CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of Chinese Patent Application No. 202110468908.5 filed on Apr. 28, 2021. The contents of the above-identified applications are hereby incorporated by reference.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The Sequence Listing is submitted as an ASCII formatted text file via EFS-Web, with a file name of “Sequence_Listing.txt”, a creation date of Sep. 29, 2021, and a size of 12,467 bytes. The Sequence Listing filed via EFS-Web is part of the specification and is incorporated in its entirety by reference herein.

TECHNICAL FIELD

The present disclosure belongs to the technical field of genetic engineering, and in particular relates to an innovative method for improving the enzyme activity of an NMN biosynthetic enzyme Nampt.

BACKGROUND

Nicotinamide mononucleotide (NMN) is an organic molecule as well as a nucleotide, which has the functions of reversing aging and extending life.

At present, the synthesis of nicotinamide mononucleotide is realized mostly through an enzymatic reaction. Natural niacinamide phosphoribosyltransferase (Nampt) has a shortcoming of relatively low enzyme activity, which results in a high cost of a traditional enzymatic reaction, harsh reaction conditions, an unstable production process, a great difference in index among each batch of products, and low productivity of the reaction; and consequently, large-scale industrial production is hard to realize and large-scale application of NMN is restricted.

SUMMARY

To this end, an objective of the present disclosure is to provide an innovative method for improving the enzyme activity of an NMN biosynthetic enzyme Nampt, and specifically to provide a recombinant expression vector encoding a mutant protein of Nampt, recombinant bacteria and a Nampt mutant protein, in which after the Nampt mutant protein is expressed by the recombinant bacteria, the enzyme activity is significantly improved, and large-scale industrial production can be realized.

To achieve the above-mentioned objective, the present disclosure provides the following technical solution:

The present disclosure provides a mutant protein of a nicotinamide phosphoribosyltransferase Nampt, which is configured to perform a point mutation on an amino acid sequence of the nicotinamide phosphoribosyltransferase Nampt. The amino acid sequence of the nicotinamide phosphoribosyltransferase Nampt includes a sequence shown in SEQ ID NO:1; and

sites of the point mutation include: N67K, N164L, R166W, A208G, A245T, S248A, V365L or S382M.

The present disclosure further provides a recombinant expression vector including a nucleotide sequence encoding the above-mentioned mutant protein.

Preferably, a basic vector of the recombinant expression vector includes a pPSUMO vector.

The present disclosure further provides recombinant bacteria expressing the mutant protein or including the above-mentioned recombinant expression vector.

Preferably, a basic strain of the recombinant bacteria includesEscherichia coli.

The present disclosure further provides a construction method for the recombinant bacteria, including the following steps: (1) performing codon optimization on a gene encoding the amino acid sequence shown in SEQ ID NO:1, to obtain an optimized gene;

(2) performing a PCR amplification using a site-directed mutation primer and a high-fidelity enzyme respectively by taking the optimized gene as a template, to obtain amplification products;

(3) digesting the amplification products with a DpnI enzyme respectively, and then connecting to the pPSUMO vector respectively, to obtain a recombinant expression vector; and

(4) transforming the recombinant expression vector intoE. colicompetent cells respectively, and picking positive clones, to obtain the recombinant bacteria.

Preferably, a nucleotide sequence of the optimized gene in step (1) includes a sequence shown in SEQ ID NO:2.

Preferably, the site-directed mutation primers in step (2) include: site-directed mutation primers N67K-F and N67K-R directed at a point mutation of N67K, a nucleotide sequence of the N67K-F shown in SEQ ID NO:3, and a nucleotide sequence of the N67K-R shown in SEQ ID NO:4;

site-directed mutation primers N164L-F and N164L-R directed at a point mutation of N164L, a nucleotide sequence of the N164L-F shown in SEQ ID NO:5, and a nucleotide sequence of the N164L-R shown in SEQ ID NO:6;

site-directed mutation primers R166W-F and R166W-R directed at a point mutation of R166W, a nucleotide sequence of the R166W-F shown in SEQ ID NO:7, and a nucleotide sequence of the R166W-R shown in SEQ ID NO:8;

site-directed mutation primers A208G-F and A208G-R directed at a point mutation of A208G, a nucleotide sequence of the A208G-F shown in SEQ ID NO:9, and a nucleotide sequence of the A208G-R shown in SEQ ID NO:10;

site-directed mutation primers A245T-F and A245T-R directed at a point mutation of A245T, a nucleotide sequence of the A245T-F shown in SEQ ID NO:11, and a nucleotide sequence of the A245T-R shown in SEQ ID NO:12;

site-directed mutation primers S248A-F and S248A-R directed at a point mutation of S248A, a nucleotide sequence of the S248A-F shown in SEQ ID NO:13, and a nucleotide sequence of the S248A-R shown in SEQ ID NO:14;

site-directed mutation primers V365L-F and V365L-R directed at a point mutation of V365L, a nucleotide sequence of the V365L-F shown in SEQ ID NO:15, and a nucleotide sequence of the V365L-R shown in SEQ ID NO:16; and

site-directed mutation primers S382M-F and S382M-R directed at a point mutation of S382M, a nucleotide sequence of the S382M-F shown in SEQ ID NO:17, and a nucleotide sequence of the S382M-R shown in SEQ ID NO:18.

Preferably, a procedure of the PCR amplification in step (2) includes: initial denaturation at 94° C. for 2 min, denaturation at 98° C. for 10 s, annealing at 55-65° C. for 30 s, and extension at 68° C. for 4 min, 30 cycles; and extension at 68° C. for 4 min.

Preferably, picking positive clones in step (4) includes: performing a bacterial liquid PCR using Nampt-F and Nampt-R, a nucleotide sequence of the Nampt-F shown in SEQ ID NO: 23, and a nucleotide sequence of the Nampt-R shown in SEQ ID NO: 24.

The present disclosure provides a mutant protein of a nicotinamide phosphoribosyltransferase. The mutant protein is obtained by firstly analyzing a target protein Nampt using two softwares FoldX and DeepDDG, and then predicting multiple key sites influencing the enzyme functions, and finally performing the semi-rational design of the enzyme. In the examples of the present disclosure, 10 mutant strains constructed with the designed primers according to the principle of point mutation indicates by sequencing and verifying that they all mutate successfully on a given site, thereby indicating successful cloning of the 10 mutant strains. Among the 10 cloned mutants, 8 mutants have higher activity than a wild-type (E. coliDH5 α-ppsumo-Nampt) strain, and the mutant Nampt-V365L has the highest activity, with an NMN yield at 45.42 mg/L, an increase of 62% from that (28.11 mg/L) of the wild-type strain. The NMN yields of mutants Nampt-S248A, Nampt-N164L, Nampt-S382M, Nampt-A245T and Nampt-A208G are increased by 34%, 27%, 27%, 22% and 17% compared to the wild-type strain, while the NMN yields of Nampt-V467L and S155I are reduced by 53% and 31% respectively.

