Patent Application
20190153407
The present invention relates to technical field of molecular biology and biotechnology, and particularly a nicotinamide phosphoribosyltransferase (Nampt) mutant artificially obtained through Site-directed mutagenesis.
Nampt, also known as visfatin or pre-B cell colony enhancer factor (PBEF), is a protein indispensable for the life activities of mammalians that is widely distributed in various tissues, particularly in adipose tissue, liver, muscle and bone marrow of human.
At present, Nampt is considered to have mainly the following 3 functions. 1. Nampt functions to have an enzyme activity, by which nicotinamide and 5′-phosphoribosyl-1′-pyrophosphate (PRPP) are catalyzed to convert into nicotinamide mononucleotide (NMN) and pyrophosphate, and NMN is then catalytically converted, by nicotinamide mononucleotide adenylyltransferase (Nmnat), into nicotinamide adenine dinucleotide (NAD), a energy substance essential to the cells. 80% of NAD in the mammalian cells is synthesized through this biosynthetic pathway, and Nampt is the rate limiting enzyme of the pathway, thus regulating the intracellular level of NAD. 2. Nampt functions as a pre-B cell colony enhancer factor (PBEF) secreted by lymphocytes, which stimulates the formation of B cell precursors in the presence of interleukin-7 (IL-7) and stem cell factor (SCF). 3. Nampt functions as an adipokine secreted by adipose cells and is involved in various biological functions. In 2005, Fukuhara et al. found that Nampt can activate the insulin receptor signaling by direct interaction with the insulin receptor. Since Nampt is highly expressed in visceral adipocytes, it is referred to as Visfatin.
With the increasing insight into the medicinal and health care effects of NMN, the market demand for NMN grows increasingly. The most environmentally friendly and pollution free method for producing NMN is a biocatalytic method in which nicotinamide and 5′-phosphoribosyl-1′-pyrophosphate (PRPP) are catalytically converted into NMN by Nampt. However, due to the low enzyme activity of the existing wild-type Nampt, the yield is low when Nampt is used in large-scale industrial catalytic production of NMN. As a result, the production cost is high, and the product is less competitive in the market, which severely restrict the industrial application of the biocatalytic production technology of NMN
Therefore, increasing the catalytic activity of Nampt is a key factor in reducing the biocatalytic synthesis cost of NMN, increasing the industrial application value of Nampt, and promoting the application of biocatalytic technology in industrial production of NMN.
In view of the problems of low catalytic activity and low industrial application value of the existing Nampt mentioned in the background art, an object of the present invention is to provide a Nampt mutant having a catalytic activity which is higher than that of a conventional wild type parent, by which nicotinamide and PRPP are efficiently catalyzed to convert into NMN, thus greatly reducing the cost for biocatalytic production of NMN in the industry. Therefore, the mutant has a high industrial application value.
In order to achieve the above object, site-directed mutagenesis is made by the present inventors to the gene of parent Nampt having a nucleotide sequence as shown in SEQ ID NO: 1, and the gene is then amplified by PCR, inserted into an appropriate vector, and then screened in an LB medium, to obtain a series of Nampt mutants having high catalytic activity. Accordingly, the present invention provides a Nampt mutant, which is a protein of (a) or (b) shown below:
(a) a protein having an amino acid sequence as shown in SEQ ID NO: 3; and
(b) a protein derived from (a) by substitution, deletion, or insertion of one or more amino acids in the amino acid sequence as defined in (a), and having Nampt catalytic activity for the substrates nicotinamide and PRPP that is higher than the parent having an amino acid sequence as shown in SEQ ID NO: 2.
The parent as used herein refers to Nampt derived from Meiothermus ruber DSM 1279, the nucleotide sequence is as shown in SEQ ID NO: 1 and the amino acid sequence is as shown in SEQ ID NO: 2.
Preferably, the mutant has at least one mutation at at least one position selected from positions 180, 182, 231, 298, 338, and 377, compared with the amino acid sequence as shown in SEQ ID NO: 2.
More preferably, the mutant has at least one of the mutations F180A, F180W, A182Y, E231A, E231Q, D298A, D298N, D298E, D338N, D338E, D377A, D377N, and D377E.
The present invention further provides a nucleotide sequence encoding the Nampt mutant.
The present invention further provides an expression vector comprising the nucleotide sequence.
The present invention further provides a viable cell transformed with the nucleotide sequence and capable of expressing and secreting the Nampt mutant.
The present invention further provides use of the Nampt mutant in a process for preparing nicotinamide mononucleotide, where the process may be a biocatalytic process or a fermentation process.
The biocatalytic process for the preparation of nicotinamide mononucleotide (NMN) described above specifically refers to a process in which the substrates are catalyzed to convert into NMN by a biological enzyme, where the biological enzymes is the Nampt mutant according to the present invention or a combination of the Nampt mutant according to the present invention with one or more other enzymes; the substrates may be PRPP and nicotinamide or precursors able to be converted into PRPP or nicotinamide.
For example, nicotinamide and PRPP are used as raw materials for catalytically preparing NMN in the presence of the Nampt mutant of the present invention;
nicotinamide, ATP, and xylose are used as raw materials for catalytically preparing NMN in the presence of the Nampt mutant of the present invention, ribose phosphate pyrophosphokinase, ribose-5-phosphate isomerase, ribulose-3-phosphate isomerase, xylulose kinase and xylose isomerase;
nicotinamide, ATP, and ribose are used as raw materials for catalytically preparing NMN in the presence of the Nampt mutant of the present invention, ribose phosphate pyrophosphokinase, and ribokinase;
nicotinamide, pyrophosphate or a salt thereof and AMP are used as raw materials for catalytically preparing NMN in the presence of the Nampt mutant of the present invention and adenine phosphoribosyltransferase;
nicotinamide, ATP, and AMP are used as raw materials for catalytically preparing NMN in the presence of the Nampt mutant of the present invention, ribose phosphate pyrophosphokinase, and AMP nucleosidase; and
nicotinamide, pyrophosphate or a salt thereof and inosinic acid or a salt thereof are used as raw materials for catalytically preparing NMN in the presence of the Nampt mutant of the present invention, hypoxanthine phosphoribosyltransferase, and xanthine oxidase.
Preferably, the Nampt mutant of the present invention is used in the form of an enzyme solution, an enzyme lyophilized powder, enzyme-containing cells, an immobilized enzyme, or immobilized enzyme-containing cells.
Beneficial Effect:
Compared with the existing wild-type Nampt, the Nampt mutant provided in the present invention has a considerably increased enzymatic activity. As shown in an enzyme activity assay with nicotinamide and PRPP as substrates, the enzymatic activity of the
Narnpt mutant provided in the present invention is 1.2-6.9 times of the enzymatic activity of the parent. Such a high catalytic activity allows the mutant to be used in the form of a crude enzyme without purification, or be used merely after partial purification. This results in a greatly reduced cost in the catalytic production of NMN by using the Nampt mutant provided in the present invention, thus bringing about high market competitiveness, and enabling the method for producing NMN by biocatalysis to be applicable to large-scale industrial production.
The present invention will be described in further detail with reference to specific examples. The following examples are illustrative of the present invention and the present invention is not limited thereto. Where no specific conditions are given in the examples, conventional conditions or conditions recommended by a manufacturer are adopted.
A process for preparing the Nampt mutant provided in the present invention was substantially as follows. A plasmid vector containing parent Nampt gene was constructed. Then a site for site-directed mutation and the type of the amino acid after mutation were determined. Suitable primers were synthesized, DNA fragments were amplified by PCR using the plasmid vector containing parent Nampt gene as a template, the amplified DNA fragments were assembled, and the full-length mutant gene was amplified by PCR. Then, the full-length mutant gene was cloned onto a suitable vector, then transformed into suitable host cells, and incubated, to screen out positive clones having Nampt activity. Plasmid DNA was extracted from the positive clones, and sequenced, to determine the mutation introduced. After a fragment of interest is determined to be inserted into the vector, the clones were screened in a LB+ Kanamycin medium, to obtain a series of Nampt mutants having high catalytic activity.
In the preparation method, any suitable vectors may be used, for example, prokaryotic expression vectors such as pRSET, pES21, and the like; and cloning vectors such as pUC18/19 and pBluscript-SK. In the present invention, pRSET-A is preferably used as a vector. The host cell to which the vector is transferred may be a prokaryotic cell including Escherichia coli or an eukaryotic cell including Saccharomyces cerevisiae and Pichia pastoris.
For the enzymes used in the following examples, except that the Nampt mutant is obtained through artificially induced site-directed mutation of parent Nampt gene derived from Meiothermus ruber DSM 1279 and having a nucleotide sequence as shown in SEQ ID NO: 1, the remaining enzymes are all enzyme lyophilized powders directly purchased from the market.
Whole sequence artificial synthesis was performed on the parent Nampt gene sequence publicized in the Genebank (GenBank Accession No.: CP001743.1) derived from Meiothermus ruber DSM 1279 (by a commercial synthesis company). The synthesized product was enzymatically cleaved by the restriction endonucleases NdeI and BamHI, and then ligated to the vector pRSET-A (available from Invitrogen, USA) that was also enzymatically cleaved by the restriction endonucleases NdeI and BamHI, to obtain plasmid pRSET-nampt. After DNA sequencing, it is determined that the nucleotide sequence of the cloned parent Nampt gene is as shown in SEQ ID NO: 1, and the amino acid sequence is as shown in SEQ ID NO: 2.
PCR amplification reaction system: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 1.5 U Pfu DNA polymerase (Promega, USA), 20 ng DNA template, and 400 nM upstream primer, and 400 nM downstream primer, where the reaction volume was adjusted to 50 μl with sterile water.
PM amplification reaction conditions: 3 min at 95° C.; 35 cycles of: 50 s at 95° C., 30 s at 52° C., and 3 min at 72° C.; and finally 5 min at 72° C.
1. Preparation of F180A Mutant
The primer pair SEQ ID NO: 4_F180A-F: 5′ GTTCAAACTGCACGACGCGGGTGCTCGTGGTGTTTC 3′ and SEQ ID NO: 5_F180A-R: 5′ GAAACACCACGAGCACCCGCGTCGTGCAGTTTGAAC 3′ were used. The plasmid pRSET-nampt constructed in Example 1 was used as a template. The F180A mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-F180A. The plasmid pRSET-F180A was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-F180A DNA was extracted from the clones, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the F180A mutant is as shown in SEQ ID NO: 3, and has a mutation of Phe (F) to Ala (A) at position 180.
2. Preparation of F180W Mutant
The primer pair SEQ ID NO: 6_F180W-F: 5′ GTTCAAACTGCACGACTGGGGTGCTCGTGGTGTTTC 3′ and SEQ ID NO: 7_F180W-R: 5′ GAAACACCACGAGCACCCCAGTCGTGCAGTTTGAAC 3′ were used. The plasmid pRSET-nampt constructed in Example 1 was used as a template. The F180W mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-F180W. The plasmid pRSET-F180W was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-F180W DNA was extracted from the clones, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the F180W mutant as shown in SEQ ID NO: 30 has a mutation of Phe (F) to Trp (W) at position 180.
3. Preparation of A182Y mutant
The primer pair SEQ ID NO: 8_A182Y-F: 5′ CAAACTGCACGACTTCGGTTATCGTGGTGTTTCTTCTCTG 3′ and SEQ ID NO: 1_A182Y-R: 5′ CAGAGAAGAAACACCACGATAACCGAAGTCGTGCAGTTTG 3′ were used. The plasmid pRSET-nampt constructed in Example 1 was used as a template. The A182Y mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-A182Y. The plasmid pRSET-A182Y was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-A182Y DNA was extracted from the clones, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of A182Y mutant as shown in SEQ ID NO: 31 has a mutation of Ala (A) to Tyr (Y) at position 182.
4. Preparation of E231A Mutant
The primer pair SEQ ID NO: 10_E231A-F: 5′ CTATCCCGGCTATGGCGCACTCTACCGTTAC 3′ and SEQ ID NO: 11_E231A-R: 5′ GTAACGGTAGAGTGCGCCATAGCCGGGATAG 3′ were used. The plasmid pRSET-nampt constructed in Example 1 was used as a template. The E231A mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-E231A. The plasmid pRSET-E231A was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-E231A DNA was extracted from the clones, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the E231A mutant as shown in SEQ ID NO: 32 has a mutation of Glu (E) to Ala (A) at position 231.
5. Preparation of E231Q Mutant
The primer pair SEQ ID NO: 12 E231Q-F: 5′ CTCTATCCCGGCTATGCAGCACTCTACCGTTACC 3′ and SEQ ID NO: 13_E231Q-R: 5′ GGTAACGGTAGAGTGCTGCATAGCCGGGATAGAG 3′ were used. The plasmid pRSET-nampt constructed in Example 1 was used as a template. The E231Q mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-E231Q. The plasmid pRSET-E231Q was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-E231Q DNA was extracted from the clones, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of E231 Q mutant as shown in SEQ ID NO: 33 has a mutation of Glu (E) to Gln (Q) at position 231.
6. Preparation of D298A Mutant
The primer pair SEQ ID NO: 14_D298A-F: 5′ TATCCGTCCGGCGTCTGGTGACCC 3′ and SEQ ID NO: 15_D298A-R: 5′ GGGTCACCAGACGCCGGACGGATA 3′ were used. The plasmid pRSET-nampt constructed in Example 1 was used as a template. The D298A mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-D298A. The plasmid pRSET-D298A was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-D298A DNA was extracted from the clones, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the D298A mutant as shown in SEQ ID NO: 34 has a mu to on of Asp (D) to Ala (A) at position 298.
7. Preparation of D298N Mutant
The primer pair SEQ ID NO: 16_D298N-F: 5′ GTTGTTATCCGTCCGAATTCTGGTGACCCGCCG 3′ and SEQ ID NO: 17_D298N-R: 5′ CGGCGGGTCACCAGAATTCGGACGGATAACAAC 3′ were used. The plasmid pRSET-nampt constructed in Example 1 was used as a template. The D298N mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-D298N. The plasmid pRSET-D298N was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-D298N DNA was extracted from the clones, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the D298N mutant as shown in SEQ ID NO: 35 has a mutation of Asp (D) to Asn (N) at position 298.
8. Preparation of D298E Mutant
The primer pair SEQ ID NO: 18_D298E-F: 5′ GTTGTTATCCGTCCGGAATCTGGTGACCCGCCGTTC 3′ and SEQ ID NO: 19_D298E-R: 5′ GAACGGCGGGTCACCAGATTCCGGACGGATAACAAC 3′ were used. The plasmid pRSET-nampt constructed in Example 1 was used as a template. The D298E mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-D298E. The plasmid pRSET-D298E was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-D298E DNA was extracted from the clones, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the D298E mutant as shown in SEQ ID NO: 36 has a mutation of Asp (D) to Glu (E) at position 298.
9. Preparation of D338N Mutant
The primer pair SEQ NO: 20_D338N-F: 5′ GTTCGTGTTATCCAGGGTAATGGTGTTAACGCTGACTC 3′ and SEQ ID NO: 21_D338N-R: 5′ GAGTCAGCGTTAACACCATTACCCTGGATAACACGAAC 3′ were used. The plasmid pRSET-nampt constructed in Example 1 was used as a template. The D338N mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-D338N. The plasmid pRSET-D338N was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-D338N DNA was extracted from the clones, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the D338N mutant as shown in SEQ ID NO: 37 has a mutation of Asp (D) to
Asn (N) at position 338.
10. Preparation of D338E Mutant
The primer pair SEQ ID NO: D338E-F: 5′ GTTATCCAGGGTGAAGGTGTTAACGCTGAC 3′ and SEQ ID NO: 23_D338E-R: 5′ GTCAGCGTTAACACCTTCACCCTGGATAAC 3′ were used. The plasmid pRSET-nampt constructed in Example 1 was used as a template. The D338E mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-D338E. The plasmid pRSET-D338E was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-D338E DNA was extracted from the clones, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the D338E mutant as shown in SEQ ID NO: 38 has a mutation of Asp (D) to Glu (E) at position 338.
11. Preparation of D377A Mutant
The primer pair SEQ NO: 24_D377A-F: 5′ CACCCGCACCGTGCGACCCAGAAATTC 3′ and SEQ ID NO: 25_D377A-R: 5′ GAATTTCTGGGTCGCACGGTGCGGGTG 3′ were used. The plasmid pRSET-nampt constructed in Example 1 was used as a template. The D377A mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-D377A. The plasmid pRSET-D377A was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-D377A DNA was extracted from the clones, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the D377A mutant as shown in SEQ ID NO: 39 has a mutation of Asp (D) to Ala (A) at position 377.
12. Preparation of D377N Mutant
The primer pair SEQ ID NO: 26_D377N-F: 5′ GCAACACCCGCACCGTAATACCCAGAAATTCGCTC 3′ and SEQ ID NO: 27_D377N-R: 5′ GAGCGAATTTCTGGGTATTACGGTGCGGGTGTTGC 3′ were used. The plasmid pRSET-nampt constructed in Example 1 was used as a template. The D377N mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-D377N. The plasmid pRSET-D377N was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-D377N DNA was extracted from the clones, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the D377N mutant as shown in SEQ ID NO: 40 has a mutation of Asp (D) to Asn (N) at position 377.
13. Preparation of D377E Mutant
The primer pair SEQ ID NO: 28_D377E-F: 5′ CCCGCACCGTGAAACCCAGAAATTCG 3′ and SEQ ID NO: 29_D377E-R: 5′ CGAATTTCTGGGTTTCACGGTGCGGG 3′ were used. The plasmid pRSET-nampt constructed in Example 1 was used as a template. The D377E mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-D377E. The plasmid pRSET-D377E was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-D377E DNA was extracted from the clones, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the D377E mutant as shown in SEQ ID NO: 41 has a mutation of Asp (D) to Glu (E) at position 377.
14. Preparation of E231Q/D338E Mutant
The primer pair SEQ ID NO: 22_D338E-F: 5′ GTTATCCAGGGTGAAGGTGTTAACGCTGAC 3′ and SEQ ID NO: 23_D338E-R: 5′ GTCAGCGTTAACACCTTCACCCTGGATAAC 3′ were used. The plasmid pRSET-E231Q constructed in Section 5 in Example 2 was used as a template. The E231Q/D338E mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-21. The plasmid pRSET-21 was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-21 DNA was extracted from the clone, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the E231Q/D338E mutant as shown in SEQ D NO: 42 has a mutation of Glu (E) to Gln (Q) at position 231, and a mutation of Asp (D) to Glu (E) at position 338.
15. Preparation of E231Q/D377E Mutant
The primer pair SEQ ID NO: 28_D377E-F: 5′ CCCGCACCGTGAAACCCAGAAATTCG 3′ and SEQ ID NO: 29_D377E-R: 5′ CGAATTTCTGGGTTTCACGGTGCGGG 3′ were used. The plasmid pRSET-E231Q constructed in Section 5 in Example 2 was used as a template. The E231Q/D377E mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-22. The plasmid pRSET-22 was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-22 DNA was extracted from the clone, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the E231Q/D377E mutant as shown in SEQ ID NO: 43 has a mutation of Glu (E) to Gln (Q) at position 231 and a mutation of Asp (D) to Glu (E) at position 377.
16. Preparation of D338E/D377E Mutant
The primer pair SEQ ID NO: 28_D377E-F: 5′ CCCGCACCGTGAAACCCAGAAATTCG 3′ and SEQ ID NO: 29_D377E-R: 5′ CGAATTTCTGGGTTTCACGGTGCGGG 3′ were used. The plasmid pRSET-D338E constructed in Section 10 in Example 2 was used as a template. The D338E/D377E mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-23. The plasmid pRSET-23 was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-23 DNA was extracted from the clone, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the D338E/D377E mutant as shown in SEQ ID NO: 44 has a mutation of Asp (D) to Glu (E) at position 338 and a mutation of Asp (D) to Glu (E) at position 377.
17. Preparation of E231Q/D338E/D377E Mutant
The primer pair SEQ ID NO: 28_D377E-F: 5′ CCCGCACCGTGAAACCCAGAAATTCG 3′ and SEQ ID NO: 29_D377E-R: 5′ CGAATTTCTGGGTTTCACGGTGCGGG 3′ were used. The plasmid pRSET-21 constructed in Section 14 in Example 2 was used as a template. The E231Q/D338E/D377E mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-31. The plasmid pRSET-31 was transformed into competent bacterial cells E. coli BL21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-31 DNA was extracted from the clone, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the E231Q/D338E/D377E mutant as shown in SEQ ID NO: 45 has a mutation of Glu (E) to Gln (Q) at position 231, a mutation of Asp (D) to Glu (E) at position 338, and a mutation of Asp (D) to Glu (E) at position 377.
18. Preparation of E231Q/D298A/D338E/D377E Mutant
The primer pair SEQ ID NO: 14_D298A-F: 5′ TATCCGTCCGGCGTCTGGTGACCC 3′ and SEQ ID NO: 15_D298A-R: 5′ GGGTCACCAGACGCCGGACGGATA 3′ were used. The plasmid pRSET-31 constructed in Section 17 in Example 2 was used as a template. The E231Q/D298A/D338E/D377E mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions. The amplified product was isolated by electrophoresis on 1% agarose gel, recovered using a commercial kit, and ligated to the vector pRSET-A (See Example 1 for details) to obtain plasmid pRSET-41. The plasmid pRSET-41 was transformed into competent bacterial cells E. coli B21, and clones having Nampt activity were screened out on a Luria broth (LB) plate (containing 50 mg/L Kanamycin). Plasmid pRSET-41 DNA was extracted from the clone, and sequenced to determine that the point mutation introduced was correct. Compared with the parent amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of the E231Q/D298A/D338E/D377E mutant as shown in SEQ ID NO: 46 has a mutation of Glu (E) to Gln (Q) at position 231, a mutation of Asp (D) to Ala (A) at position 298, a mutation of Asp (D) to Glu (E) at position 338, and a mutation of Asp (D) to Glu (E) at position 377.
The plasmid pRSET-nampt containing parent Nampt gene and the plasmid pRSET-F80A, pRSET-F180W, pRSET-A182Y, pRSET-E231A, pRSET-E231Q, pRSET-D298A, pRSET-D298N, pRSET-D298E, pRSET-D338N, pRSET-D338E, pRSET-D377A, pRSET-D377N, pRSET-D377E, pRSET-21, pRSET-22, pRSET-23, pRSET-31, and pRSET-41 containing Nampt mutant genes were respectively transformed into competent bacterial cells E. coli BL21, and incubated for 24 hrs on a Luria broth (LB) plate (containing 50 mg/L Kanamycin) at 37° C. Individual clones were inoculated in 50 ml of LB liquid medium (containing 50 mg/L Kanamycin), and incubated for 16-20 hrs at 30° C. The bacterial cells were collected by centrifugation, and the same amount of cells were weighed and suspended in a cell lysis buffer (pH 7.5) at a ratio of 1:4. The bacterial cells were ultrasonically lyzed. After centrifugation (4-10° C., 12000 rpm, 10 min), the supernatant was collected, that is, the protein supernatant of parent Nampt and a series of Nampt mutants was obtained respectively, which could be used in the enzyme activity assay and in the preparation of NMN by biocatalysis.
A substrate solution containing 60 mM nicotinamide, 25 mM PRPP, 18 mM MgCl2, 15 mM KCl, and 100 mM Tris buffer was formulated and adjusted to pH 7.5. 19 portions of the substrate solution (each 900 μl) were taken, then added respectively to 100 μl of equal concentration of the protein supernatant of parent Nampt and a series of Nampt mutants obtained in Example 3, and reacted for 10 min at 37° C. The reaction was terminated by adding 100 μL of 25% trichloroacetic acid. The NMN content in the reaction solution was determined by HPLC, and the specific activity of each enzyme was calculated. Where the specific activity of parent Nampa was assumed to be 100, the relative specific activity of the parent and the mutants are as shown in Table 1.
A substrate solution containing 30 mM nicotinamide, 20 mM ATP, 30 mM xylose, 12 mM MgCl2, 10 mM KCl, 10 mM ZnCl2, and 100 mM Tris-HCl buffer was added to a reactor, and adjusted to pH 70-7.5. Then, various catalytic enzymes were added, in amounts of 10 ml of the protein supernatant of Nampt mutant (F180A) prepared in Example 3/L of substrate solution, 6 g of ribose phosphate pyrophosphokinase/L of substrate solution, 10 g of ribose-5-phosphate isomerase/L of substrate solution, 11 g of ribulose-3-phosphate isomerase/L of substrate solution, 10 g of xylulose kinase/L of substrate solution, 10 g of xylose isomerase/L of substrate solution. The system was stirred until uniform and then reacted. During the reaction process, stirring was continued (at a stirring speed of 50 rpm), the reaction temperature was controlled at 37° C., and the pH was maintained at 7.0-7.5. After 6 hrs of reaction, a crude nicotinamide mononucleotide product solution (containing 10 mM NMN) was obtained, which was filtered, purified, and dried, to obtain nicotinamide mononucleotide.
A substrate solution containing 30 mM nicotinamide, 20 mM ATP, 30 mM ribose, 15 mM MgCl2, 15 mM KCl, and 100 mM Tris-HCl buffer was added to a reactor, and adjusted to pH 7.0-7.5. Then, various catalytic enzymes were added to the substrate solution in amounts of: 10 ml of the protein supernatant of Nampt mutant (F180A) prepared in Section III of Example 6L of substrate solution, 20 g of ribose phosphate pyrophosphokinase/L of substrate solution, and 20 g of ribokinase/L of substrate solution. The system was stirred until uniform and then reacted. During the reaction process, stirring was continued (at a stirring speed of 50 rpm), the reaction temperature was controlled at 37° C., and the pH was maintained at 7.0-7.5. After 4 hrs of reaction, a crude nicotinamide mononucleotide product solution (containing 10 mM NMN) was obtained, which was filtered, purified, and dried, to obtain a product nicotinamide mononucleotide.
A substrate solution containing 75 mM nicotinamide, 75 mM disodium pyrophosphate, 50 mM AMP, 15 mM MgCl2, 10 mM KCl, and 100 mM Tris-HCl buffer was added to a reactor, and adjusted to pH 7.0-7.5. Then, various catalytic enzymes were added in amounts of: 10 ml of the protein supernatant of Nampt mutant (F180A) prepared in Section III of Example 6/L of substrate solution, and 20 g of adenine phosphoribosyltransferase/L of substrate solution. The system was stirred until uniform and then reacted. During the reaction process, stirring was continued (at a stirring speed of 50 rpm), the reaction temperature was controlled at 37° C., and the pH was maintained at 7.0-8.0. After 5 hrs of reaction, a crude nicotinamide mononucleotide product solution (containing 49.6 mM NMN) was obtained, which was filtered, purified, and dried, to obtain a product nicotinamide mononucleotide.
A substrate solution containing 60 mM nicotinamide, 10 mM ATP, 20 mM AMP, 15 MgCl2, 15 mM KCl, and 100 mM Tris-HCl buffer was added to a reactor, and adjusted to pH 7.0-7.5. Then, various catalytic enzymes were added to the substrate solution in amounts of: 10 ml of the protein supernatant of Nampt mutant (F180A) prepared in Section III of Example 6/L of substrate solution, 15 g of ribose phosphate pyrophosphokinase/L of substrate solution, and 15 g of AMP nucleosidase/L of substrate solution. The system was stirred until uniform and then reacted. During the reaction process, stirring was continued (at a stirring speed of 50 rpm), the reaction temperature was controlled at 37° C., and the pH was maintained at 7.0-7.5. After 3 hrs of reaction, a crude NMN product solution (containing 9.8 mM NMN) was obtained, which was filtered, purified, and dried, to obtain a product NMN.
A substrate solution containing 60 mM nicotinamide, 30 mM disodium pyrophosphate, 20 mM disodium inosinate, 20 mM MgCl2, 20 mM ZnCl2, 35 mM sodium bisulfite and 100 mM Tris-HCl buffer was added to a reactor, and adjusted to pH 7.0-7.5. Then, various catalytic enzymes were added to the substrate solution in amounts of: 10 ml of the protein supernatant of Nampt mutant (F180A) prepared in Section III of Example 6/L of substrate solution/L of substrate solution, 10 g of hypoxanthine phosphoribosyltransferase/L of substrate solution, 20 g of xanthine oxidase/L of substrate solution. The system was stirred until uniform and then reacted. During the reaction process, stirring was continued (at a stirring speed of 50 rpm), the reaction temperature was controlled at 37° C., and the pH was maintained at 7.0-7.5. After 5 hrs of reaction, a crude nicotinamide mononucleotide product solution (containing 19.8 mM NMN) was obtained, which was filtered, purified, and dried, to obtain a product nicotinamide mononucleotide.
This application is a continuation application of application Ser. No. 15/574,849, filed on Nov. 17, 2017, which is the national phase entry of International Application No. PCT/CN2016/092456, filed on Jul. 30, 2016. The contents of these related applications are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15574849 | Nov 2017 | US |
| Child | 16198871 | US |
