence was correct. In this way, the constructed pEHC4 recombinant vector was obtained, and the map of the recom...

Embodiment 3

[0030] Then, the verified recombinant vector was transformed into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli BL21(DE3) / pEHC4. Embodiment 3: Fermentation experiment of recombinant escherichia coli BL21(DE3) / pEHC4

[0031] Seed culture: Pick a single colony of recombinant Escherichia coli BL21(DE3) / pEHC4 on the Amp resistant plate, inoculate it into LB liquid medium (ampicillin 100 μg / mL), and culture it at 37°C and 220rpm for 6-8h.

[0032] Shake flask fermentation: Inoculate 6% inoculum into a 250mL shake flask containing 30mL GT medium, culture in a rotary shaker at 25°C and 220rpm for 48h, then take the fermentation broth to detect cis-4-hydroxy-L- Proline concentration.

[0033] Yield determination: For the determination method of cis-4-hydroxy-L-proline, please refer to the general description of the examples.

[0034] The fermentation results showed that the cis-4-hydroxyl-L-proline yield of the recombinant Escherichia coli BL21(DE3) / pEHC4 reached...

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Abstract

The invention belongs to the field of genetic engineering and discloses a method for producing cis-4-hydroxy-L-proline through recombinant escherichia coli. The recombinant escherichia coli is constructed by a method comprising the following steps: optimizing the known gene sequence of L-proline-cis-4-hydroxylase; performing total gene synthesis of the optimized L-proline-cis-4-hydroxylase gene; connecting the gene to a carrier containing a proper promoter to construct a recombinant plasmid which can over-express L-proline-cis-4-hydroxylase; then introducing the recombinant plasmid into escherichia coli to obtain recombinant escherichia coli which can convert free L-proline into cis-4-hydroxy-L-proline. The invention further discloses application of the recombinant escherichia coli in production of cis-4-hydroxy-L-proline. The shake-flask fermentation result shows that the output of cis-4-hydroxy-L-proline produced by the recombinant escherichia coli is up to 47.33mg / L.

Description

technical field [0001] The invention discloses a method for producing cis-4-hydroxyl-L-proline by recombinant Escherichia coli, which belongs to the field of microbial genetic engineering. Background technique [0002] Hydroxyproline (Hyp) is an imino acid, which is the product of hydroxylation of proline. According to its hydroxylation position and spatial structure, there are 8 isomers in total. Among them, cis-4-hydroxy-L-proline is a rare amino acid in nature, which can be incorporated into the polypeptide chain during the synthesis of procollagen, resulting in the misfolding of procollagen, thus in Reduced collagen deposition during fibrosis and tumor cell growth. cis-4-hydroxy-L-proline is used as a precursor for the production of antifibrotic, anticancer and antihypertensive drugs. [0003] The production methods of cis-4-hydroxyl-L-proline include chemical synthesis and biotransformation. Among them, the starting material of the chemical synthesis method is trans-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/53C12P13/24
Inventor 张震宇王付才张胜利王晓姣夏紫薇
Owner JIANGNAN UNIV
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