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A kind of optimized sequence of trehalose synthase gene and its application

A technology of trehalose synthase and gene, applied in application, genetic engineering, glycosyltransferase, etc., can solve the problems of low expression efficiency and low content of trehalose synthase, so as to increase industrial output value, reduce production cost, improve The effect of gene translation efficiency

Active Publication Date: 2021-09-17
SHANDONG ACADEMY OF PHARMACEUTICAL SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the low content of trehalose synthase in Streptomyces griseus and the low expression efficiency of the trehalose synthase gene derived from Streptomyces griseus in Escherichia coli, the present invention provides a trehalose synthesis method with highly soluble expression in Escherichia coli Enzyme Gene Optimized Sequence

Method used

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  • A kind of optimized sequence of trehalose synthase gene and its application
  • A kind of optimized sequence of trehalose synthase gene and its application
  • A kind of optimized sequence of trehalose synthase gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Recombinant expression vector pET-26b(+)- tres1 construction and identification.

[0028] The Streptomyces griseus trehalose synthase ( tres ) sequence (SEQ ID No.3) as a reference, according to the degeneracy of codons, without changing the amino acid composition, the high-frequency or sub-high-frequency codons of E. Some rare codons of Bacteria were replaced by E. coli preferred codons, and the optimized sequence of trehalose synthase gene was finally obtained as shown in SEQ ID NO.1; its CAI was 0.97, GC content was 54.4%, CFD> 50%.

[0029] Trehalose synthase gene optimized sequence ( tres1 ) at the 5’ end plus Nco I restriction site (CCATGG), 3' end plus xho I enzyme cleavage site (CTCGAG) for whole gene synthesis. The optimized sequence of the trehalose synthase gene was double digested and connected to the pET-26b(+) plasmid (Novagen Company) after the same double digestion. The ligation product was transformed into Escherichia coli BL21 com...

Embodiment 2

[0031] Example 2 Recombinant strain BL21 / pET-26b(+)- tres1 construction and inducible expression.

[0032] The Escherichia coli expression plasmid pET-26b(+)- tres1 and pET-26b(+)- tres respectively through CaCl 2 Heat shock method was used to transfer Escherichia coli BL21 (DE3) (Beijing Quanshijin Biotechnology Co., Ltd.), adding kanamycin to 50 mg / L in the LB plate medium for screening, picking a single colony for sequencing identification, and constructing Successful ones are BL21 / pET-26b(+)- tres1 or BL21 / pET-26b(+)- tres .

[0033]Pick a single colony containing the correct recombined positive plasmid, inoculate it into LB liquid medium containing 50mg / L kanamycin, shake it at 37°C and 220 rpm overnight, and inoculate the overnight cultured bacterial solution at a ratio of 1%. Inoculate into fresh LB liquid medium containing 50mg / L kanamycin, culture at 37°C, shake at 220rpm until the bacterial concentration is OD 600 When it is about 1.0, immediately add t...

Embodiment 3

[0035] Example 3 Activity Comparison of Trehalose Synthase

[0036] The enzyme activity of the two crude enzyme solutions prepared in Example 2 was measured, and 200 μL of the enzyme solution was added to an equal amount of 10% maltose solution prepared with 0.1 mol / L pH 6.5 phosphate buffer solution, and reacted in a water bath at 24 °C for 1 h , the reaction was terminated by inactivating the enzyme in a boiling water bath for 10 min. The enzyme reaction product was analyzed by HPLC (Agilent 1200 type). HPLC conditions: separation column ZORBAXNH2, 4.6×250 mm, 5 μm, mobile phase is acetonitrile:water=75:25 (v / v), column temperature: 50 ℃, flow rate 1.0 mL / min. Trehalose synthase enzyme activity 1 U Definition: Under the reaction conditions of 24 ℃ and pH 6.5, with 10% maltose solution as the substrate, the amount of enzyme required to produce 1 μmol trehalose per 1 min. The result is as image 3 Shown: After sequence optimization, the enzyme activity of the crude enzyme s...

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Abstract

The present invention provides an optimized sequence of trehalose synthase gene, which comprises the DNA sequence shown in SEQ ID No.1. The amino acids expressed in the optimized sequence of the trehalose synthase gene include the sequence shown in SEQ ID No.2. The optimized sequence of the trehalose synthase gene of the present invention, through codon optimization, without changing the final amino acid sequence, rationally optimizes the rare codons or low-frequency codons of Escherichia coli in the trehalose synthase gene sequence derived from Streptomyces griseus Optimization, breaking through the bottleneck of gene translation of heterologous genes in E. coli, improving gene translation efficiency, realizing high-yield soluble expression in E. coli, solving the problem of low efficiency of heterologous expression in E. coli, and greatly reducing trehalose synthase production cost. The invention can increase the industrial output value of the trehalose synthase and reduce the production cost.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to an optimized sequence of a trehalose synthase gene and a production method and application thereof. Background technique [0002] Trehalose synthase (Trehalose Synthase) can catalyze the conversion of two glucose molecules α,α-1,4-glucosidic bonds in maltose into glucose α,α-1,1-glucosidic bonds through intramolecular transglycosidation, thereby generating seaweed sugar. It has been reported that there are five pathways for the synthesis of trehalose by biological enzymatic methods, including TPS / TPP, TS, TreY / TreZ, TreP, and TreT. Among them, the trehalose synthase pathway (TS pathway, also known as the Tres pathway) only requires one enzyme for one-step enzymatic reaction. In the industrial production of trehalose, compared with the traditional yeast cell extraction method and other biosynthetic pathways, the substrate is cheap and the process is Simple, easy to regu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/70C12N1/21C12R1/19
CPCC12N9/1051C12N15/70C12N2800/22C12Y204/01245
Inventor 张晓元刘飞郝荣华袁丹丹陈勉凌沛学张辉解荣利王羽高亮
Owner SHANDONG ACADEMY OF PHARMACEUTICAL SCIENCES
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