Engineered ribokinase, coding gene, expression vector, engineering bacterium and application thereof

文档序号：1811069 发布日期：2021-11-09 浏览：34次中文

阅读说明：本技术 一种工程化核糖激酶、其编码基因、表达载体、工程菌及其应用 (Engineered ribokinase, coding gene, expression vector, engineering bacterium and application thereof ) 是由李桂东王剑峰于 2021-04-30 设计创作，主要内容包括：本发明公开了一种分子改造的工程化核糖激酶、其编码基因、表达载体、工程菌及其应用。采用理性设计的定向进化技术筛选到一株最优突变株(G42F)。其制备方法为以pet28a为载体,利用定点突变技术将42位的甘氨酸突变成苯丙氨酸,并在大肠杆菌中培养表达。与野生型核糖激酶相比,本发明提供的工程化核糖激酶具有更高的反应活性。(The invention discloses a molecular modified engineered ribokinase, a coding gene, an expression vector, an engineering bacterium and application thereof. An optimal mutant strain is screened by a rational directed evolution technology (G42F). The preparation method comprises the steps of using pet28a as a vector, mutating glycine at position 42 into phenylalanine by using a site-directed mutagenesis technology, and culturing and expressing in escherichia coli. Compared with wild type ribokinase, the engineered ribokinase provided by the invention has higher reactivity.)

1. An engineered ribokinase, wherein the amino acid sequence of the engineered ribokinase is shown in SEQ ID No. 2.

2. A coding gene characterized by having a nucleotide sequence corresponding to the amino acid sequence of said engineered ribokinase.

3. A recombinant expression vector comprising the vector pet28a and the coding gene of claim 2.

4. A genetically engineered bacterium comprising a host cell and a target gene transferred into the host cell, wherein the target gene comprises a gene encoding the (R, S) -carbonyl reductase according to claim 2.

5. The genetically engineered bacterium of claim 4, wherein the host cell is Escherichia coli.

6. A method for preparing ribose 5-phosphate, comprising:

ribose reacts with ATP to give ribose 5-phosphate catalyzed by the engineered ribokinase of claim 1.

7. A process for producing ribose 5-phosphate according to claim 6, wherein the reaction temperature is 25 to 35 ℃ and the reaction time is 10 to 15 hours.

8. The method for producing ribose 5-phosphate according to claim 6, wherein the reaction solvent is acetonitrile.

Technical Field

The invention belongs to the fields of biological pharmacy and biological transformation, and particularly relates to an engineered ribokinase and application thereof.

Background

Nicotinamide Mononucleotide (NMN) is involved in the synthesis of Nicotinamide Adenine Dinucleotide (NAD), an important coenzyme in human cells, and plays an important role in cellular energy production. In recent years, NMN has attracted much attention for its applications in anti-aging and age-related degenerative diseases, and its preparation method has also become a research hotspot of various pharmaceutical companies. From the perspective of the catalyst, the synthesis of NMN is mainly divided into chemical catalysis and biological catalysis, wherein the biological catalysis has the characteristics of environmental protection, high efficiency, few byproducts and the like, and occupies an advantage position.

Various companies have also reported methods for the biocatalytic synthesis of NMN. For example, the Shangke (CN108949865A) discloses a method for producing NMN by catalyzing the reaction of 5-phosphoribose and nicotinamide by immobilized cells, wherein the immobilized cells can be recycled, the reaction concentration can reach 13.3g/L, but the price of the 5-phosphoribose is higher. The buntah organism (US2018162895A1) discloses a three-enzyme tandem one-pot method for synthesizing NMN by ribokinase, pyrophosphate synthase and nicotinamide phosphoribosyltransferase, and molecular modification is carried out on the nicotinamide phosphoribosyltransferase in the NMN to obtain better reaction yield, but no further research is carried out on the first step (the ribose kinase catalyzes and generates 5-phosphoribosyl) of the tandem reaction.

Therefore, the method, whether the step method or the one-pot method, has important significance for the research of improving the catalytic activity of the ribokinase.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides an engineered nucleoside kinase (Rbk) through molecular modification, also provides a gene of the engineered ribokinase, an expression vector containing the gene, an engineered strain and a preparation method thereof, and a reaction process for preparing 5-phosphoribosyl by using the engineered ribokinase.

An engineered ribokinase, the amino acid sequence of which is shown in SEQ ID NO. 2. The experimental object of the inventor is a wild type ribokinase derived from Escherichia coli, and the amino acid sequence of the wild type ribokinase is shown as a sequence 1. However, the catalytic activity of the wild type is not ideal. The inventor modifies the ribokinase through directed evolution technology to obtain an engineered ribokinase, and unexpectedly finds that the mutant G42F has better catalytic activity after the glycine at the 42 th position is mutated into the phenylalanine, and the amino acid sequence of the mutant is shown as sequence 2.

The invention carries out molecular modification on the ribokinase by combining rational design with a directed evolution method, and the improved enzyme is improved in catalytic activity. Firstly, screening out a plurality of key amino acids near an active center by analyzing the crystal structure of ribokinase, and respectively mutating the key amino acids into alanine, leucine and phenylalanine with different volumes by primer design and PCR amplification sequence; then, the sequence is connected with pET28a plasmid by using genetic engineering technology; and finally, transforming and constructing engineering bacteria to express the ribokinase.

The preparation method of the engineered ribokinase comprises the following steps:

designing a ribokinase gene sequence primer and carrying out PCR amplification;

b, connecting the ribokinase gene fragment with a cloning vector to obtain a recombinant expression vector carrying a ribokinase gene;

c, transforming the recombinant expression vector into host bacteria to obtain recombinant bacteria;

d recombinant bacteria culture and extraction to obtain crude enzyme solution containing ribokinase

The invention also provides a coding gene, and the nucleotide sequence has a corresponding relation with the amino acid sequence of the engineered ribokinase.

The invention also provides a recombinant expression vector, which comprises the vector pet28a and the coding gene.

The invention also provides a gene engineering bacterium, which comprises a host cell and a target gene transferred into the host cell, wherein the target gene comprises the coding gene of the (R, S) -carbonyl reductase.

The expression vector is suitable for expression in E.coli, and therefore, the host cell is preferably E.coli.

The invention further provides a preparation method of 5-ribose phosphate, which comprises the following steps:

under the catalysis of the engineered ribokinase, ribose reacts with ATP to obtain 5-phosphoribose. The process route is as follows:

preferably, the reaction temperature is 25-35 ℃, and the reaction time is 10-15 hours.

Preferably, the reaction solvent is acetonitrile.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, through modification of wild type ribokinase and a large amount of screening, after 42-position glycine is mutated into phenylalanine, the mutant strain G42F has better catalytic activity, and the yield of catalytic reaction is improved.

Drawings

FIG. 1 is a schematic diagram of the ligation mode of the plasmid of the present invention.

Detailed Description

The invention is further illustrated below with reference to specific embodiments and the accompanying drawings. The following examples are illustrative of the present invention, but the present invention is not limited to the following examples.

Example 1: gene cloning and construction of expression vectors

The amino acid sequence of wild-type ribokinase derived from Escherichia coli can be retrieved from NCBI as shown in sequence 1, and the corresponding nucleic acid is synthesized by techniques common in the art and cloned into the expression vector pet28 a. The recombinant expression plasmid is transformed into competent cells of E.coil BL21(DE3), the transformation condition is 42 ℃, the heat shock is 90 seconds, the transformation solution is coated on an LB plate containing chloramphenicol, and the inversion culture is carried out at 37 ℃ overnight, thus obtaining the recombinant transformant.

Example 2: expression of ribokinase and preparation of enzyme solution

5 mu L of the frozen RK bacterial liquid is transferred into 5mL (containing 50 mu L/mL kanamycin sulfate) LB culture medium and activated overnight.

The activated bacterial liquid is inoculated into 500mL LB culture medium, cultured at 37 ℃ until the OD600 is about 0.7, and precooled to 20 ℃.

An IPTG inducer was added to a final concentration of 0.2mM, followed by culturing in a shaker at 20 ℃ for 20 h.

The cultured broth was centrifuged and washed three times with 50mM Tris-His buffer (containing 100mM NaCl) at pH 8.0

The washed bacteria were resuspended in 50mM Tris-His buffer (containing 100mM NaCl) at pH 8.0 and lysed by sonication. Taking out the cracked broken bacteria liquid for centrifugation, and taking the supernatant for subsequent reaction.

Example 3: construction of Ribose kinase mutation library

Based on the crystal structure of ribokinase, key amino acids around the active center (14, 16, 42, 46, 143, 265, 273) were selected and mutated to alanine (a), leucine (L), phenylalanine (F), respectively, which typically represent small-medium-large volumes. The specific method comprises the following steps:

the PCR system is as follows: 10xBuffer 5. mu.L, 2mM dNTPs 5. mu.L, plasmid DNA template 1. mu.L (50 ng/. mu.L), 2. mu.L each of upstream and downstream primers (10. mu.M), high fidelity enzyme 0.5. mu.L, DMSO 1. mu.L, ddH2O 34. mu.L. The codons of the PCR primers at the mutated positions are as follows:

mutant strain	Primer sequences
		N14A	CTGGGTAGCGTTGCGGCAGATCATGTTC
N14L	CTGGGTAGCGTTCTGTGCAGATCATGTTC
		N14F	CTGGGTAGCGTTTTTGCAGATCATGTTC
D16A	GTTAATGCAGCGCATGTTCTG
		D16L	GTTAATGCACTGCATGTTCTG
D16F	GTTAATGCATTTCATGTTCTG
		G42A	GTTATTCCGGGTGCGAAAGGCGCAAATC
G42L	GTTATTCCGGGTCTGAAAGGCGCAAATC
		G42F	GTTATTCCGGGTTTTAAAGGCGCAAATC
K43A	ATTCCGGGTGGTGCGGGCGCAAATCAGGC
		K43L	ATTCCGGGTGGTCTGGGCGCAAATCAGGC
K43F	ATTCCGGGTGGTTTTGGCGCAAATCAGGC
		E143A	CTGATGCAGCTGGCGACCCCGCTGGATGG
E143L	CTGATGCAGCTGCTGACCCCGCTGGATGG
		E143F	CTGATGCAGCTGTTTACCCCGCTGGATGG
D255A	CTGCAGCCGGTGCGACCTTTAATGGTG
		D255L	CTGCAGCCGGTCTGACCTTTAATGGTG
D255F	CTGCAGCCGGTTTTACCTTTAATGGTG
		S273A	TGCCTCTGGAAGCGGCAATTAAATTTG
S273L	TGCCTCTGGAACTGGCAATTAAATTTG
		S273F	TGCCTCTGGAATTTGCAATTAAATTTG

The PCR amplification step is as follows: (1) pre-denaturation at 94 ℃ for 3 min; (2) denaturation at 98 ℃ for 10 s; (3) annealing at 64 ℃ for 30 s; (4) extending for 4min at 68 ℃; repeating the steps (2) to (3) 29 times; (5) extension was continued for 10min at 72 ℃ and cooled to 4 ℃. Add 2. mu.L of LDpnI to the PCR product and digest overnight at 37 ℃ to eliminate the plasmid template. The digested PCR product was transformed into E.coli BL21(DE3) competent cells by electroporation, and spread on LB plate containing kanamycin sulfate to obtain a library of saturation mutations at the target residue position.

Example 4: catalytic generation of 5-phosphoribosyl by engineered ribokinase

Reaction conditions are as follows: 50. mu.L of acetonitrile containing 5mg of ribose was added to 1mL of the crude enzyme solution, and the reaction was carried out at 30 ℃ for 12 hours.

Mutant strain	Yield (%)^a
		WT	71.6
N14A	10.0
		N14L	22.8
N14F	25.2
		D16A	19.8
D16L	59.2
		D16F	41.7
G42A	35.1
		G42L	69.9
G42F	87.3
		K43A	30.5
K43L	29.6
		K43F	45.9
E143A	26.3
		E143L	39.4
E143F	43.7
		D255A	23.9
D255L	41.7
		D255F	50.4
S273A	37.6
		S273L	42.1
S273F	62.5

^aThe above yields are HPLC yields.

From the above results, it was found that the reaction yield of the best mutant strain G42F was increased from 71.6% to 87.3% of the wild type.

Sequence listing

<110> Hangzhou Ling rhinoceros health science and technology Limited

<120> engineered ribokinase, coding gene, expression vector, engineering bacterium and application thereof

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Gly Thr Ala Thr Thr Ala Ala Thr Ala Thr Cys Cys Gly Thr Gly Ala

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Gly Gly Thr Ala Thr Thr Ala Ala Thr Ala Cys Ala Gly Cys Ala Gly

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Gly Thr Gly Thr Gly Ala Ala Ala Cys Thr Gly Cys Ala Gly Cys Cys

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Gly Ala Ala Thr Thr Gly Thr Cys Cys Ala Ala Cys Cys Gly Gly Thr

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Ala Thr Thr Gly Cys Gly Ala Thr Gly Ala Thr Thr Cys Ala Gly Gly

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Thr Thr Ala Gly Thr Gly Ala Thr Ala Gly Cys Gly Gly Thr Gly Ala

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Ala Ala Ala Thr Ala Gly Cys Ala Thr Thr Thr Gly Thr Ala Thr Thr

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Ala Gly Cys Gly Cys Ala Gly Ala Ala Gly Cys Ala Ala Ala Thr Gly

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Cys Thr Ala Ala Ala Cys Thr Gly Ala Cys Cys Gly Cys Ala Gly Cys

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Ala Gly Cys Ala Ala Thr Thr Gly Ala Ala Cys Cys Gly Gly Ala Thr

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Cys Thr Gly Gly Cys Ala Gly Cys Cys Ala Thr Thr Cys Gly Thr Gly

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Ala Thr Gly Cys Ala Cys Gly Cys Thr Ala Thr Cys Thr Gly Cys Thr

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Gly Ala Thr Gly Cys Ala Gly Cys Thr Gly Gly Ala Ala Ala Cys Cys

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Thr Gly Ala Ala Ala Gly Cys Ala Gly Cys Ala Cys Ala Gly Gly Ala

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Ala Cys Cys Ala Ala Thr Gly Thr Ala Ala Thr Cys Cys Thr Gly Ala

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Ala Thr Cys Cys Gly Gly Cys Ala Cys Cys Gly Gly Cys Ala Cys Gly

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Cys Gly Ala Ala Cys Thr Gly Cys Cys Gly Gly Ala Thr Gly Ala Ala

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Cys Thr Gly Cys Thr Gly Ala Ala Ala Thr Gly Thr Gly Thr Ala Gly

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Ala Thr Cys Thr Gly Ala Thr Cys Ala Cys Cys Cys Cys Gly Ala Ala

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Thr Gly Ala Ala Ala Cys Cys Gly Ala Ala Gly Cys Ala Gly Ala Ala

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Gly Thr Cys Cys Thr Gly Ala Cys Cys Gly Gly Cys Ala Thr Thr Ala

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Cys Cys Gly Thr Cys Thr Ala Thr Gly Ala Thr Gly Ala Thr Ala Gly

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Cys Thr Cys Thr Gly Cys Ala Cys Ala Gly Cys Ala Gly Gly Cys Ala

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Gly Cys Cys Gly Ala Thr Gly Cys Ala Cys Thr Gly Cys Ala Thr Thr

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Gly Thr Ala Ala Ala Gly Gly Thr Ala Thr Thr Gly Ala Ala Ala Thr

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Thr Gly Thr Thr Ala Thr Cys Ala Thr Cys Ala Cys Cys Cys Thr Gly

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Gly Gly Thr Ala Gly Cys Ala Ala Ala Gly Gly Thr Gly Thr Thr Thr

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Gly Gly Cys Thr Gly Ala Gly Thr Cys Ala Gly Ala Ala Cys Gly Gly

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Thr Cys Gly Cys Gly Gly Cys Cys Ala Gly Cys Gly Cys Ala Thr Thr

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Cys Cys Gly Gly Gly Thr Thr Thr Thr Gly Thr Thr Gly Thr Thr Ala

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Ala Ala Gly Cys Ala Ala Cys Ala Gly Ala Thr Ala Cys Cys Ala Cys

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Thr Gly Cys Ala Gly Cys Cys Gly Gly Thr Gly Ala Thr Ala Cys Cys

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Thr Thr Thr Ala Ala Thr Gly Gly Thr Gly Cys Cys Cys Thr Gly Gly

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Thr Thr Ala Cys Thr Gly Gly Thr Cys Thr Gly Cys Thr Gly Cys Ala

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Gly Gly Ala Ala Ala Thr Gly Cys Cys Thr Cys Thr Gly Gly Ala Ala

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Ala Gly Cys Gly Cys Ala Ala Thr Thr Ala Ala Ala Thr Thr Thr Gly

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Cys Ala Cys Ala Thr Gly Cys Ala Gly Cys Ala Gly Cys Cys Gly Cys

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Ala Ala Thr Thr Ala Gly Cys Gly Thr Thr Ala Cys Cys Cys Gly Thr

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Thr Thr Thr Gly Gly Cys Gly Cys Ala Cys Ala Gly Ala Cys Cys Thr

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Cys Ala Ala Thr Thr Cys Cys Gly Ala Cys Gly Cys Gly Thr Gly Cys

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Ala Gly Ala Ala Gly Thr Thr Gly Ala Ala Gly Cys Ala Thr Thr Thr

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Cys Thr Gly Gly Cys Ala Gly Ala Ala Cys Ala Thr Thr Cys Cys Thr

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Ala Ala

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