CN117230038A - Mutant modified phosphoribosyl pyrophosphate synthetase and application thereof - Google Patents

Abstract

The invention provides phosphoribosyl pyrophosphate synthetase (PRS) derived from hot spring corynebacterium (Pyrobaculum calidifontis) and a mutant thereof, and application of the mutant in enzyme-catalyzed synthesis of beta-NMN.

Description

Mutant modified phosphoribosyl pyrophosphate synthetase and application thereof

Technical Field

The invention belongs to the field of artificial modification enzyme proteins, and particularly relates to mutation modification of phosphoribosyl pyrophosphate synthetase and application of a mutant in enzyme catalytic synthesis of beta-nicotinamide mononucleotide.

Background

Beta-nicotinamide mononucleotide (beta-NMN) is converted in humans by conversion to Nicotinamide Adenine Dinucleotide (NAD) ⁺ ) Playing a role. NAD (NAD) ⁺ (also called coenzyme I), which is an important coenzyme in the human body, is present in all living cells and plays a vital role in regulating cell senescence and maintaining normal functions of the body. NAD during aging ⁺ Is considered to be one of the leading causes of disease and disability, such as hearing and vision loss, cognitive and motor dysfunction, immunodeficiency, arthritis caused by autoimmune inflammatory response disorder, metabolic disorder and cardiovascular disease. Thus, supplementation with β -NMN may increase NAD in vivo ⁺ Content, thereby delaying, ameliorating, preventing various phenotypes associated with aging, or age-induced metabolic turbulenceDisorder, senile diseases, etc.

The existing synthesis method of beta-NMN mainly comprises a chemical method and an enzyme method, the cost of synthesizing beta-NMN by the chemical method is high, the environmental pollution is large, and the cost of substrate is high by mainly synthesizing nicotinamide ribose as a raw material by the enzyme method. The enzymatic synthesis of beta-NMN by using D-ribose as a raw material is limited by lower enzyme activity of phosphoribosyl pyrophosphate synthetase, and higher enzyme dosage, so that the cost is higher and industrialization is not possible.

Disclosure of Invention

In order to solve the problems, the invention provides phosphoribosyl pyrophosphate synthetase (PRS) derived from hot spring corynebacterium (Pyrobaculum calidifontis) and a mutant thereof, and application of the mutant in enzyme-catalyzed synthesis of beta-NMN.

The phosphoribosyl pyrophosphate synthetase mutant is obtained by carrying out D2G, K3D single-site mutation or D2G/K3D double-site mutation on the amino acid sequence shown in SEQ ID No. 2.

The coding genes of the single-point mutants disclosed by the invention have nucleotide sequences disclosed by SEQ ID No.3, SEQ ID No.5 or SEQ ID No.7, and also belong to the protection scope of the invention.

Another aspect of the present invention provides a recombinant expression vector or a recombinant host cell containing the gene encoding the phosphoribosyl-pyrophosphate synthetase mutant; the recombinant expression vector can be a recombinant prokaryotic expression vector or a recombinant eukaryotic vector.

In another aspect, the invention provides the use of the phosphoribosyl-pyrophosphate synthetase mutants for the enzyme-catalyzed synthesis of beta-NMN.

In another aspect, the present invention provides a method for enzymatic synthesis of β -NMN, comprising the steps of:

step one: d-ribose is catalyzed and acidified by ribose kinase to obtain D-ribose-5-phosphate;

step two: catalyzing D-ribose-5-phosphate with the phosphoribosyl-pyrophosphate synthetase mutants to obtain 5-phosphoribosyl-1-pyrophosphate;

step three: catalyzing the 5-phosphoribosyl-1-pyrophosphate with nicotinamide phosphoribosyl transferase to obtain the beta-NMN.

Preferably, the third step further comprises adding pyrophosphatase to hydrolyze byproduct pyrophosphoric acid.

Preferably, the ATP regeneration system of the enzyme-catalyzed reaction is: adenylate kinase and polyphosphate kinase are regenerated using sodium hexametaphosphate.

Preferably, the three-step reaction and ATP regeneration are reacted in a single pot.

The construction of the phosphoribosyl pyrophosphate synthetase (PRS) mutant of the invention is to obtain a protein three-dimensional structure through homologous modeling, obtain an enzyme-substrate complex structure after molecular docking with a substrate, select sites according to amino acid residues nearby the substrate, generate a gene variant library (Methods for the directed evolution of proteins. Nat Rev Genet,2015,16 (7): 379-394) from the plasmid construct by utilizing the directed evolution technology generally known to the skilled person, and obtain the dominant mutant through enzyme activity screening.

Drawings

Fig. 1: the invention relates to a flow chart of an enzyme catalytic synthesis method of beta-NMN

Detailed Description

The present invention can be achieved by the following specific embodiments, but the present invention is not limited to the following examples.

Example 1: gene cloning and construction of expression vectors

The amino acid sequences of wild-type phosphoribosyl-pyrophosphate synthases (PRSs) (from hot spring corynebacterium (Pyrobaculum calidifontis), SEQ ID No. 2), nicotinamide phosphoribosyl-transferase (NAMPRT) (from Mannheimia sp.) (SEQ ID No. 10), ribokinase (RK, from Escherichia coli), SEQ ID No. 12), pyrophosphorohydrolase (PPA, SEQ ID No. 14), adenylate kinase (AK, from Acinetobacter (Acinetobacter johnsonii), SEQ ID No. 16) and polyphosphokinase (PPK, from Acinetobacter (Acinetobacter johnsonii)), SEQ ID No. 18) can be obtained in NCBI databases, pET30a expression plasmids containing the gene of interest can be constructed by gene synthesis and ordinary molecular cloning means, wherein the nucleic acid sequence of PRS is SEQ ID No.1, NAMPRT is SEQ ID No.9, the nucleic acid sequence of RK is SEQ ID No.11, the nucleic acid sequence of PPA is SEQ ID No.13, the nucleic acid sequence of AK is SEQ ID No.15, and the nucleic acid sequence of AK is SEQ ID No.17. The recombinant plasmid is transformed into E.coli BL21 (DE 3) cells to obtain recombinant bacteria. A library of gene variants was generated from this plasmid construct using directed evolution techniques generally known to those skilled in the art (Methods for the directed evolution of proteins. Nat Rev Genet,2015,16 (7): 379-394).

Example 2: recombinant enzyme expression

The recombinant strain of example 1 was inoculated into LB medium (peptone 10g/L, yeast powder 5g/L, naCl 10g/L, pH 7.0) containing 50. Mu.g/mL kanamycin, and cultured overnight at 37 ℃. The overnight culture was transferred to TB medium (peptone 12g/L, yeast extract 24g/L, glycerol 4mL/L, potassium dihydrogen phosphate 2.31g/L, dipotassium hydrogen phosphate 12.54 g/L) and incubated at 37℃to OD ₆₀₀ To 0.6-0.8, IPTG was added at a final concentration of 0.4mM and expression was induced overnight at 25 ℃. After cells were collected by centrifugation, they were resuspended in 20mM phosphate buffer pH7.0, disrupted by sonication, centrifuged, and the supernatant was either reacted or frozen at-20 ℃.

Example 3: high throughput cell culture and assay

Transformed E.coli cells were selected by plating on LB agar plate medium containing 50. Mu.g/mL kanamycin. After overnight incubation at 37 degrees, colonies were placed into wells of a 96-well shallow well plate filled with 100. Mu.L/well LB medium containing 50. Mu.g/mL kanamycin. Cultures were grown overnight in a shaker for 18 hours (250 rpm,30 degrees and 85% relative humidity, kuhner). Overnight grown samples (10. Mu.L) were transferred to 96-well deep well plates filled with 180. Mu.L of TB medium containing 50. Mu.g/mL kanamycin. Plates were incubated at 30℃for 2 hours, then induced with addition of IPTG at a final concentration of 0.4mM, and incubated in a shaker at 25℃overnight for 20 hours. Cells were collected by centrifugation, and 300. Mu.L of lysate (20 mM phosphate buffer pH7.0,1mg/mL lysozyme and 0.5mg/mL polymyxin B sulfate) was added to the cells. The mixture was shaken at room temperature for 2h, centrifuged and the enzymatic activity of the supernatant was determined.

Phosphoribosyl cokeScreening method of Phosphosynthase (PRS) mutants: d-ribose, nicotinamide, adenosine Triphosphate (ATP) and sodium hexametaphosphate are used as raw materials, beta-nicotinamide mononucleotide (beta-NMN) is generated through enzyme catalytic reaction, and the generation amount of the beta-NMN is detected through liquid chromatography. The reaction system was 300. Mu.L and contained 10g/L D-ribose, 9.76g/L nicotinamide, 44.88g/L sodium hexametaphosphate, 0.67mM ATP, 100mM MgCl ₂ 1% ribokinase, 1% pyrophosphorohydrolase, 5% nicotinamide phosphoribosyl transferase, 1% adenylate kinase, 1% polyphosphate kinase, phosphoribosyl pyrophosphate synthetase mutant well plate enzyme solution 30. Mu.l and 0.2mol/L Tris-HCl pH8.5, and each reaction is added into 96 well plates and subjected to shaking reaction at 35℃for 30min. After the completion of the reaction, 100. Mu.l of the reaction mixture was taken, 400. Mu.l of acetonitrile was added to terminate the reaction, and 500. Mu.l of 0.085% H was added ₃ PO ₄ Mixing, passing through membrane, and loading into HPLC.

Example 4: HPLC analysis method

High throughput screening of liquid phase method: using Agilent SB-AQ 4.6mm by 250mm by 5 μm; the mobile phase was buffer A (50 mM KH) ₂ PO ₄ ): acetonitrile=100:0 (0-4 min), 100:0→70:30 (4-8 min), 70:30→100:0 (8-9 min), 100:0 (9-13 min), detection wavelength 254nm, flow rate 1.0ml/min, column temperature 30 ℃. NMN (beta-nicotinamide mononucleotide) retention time is about 3.8min, and NAM (nicotinamide) retention time is about 10.8 min.

Example 5: assessment of phosphoribosyl pyrophosphate synthetase (PRS) for beta-nicotinamide mononucleotide synthesis

The activity of wild-type phosphoribosyl pyrophosphate synthetase and of a plurality of mutants based on Seq ID No.2 is evaluated as described in example 3. Table 1 shows the results of the improvement of the activity of the mutant corresponding to SEQ ID NO.2 of wild type phosphoribosyl pyrophosphate synthetase.

TABLE 1

SEQ ID No.	From the mutation or species source of SEQ ID No.2	Activity(s)
			2	Pyrobaculum calidifontis
4	D2G	+
			6	K3D	++
8	D2G，K3D	+++

^a +: 1-1.5 times higher than PRS enzyme activity with SEQ ID No. 2; ++: 1.5-2 times higher than PRS enzyme activity with SEQ ID No. 2; ++ is 2-3 times higher than PRS enzyme activity with SEQ ID No. 2.

Example 6: preparation of beta-nicotinamide mononucleotide and Large Scale production

Into a 3L reaction flask, 10g D-ribose, 9.74g nicotinamide, 0.366g ATP,44.755g sodium hexametaphosphate, and 20.3g MgCl were added sequentially ₂ *6H ₂ O, adding 500mL0.1M pH9.0 TrisHCl buffer solution, stirring for dissolving, adding 1% ribose kinase, 1% pyrophosphorohydrolase, 5% nicotinamide phosphoribosyl transferase, 1% adenylate kinase, 1% polyphosphate kinase and 3% phosphoribosyl pyrophosphate synthetase (PRS) (SEQ ID No. 2), finally, using pure water to fix the volume to 1L, stirring at 35 ℃ for reaction overnight, controlling the pH value to 8-8.5 by NaOH, and finally obtaining the final molar yield of 63%.

Example 7: preparation of beta-nicotinamide mononucleotide and Large Scale production

Into a 3L reaction flask, 10g D-ribose, 9.74g nicotinamide, 0.366g ATP,44.755g sodium hexametaphosphate, and 20.3g MgCl were added sequentially ₂ *6H ₂ O, adding 500mL0.1M pH9.0 TrisHCl buffer solution, stirring for dissolving, adding 1% ribose kinase, 1% pyrophosphorohydrolase, 5% nicotinamide phosphoribosyl transferase, 1% adenylate kinase, 1% polyphosphate kinase and 3% phosphoribosyl pyrophosphate synthetase (PRS) mutant SEQ ID No.8, finally, using pure water to constant volume to 1L, stirring at 35 ℃ for reaction overnight, controlling pH to 8-8.5 by NaOH, and finally obtaining the final molar yield of 88%.

The above-described embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. All insubstantial changes and modifications made by those skilled in the art based on the teachings herein are intended to be within the scope of the teachings herein as claimed.

Claims (10)

1. A phosphoribosyl pyrophosphate synthetase mutant is obtained by carrying out D2G, K3D single-site mutation or D2G/K3D double-site mutation on the amino acid sequence shown in SEQ ID No. 2.

2. The phosphoribosyl-pyrophosphate synthetase mutant encoding gene according to claim 1, which has the nucleotide sequences as shown in SEQ ID No.3, SEQ ID No.5 and SEQ ID No. 7.

3. A recombinant expression vector or recombinant host cell comprising a gene encoding the phosphoribosyl pyrophosphate synthetase mutant of claim 2.

4. The recombinant expression vector of claim 2, which is a recombinant prokaryotic expression vector or a recombinant eukaryotic vector.

5. Use of a phosphoribosyl pyrophosphate synthetase mutant according to claim 1 for the enzymatic synthesis of beta-NMN.

6. An enzymatic synthesis method of beta-NMN, comprising the following steps:

step one: d-ribose is catalyzed and acidified by ribose kinase to obtain D-ribose-5-phosphate;

step two: catalyzing D-ribose-5-phosphate with the phosphoribosyl-pyrophosphate synthetase mutant according to claim 1 to obtain 5-phosphoribosyl-1-pyrophosphate;

step three: catalyzing the 5-phosphoribosyl-1-pyrophosphate with nicotinamide phosphoribosyl transferase to obtain the beta-NMN.

7. The method of claim 6, wherein the third step further comprises adding pyrophosphatase to hydrolyze byproduct pyrophosphoric acid.

8. The method of claim 6 or 7, wherein the ATP regeneration system of the enzyme-catalyzed reaction is adenylate kinase and polyphosphate kinase regeneration using sodium hexametaphosphate.

9. The method of claim 6 or 7, wherein the three-step reaction is carried out in a single pot.

10. The method of claim 8, wherein the three-step reaction and ATP regeneration are reacted in a single pot.