Method for production of L-4-hydroxyproline by using recombinant escherichia coli fermentation

A technology for recombining Escherichia coli and hydroxyproline, applied in the field of microbial genetic engineering, can solve the problems of high cost, low extraction rate, and large pollution of L-4-hydroxyproline, and achieve good industrial development prospects

Inactive Publication Date: 2014-01-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the defects of high cost, high pollution and low extraction rate of L-4-hydroxyproline obtained by the current biological extraction method,

Method used

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  • Method for production of L-4-hydroxyproline by using recombinant escherichia coli fermentation
  • Method for production of L-4-hydroxyproline by using recombinant escherichia coli fermentation
  • Method for production of L-4-hydroxyproline by using recombinant escherichia coli fermentation

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Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Design of tryptophan tandem promoter sequence

[0036] The tryptophan tandem promoter is derived from the ptrpL1 plasmid (purchased from Stratagene, Germany), and the plasmid map is attached to the instructions. figure 2The tryptophan promoter of this plasmid is derived from the tryptophan operon of Escherichia coli K12 strain. It is a strong promoter suitable for industrial production applications. There is a Cla I restriction site between the SD sequence and the start codon sequence At the same time, there is a HindIII restriction site upstream of the -35 region of the tryptophan promoter, which is convenient for connecting the promoter to other expression vectors. Since the two tryptophan promoters are connected in series, the expression intensity can be improved, so , based on the tryptophan promoter of the ptrpL1 plasmid, a tryptophan tandem promoter was synthesized by total gene synthesis.

[0037] By optimizing the tryptophan tandem promoter, the Hin...

Embodiment 2

[0039] Embodiment 2: the design of proline-4-hydroxylase gene sequence

[0040] According to the codon usage frequency of Escherichia coli to optimize the gene codon, eliminate the codon with low usage rate, and use the synonymous transformation method to eliminate HindIII, BamH I, EcoR I restriction sites, so the original sequence and The terminator was changed to TAAT strong terminator. In order to facilitate the connection of the proline-4-hydroxylase gene to other plasmid vectors, a restriction site BamH I (GGATCC) was inserted behind the terminator.

[0041] The secondary structure of mRNA should also be taken into consideration. First of all, it is necessary to ensure that the codon translation pocket consisting of several bases after the AUG start codon is open to reduce the energy potential of ribosomes binding to mRNA, so that ribose The body can be smoothly translated backwards along the start codon.

[0042] For the original sequence of the proline-4-hydroxylase ge...

Embodiment 3

[0043] Embodiment 3: Construction of proline-4-hydroxylase gene expression vector and recombinant Escherichia coli

[0044] Synthesize the designed tryptophan tandem promoter and proline-4-hydroxylase gene respectively first, since the 5-terminal sequence of the tryptophan tandem promoter gene sequence has an EcoR I (GAATTC) restriction site, the 3' There is a HindIII (AAGCTT) restriction site at the end, and the 5' end sequence of the proline-4-hydroxylase gene has a HindIII (AAGCTT) restriction site, and there is a BamH I (GGATCC) restriction site at the 3' end point. The tryptophan tandem promoter gene sequence was double-digested with restriction endonucleases EcoR I and HindIII, and the proline-4-hydroxylase gene was double-digested with HindIII and BamH I. At the same time, the plasmid vector pAMP was also digested They were digested with EcoR I and BamH I, respectively. Recover the tryptophan tandem promoter gene sequence, proline-4-hydroxylase gene and pAMP plasmid v...

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Abstract

The invention belongs to the field of gene engineering, and discloses a method for production of L-4-hydroxyproline by using recombinant escherichia coli fermentation. Recombinant escherichia coli is constructed by the following method: according to a published proline-4-hydroxylase gene and tryptophan promoter sequence, first optimally designing a tryptophan tandem promoter and a proline-4-hydroxylase structure gene, then after the total gene synthesis of the tryptophan tandem gene promoter and the proline-4-hydroxylase structure gene, connecting the promoter and the structure gene to pAMP plasmid to construct recombinant plasmid pAMP-P2trp-Hyp for overexpression of proline-4-hydroxylase. The invention also discloses the application of the escherichia coli in the production of the 4-hydroxyproline, the shake flask fermentation results show that the 4-hydroxyproline prepared by the recombinant escherichia coli has a yield reached 0.31g / L, and suggest that the recombinant escherichia coli has good industrial development prospects.

Description

technical field [0001] The invention discloses a method for fermenting and producing L-4-hydroxyproline 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 the position of its hydroxylation, it can be divided into 3-hydroxyproline (3-Hyp) or 4-hydroxyproline amino acid (4-Hyp). Trans-4-hydroxyproline is more common in nature, and trans-4-hydroxyproline is an important component of animal tissue proteins such as collagen. 4-Hydroxyproline is widely used in medicine, chemical industry, animal feed, nutrition and beauty industry, etc. In medicine, 4-hydroxyproline can be used as a raw material for the synthesis of anti-inflammatory drugs, carbapenems, and angiotensin-converting enzyme inhibitors. In terms of chemical synthesis, 4-hydroxyproline is an indispensable chiral raw material for the synthesis of...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P13/24C12R1/19
Inventor 张震宇刘合栋袁春伟
Owner JIANGNAN UNIV
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