Method for effectively improving proline conversion rate in system for producing Cis-3-hydroxy-L-proline according to biosynthesis method

A technology of proline conversion rate and biosynthesis, which can be used in biochemical equipment and methods, microorganisms, nucleic acid carriers, etc., and can solve problems such as low content

Inactive Publication Date: 2016-05-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The hydrolysis method is mainly used to extract trans-4-hydroxyproline in animal bone collagen[16]. Since the content of cis-3-hydroxyproline in bone collagen is very small, it cannot be obtained by common hydrolysis methods

Method used

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  • Method for effectively improving proline conversion rate in system for producing Cis-3-hydroxy-L-proline according to biosynthesis method
  • Method for effectively improving proline conversion rate in system for producing Cis-3-hydroxy-L-proline according to biosynthesis method
  • Method for effectively improving proline conversion rate in system for producing Cis-3-hydroxy-L-proline according to biosynthesis method

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Experimental program
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Effect test

Embodiment 1

[0023] Example 1: Design of tryptophan tandem promoter sequence

[0024] The tryptophan tandem promoter is derived from the ptrpL1 plasmid, and the plasmid map is attached to the instructions figure 2 The 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 ClaI restriction site between the SD sequence and the start codon sequence , At the same time, there is a HindⅢ enzyme cutting site upstream of the -35 region of the tryptophan promoter, which is convenient for connecting the promoter to other expression vectors. Since two tryptophan promoters can be connected in tandem to increase the expression intensity, according to the tryptophan promoter sequence of the ptrpL1 plasmid, a tryptophan tandem promoter was synthesized in the whole gene.

[0025] By optimizing the tryptophan tandem promoter, the HindⅢ restriction site AAGCTT was modified...

Embodiment 2

[0027] Embodiment 2: the design of proline-3-hydroxylase gene sequence

[0028] According to the codon usage frequency of Escherichia coli to optimize gene codons, eliminate codons with low usage rate, and use the synonymous transformation method to eliminate the EcoRI restriction site, modify the terminator in the original sequence to a TAAT strong terminator, in order to It is convenient to connect the proline-3-hydroxylase gene to other plasmid vectors, so a restriction site BamHI (GGATCC) was inserted behind the terminator.

[0029] The secondary structure of mRNA should also be taken into consideration. First, ensure that the codon translation pocket consisting of the ATG initiation codon and several bases thereafter is open, reducing the energy potential of ribosomes binding to mRNA, making ribosomes Can be smoothly translated backwards along the start codon.

[0030] The optimized proline 3-hydroxylase gene sequence in this embodiment is artificially synthesized; see S...

Embodiment 3

[0031] Embodiment 3: the construction of proline 3-hydroxylase gene expression vector

[0032] First, synthesize the designed tryptophan tandem promoter and proline 3-hydroxylase gene respectively. Since the 5' end sequence of the tryptophan tandem promoter gene sequence has an EcoRI (GAATTC) restriction site, the 3' end There is a HindⅢ (AAGCTT) restriction site, and the 5' end sequence of the proline 3-hydroxylase gene has a HindⅢ (AAGCTT) restriction site, while the 3' end has a BamHI (GGATCC) restriction site.

[0033] The tryptophan tandem promoter gene sequence was double-digested with restriction endonucleases EcoRI and HindⅢ, and the proline 3-hydroxylase gene was double-digested with HindⅢ and BamHI. At the same time, the plasmid vector pET21a was also digested with EcoRI and BamHI digestion. The tryptophan tandem promoter gene sequence, proline 3-hydroxylase gene and pET21a plasmid vector after the double-cut enzyme were recovered with an agarose gel kit, and then t...

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Abstract

The invention discloses a method for effectively improving proline conversion rate in a system for producing Cis-3-hydroxy-L-proline according to a biosynthesis method. The system for producing the Cis-3-hydroxy-L-proline according to the biosynthesis method is a production method that in biological cells, free L-proline is converted into the Cis-3-hydroxy-L-proline by utilizing proline-4-hydroxylase under the existence of alpha-oxoglutarate and ferrous ions. The method for improving the proline conversion rate is to interrupt a degradation pathway of the proline in the biological cells. With the adoption of the method, the conversion rate of the Cis-3-hydroxy-proline-L-proline reaches 100 percent.

Description

technical field [0001] The invention relates to a method for effectively improving the conversion rate of proline in a system for producing cis-3-hydroxyl-L-proline by biosynthesis, 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 cis-3-hydroxy-L-proline is an important component of animal tissue proteins such as collagen. Cis-3-hydroxy-L-proline is widely used in medicine, nutrition and beauty industry. [0003] At present, there are three main methods for producing hydroxyproline at home and abroad: hydrolysis, chemical synthesis and biosynthesis. The hydrolysis method is mainly used to extract trans-4-hydroxyproline in animal bone collagen[16]. Since the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/71
CPCC12N15/71C12N2800/101C12N2800/22C12N2800/60
Inventor 张震宇黄建华王晓姣姚动邦魏照辉
Owner JIANGNAN UNIV
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