CN116716272A - Phosphoribosyl pyrophosphate synthetase mutant and application thereof - Google Patents
Phosphoribosyl pyrophosphate synthetase mutant and application thereof Download PDFInfo
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- CN116716272A CN116716272A CN202310605392.3A CN202310605392A CN116716272A CN 116716272 A CN116716272 A CN 116716272A CN 202310605392 A CN202310605392 A CN 202310605392A CN 116716272 A CN116716272 A CN 116716272A
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- Prior art keywords
- pyrophosphate synthetase
- nicotinamide
- mutant
- phosphoribosyl
- phosphoribosyl pyrophosphate
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/02—Pentosyltransferases (2.4.2)
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Abstract
The invention discloses a phosphoribosyl pyrophosphate synthetase mutant and application thereof, provides a cell-free catalysis method of nicotinamide mononucleotide with excellent production efficiency, and provides a mutant of phosphoribosyl pyrophosphate synthetase, which is a key enzyme in the route, wherein the enzyme activity of the mutant is improved by 0.86-7.8 times. The method of separating ribose kinase and phosphoribosyl pyrophosphate synthetase competing for the same substrate on the route is adopted to carry out catalytic reaction, and after a series of catalytic reaction conditions are optimized, the production yield by the route is high, thus having industrial application prospect.
Description
Technical Field
The invention relates to construction of a phosphoribosyl pyrophosphate synthetase Ba-PRS mutant derived from bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and development of phosphoribosyl pyrophosphate synthetase and application thereof in multienzyme coupling cell-free catalytic synthesis.
Background
Phosphoribosyl pyrophosphate synthetase is a key enzyme for synthesizing nucleotide compounds. beta-Nicotinamide Mononucleotide (NMN) is present in all organisms and is an important coenzyme NAD + Key intermediates of (a). Recent studies have shown that NMN enhances NAD + Biosynthesis and amelioration of various symptoms, such as diabetes and vascular dysfunction, NMN has been studied to find that it can ameliorate the symptoms of type two diabetes in mice, and has no other toxic side effects. As a result of these pharmaceutical effects, NMN has begun to appear on the market as a nutraceutical. Generally, nucleotide compounds such as NMN are synthesized by chemical synthesis methods at present, which require expensive substrates and catalysts, and microbial fermentation methods are characterized by low productivity and lack of practicality, so that the advantage of enzyme catalysis methods for increasing NMN production is more remarkable by increasing the activity of key enzymes.
Compared with chemical catalyst, the enzyme is used as green natural biocatalyst, has the advantages of excellent stereoselectivity, regioselectivity and the like in catalytic chemical reaction, and has the advantages of mild reaction condition, environmental friendliness, no need of harsh reaction equipment and the like. However, when many enzyme molecules catalyze non-natural substrates, the problems of low activity, poor stability, substrate product inhibition and the like often exist, and the need for molecular modification of the enzyme molecules is urgent.
The patent synthesizes NMN through three steps of reactions. The first step is to catalyze ribose to synthesize ribose-5-phosphate by ribose kinase (GenBank: EFE 0717730.1), the second and third steps are to use ribose-5-phosphate as substrate, ATP-disodium and sodium polyphosphate as auxiliary substrate, to synthesize 5-phosphoribosyl-1 pyrophosphoric acid (PRPP) by phosphoribosyl pyrophosphorylase Ba-PRS (GenBank: ADU 02858.1), then to add substrate nicotinamide, to synthesize NMN by nicotinamide ribosyl transferase (GenBank: WP_ 012788281.1), and the process needs pyrophosphatase (GenBank: CP 000383.1) for circulating ATP. The catalytic reaction is carried out by adopting a method of separating two enzymes competing with the same substrate on the route, and the NMN yield produced by the route reaches 7.4g/L through optimizing a series of catalytic reaction conditions. The invention synthesizes the determination of key enzymes on the NMN route by a multienzyme method, establishes a high-throughput screening model, constructs a high-capacity mutant library, screens and obtains mutants with high activity and high catalytic effect, and further improves the NMN yield.
Disclosure of Invention
Aiming at the problem that the existing phosphoribosyl-pyrophosphate synthetase (Ba-PRS) has low catalytic activity on ATP-disodium and ribose 5-phosphate as substrates, the invention provides a phosphoribosyl-pyrophosphate synthetase series mutant and a method for using phosphoribosyl-pyrophosphate synthetase mutant gene recombinant bacteria and crude enzyme liquid thereof as biocatalysts for biocatalysis synthesis of NMN, wherein the enzyme activity of the mutant bacteria is improved by 86-7.8 times compared with that of the original bacteria, and the highest yield reaches 7.4g/L.
ATP has strong fluorescence absorption and is in a linear relation in the concentration range of 0.0001-0.5mM, a high-throughput screening method is established by reducing the fluorescence value as a signal by using the reduction amount of substrate ATP, and the method is applied to the screening of phosphoribosyl pyrophosphate synthetase, the enzyme activity of each mutant is improved by 86-7.8 times, wherein the catalytic activity of the mutant Ba-PRSH150Q is the highest, the molecular mechanism of improving the catalytic performance of the mutant is further analyzed, the reaction process parameters are optimized, and the process for synthesizing NMN by catalyzing the Ba-PRSH150Q with multiple enzymes is constructed.
The technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a phosphoribosyl-pyrophosphate synthetase (Ba-PRS) mutant obtained by mutating histidine at position 150 of the amino acid sequence shown in SEQ ID NO.2 to glutamine, leucine or valine.
Preferably, the phosphoribosyl pyrophosphate synthetase mutant is obtained by mutating histidine at position 150 of the amino acid sequence shown in SEQ ID NO.2 into glutamine.
In a second aspect, the present invention provides an expression plasmid for the phosphoribosyl pyrophosphate synthetase mutant described above.
The expression plasmid vector described above may be any of various expression vectors commonly used in the art, and in one embodiment of the present invention, the expression plasmid is pET28a (+), specifically, the expression plasmid is obtained by inserting the nucleotide sequence of the phosphoribosyl pyrophosphate synthetase mutant between the NcoI cleavage site and XhoI cleavage site of the vector pET28a (+).
In a third aspect, the present invention provides a genetically engineered bacterium expressing the phosphoribosyl pyrophosphate synthetase mutant.
The skilled artisan is aware that the phosphoribosyl pyrophosphate synthetase mutant gene may be expressed in the form of a plasmid in a host or inserted into the host genome for expression. The host of the genetically engineered bacterium may be any of various hosts commonly used in the art, and in one embodiment of the present invention, the host of the genetically engineered bacterium is e.coli BL21 (DE 3). Specifically, the genetically engineered bacterium is obtained by the following method: inserting the nucleotide sequence of the phosphoribosyl pyrophosphate synthetase mutant between an NcoI enzyme cutting site and an XhoI enzyme cutting site of a vector pET28a (+) to obtain an expression plasmid; and transferring the expression plasmid into competent cells of E.coli BL21 (DE 3) to obtain the genetically engineered bacterium.
In a fourth aspect, the invention provides an application of the phosphoribosyl pyrophosphate synthetase mutant in catalytic preparation of beta-nicotinamide mononucleotide.
Specifically, the application is as follows: the method comprises the steps of forming a conversion system by taking a phosphoribosyl pyrophosphate synthetase mutant, nicotinamide ribosyl transferase Cp-NAMPT and pyrophosphatase Ch-PPK as catalysts, ribose and nicotinamide as substrates, adenosine disodium triphosphate and sodium polyphosphate as auxiliary substrates and a phosphate buffer solution with the pH value of 7.5 and 50mM as a reaction medium, and reacting for 30min-2h at 37 ℃ and 800rpm to obtain a reaction solution containing beta-nicotinamide mononucleotide.
Further, the amino acid sequence of the nicotinamide ribosyl transferase Cp-NAMPT is shown in SEQ ID NO. 10; the amino acid sequence of the pyrophosphatase Ch-PPK is shown as SEQ ID NO. 8.
Further, in the transformation system, the final concentration of ribose is 7.5-30g/L (preferably 15 g/L), the final concentration of disodium adenosine triphosphate is 0.551-110.2g/L (preferably 0.8265 g/L), the final concentration of nicotinamide is 1.94g/L-5.81g/L (preferably 3.39 g/L), and the final concentration of sodium polyphosphate is 1.61-12.88g/L (preferably 6.44 g/L).
Further, the phosphoribosyl pyrophosphate synthetase mutants, nicotinamide ribosyl transferase Cp-NAMPT and pyrophosphatase Ch-PPK can be added in the form of pure enzyme liquid, wet bacterial cells of genetically engineered bacteria or cell disruption liquid. In one embodiment of the present invention, the catalyst is added in the form of a cell disruption solution of wet cells of genetically engineered bacteria of the respective enzymes, and the catalyst is used in an amount of 0.1 to 4g DCW/L (DCW cell dry weight, preferably 4g DCW/L) based on the total dry weight of the cells. In one embodiment of the invention, the mass ratio of phosphoribosyl-pyrophosphate synthetase mutant, nicotinamide ribosyl transferase Cp-NAMPT to pyrophosphatase Ch-PPK, based on dry cell weight, is 1:1:1.6.
the mixed bacterial cells comprise wet bacterial cells obtained by induction culture of engineering bacteria containing phosphoribosyl pyrophosphate synthetase mutant genes, engineering bacterial cells containing ribokinase E.coli-RK genes, engineering bacterial cells containing nicotinamide ribosyl transferase Cp-NAMPT genes and wet bacterial cells obtained by induction culture of engineering bacterial cells containing pyrophosphatase Ch-PPK genes.
Further, the reaction system also comprises magnesium ions. Preferably, the concentration of magnesium ions is 20mM. In one embodiment of the invention, the magnesium ions are added in the form of magnesium chloride.
Further, the reaction system also comprises transition metal ions, wherein the transition metal ions are Ca 2+ 、K + 、Na + 、Zn 2+ 、Fe 2+ 、Fe 3+ 、Mn 2+ One or more of (preferably Fe) 3+ ). In one embodiment of the invention, the transition metal ions are in FeCl 3 Is added in a final concentration of 1mM.
When the substrates are ribose and nicotinamide and the auxiliary substrates are ATP-disodium and sodium polyphosphate, the method for synthesizing NMN by the phosphoribosyl pyrophosphate synthetase E.coli-PRS mutant under the catalysis of biological enzyme comprises the following steps: the method comprises the steps of carrying out ultrasonic crushing on thalli obtained by induced culture on engineering bacteria containing phosphoribosyl pyrophosphate synthetase E.coll-PRS mutant genes, engineering thalli containing nicotinamide ribosyl transferase Cp-NAMPT genes and engineering bacteria containing pyrophosphatase Ch-PPK genes, mixing E.coll-PRS, ch-PPK and Cp-NAMPT crushing liquid, taking crude enzyme liquid as a catalyst, and synthesizing NMN by a step method. In the first step, ribose is taken as a substrate, ATP-disodium is taken as an auxiliary substrate, polyp and pyrophosphatase are added to synthesize ATP-disodium through ribose kinase to form a circulating system of ATP, and ribose-5-phosphate is synthesized in a catalytic mode. And secondly, taking ribose-5-phosphate as a substrate and ATP-disodium as an auxiliary substrate, adding polyp and pyrophosphatase to synthesize ATP-disodium through ribose kinase to form a circulating system of ATP, and synthesizing an intermediate product 5-phosphoribosyl-1-pyrophosphate (PRPP) through phosphoribosyl pyrophosphate synthetase catalysis. After addition of nicotinamide, the product NMN is synthesized by nicotinamide ribosyl transferase. The conversion system is formed by taking phosphate buffer solution with pH of 7.5 and 50mM as a reaction medium, the reaction is carried out at 37 ℃ and 600-1000 rpm, and the reaction solution is separated and purified after the reaction is finished, thus obtaining NMN pure product.
Further, the wet cell is prepared as follows: inoculating engineering bacteria containing phosphoribosyl pyrophosphate synthetase mutant genes into LB liquid culture medium containing 50 mug/mL kanamycin at a final concentration, culturing for 10h at 37 ℃, inoculating the engineering bacteria into fresh LB liquid culture medium containing 50 mug/mL kanamycin at a final concentration of 1% by volume, culturing for 5h at 37 ℃ at 180rpm, adding Isopropyl thiogalactoside (IPTG) with a final concentration of 0.1mM into the culture medium, culturing for 12h at 28 ℃, and centrifuging for 10min at 4 ℃ at 8000rpm to obtain wet bacterial bodies containing phosphoribosyl pyrophosphate synthetase mutant genes; the preparation method of the wet bacterial strain obtained by induced culture of the engineering bacterial strain containing the ribokinase E.coll-RK gene, the engineering bacterial strain containing the phosphoribosyl pyrophosphate synthetase E.coll-PRS gene and the engineering bacterial strain containing the pyrophosphatase Ch-PPK gene is the same as that of the wet bacterial strain containing the phosphoribosyl pyrophosphate synthetase mutant gene.
The crude enzyme of the invention is prepared by the following method: the cells were resuspended in 50mM phosphate buffer, pH7.5, at a rate of 100g/L of the total amount of wet cells, and sonicated on an ice-water mixture for 6min under sonication conditions: the power is 400W, crushing is carried out for 1s, and suspending is carried out for 2s, and the crushing mixed solution is taken to obtain crude enzyme solution.
The total length of the base sequences of the phosphoribosyl-pyrophosphate synthetase Ba-PRS and the phosphoribosyl-pyrophosphate synthetase Ba-PRS mutants is 972bp, the base sequence is stopped from the first base to 972 th base, the initiation codon is ATG, and the termination codon is TAA.
The invention relates to a method for preparing phosphoribosyl pyrophosphate synthetase Ba-PRS mutant, which comprises the steps of adopting site-directed saturation mutation technology, using the technology to mutate phosphoribosyl pyrophosphate synthetase Ba-PRS gene (SEQ ID NO. 1), transferring the obtained mutant plasmid into E.coli BL21 (DE 3) competent cells in a 42 ℃ heat shock mode, inoculating, transferring, inducing and recovering thalli of the obtained strain, and using a resuspension bacterial liquid to catalyze substrates ribose and ATP-disodium to synthesize ribose-5-phosphate, ADP and AMP. The specific method comprises the following steps: the first step is to activate the original bacteria to obtain the control bacteria E.coli BL21 (DE 3)/pET 28a (+) -Ba-PRS, extract plasmid pET28a (+) -Ba-PRS and store at-20 ℃. And secondly, comparing SWISS-MODEL with Ba-PRS to obtain a template protein crystal structure of homologous modeling, modeling by utilizing Modeller 9.19 homology, performing molecular docking, selecting proper mutation sites, wherein the selected sites are mainly amino acid residues near an active center and amino acid residues on a loop of the active center, designing a mutant primer, performing site-directed saturation mutation by taking pET28a (+) -Ba-PRS as a template plasmid, obtaining mutant plasmids, converting, screening dominant mutant bacteria, and obtaining phosphoribosyl pyrophosphate synthetase Ba-PRS-H150Q (marked as M150-Q), ba-PRS-H150L (marked as M150-L) and Ba-PRS-H150V (marked as M-150V).
The culture medium can be any culture medium in the field which can enable the thalli to grow and express the target gene of the invention, preferably LB culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L NaCl, and distilled water to adjust the pH to 7.0. The culture method and the culture conditions are not particularly limited, and may be appropriately selected according to the general knowledge in the art depending on the type of host, the culture method and the like.
Compared with the prior art, the invention has the main beneficial effects that: the enzyme activity of the nicotinamide ribosyl transferase mutant M150-Q, M, 150-L, H and 150V constructed by the invention is respectively increased by 7.8 times, 3.13 times and 86.68 percent compared with that of the nicotinamide ribosyl transferase of a control group. The method is characterized in that NMN is synthesized by a stepwise method, ribose is taken as a substrate, ATP-disodium is taken as an auxiliary substrate in the first step, polyp and pyrophosphatase are added to synthesize ATP-disodium through ribose kinase so as to form a circulating system of ATP, and ribose-5-phosphate is synthesized in a catalytic mode. And secondly, taking ribose-5-phosphate as a substrate and ATP as an auxiliary substrate, adding polyp and pyrophosphatase, synthesizing ATP-disodium through ribose kinase to form a circulating system of ATP, synthesizing an intermediate product 5-phosphoribosyl-1-pyrophosphate (PRPP) through phosphoribosyl pyrophosphate synthetase catalysis, adding nicotinamide, and synthesizing a product NMN through nicotinamide ribosyl transferase. A conversion system was constituted by using a phosphate buffer solution of pH7.5 and 50mM as a reaction medium, and the reaction was carried out at 37℃and 600 to 1000 rpm. Wherein the mutant M150-Q catalyzes the production of NMN at a yield of 7.4g/L. Compared with the original strain, the yield of NMN is increased by 0.49g/L, 88.5%, and the space-time yield reaches 1.85g/L h. The maximum yield of NMN produced by using the catalysis of the control group Ba-PRS reaches 6.91g/L, so that the phosphoribosyl pyrophosphate synthetase Ba-PRS-H150Q has more industrial application prospect.
Drawings
FIG. 1 is a schematic diagram showing the reaction of phosphoribosyl pyrophosphate synthetase mutant Ba-PRS-H150Q with engineering bacteria containing ribokinase E.coll-RK gene, engineering bacteria containing nicotinamide ribosyl transferase Cp-NAMPT gene and engineering bacteria containing pyrophosphatase Ch-PPK gene, catalyzing ribose and nicotinamide to prepare NMN through two steps of reaction.
FIG. 2 is a SDS-PAGE electrophoresis of phosphoribosyl pyrophosphate synthetase supernatant and pure enzyme. Lane 1: m: a standard protein molecule; lane 2: control Ba-PRS supernatant; lane 3: control Ba-PRS pure enzyme; lane 4: ba-PRSH150Q supernatant; lane 5: ba-PRSH150Q pure enzyme; lane 6: ba-PRSH150L supernatant; lane 7: ba-PRSH150L pure enzyme.
FIG. 3 relative enzyme activities of phosphoribosyl pyrophosphate synthetase Ba-PRSH150Q at different pH buffer systems.
FIG. 4 shows the application of phosphoribosyl pyrophosphate synthetase mutant Ba-PRSH150Q to NMN synthesis at various concentrations of nicotinamide substrate.
FIG. 5 shows the application of phosphoribosyl pyrophosphate synthetase mutant Ba-PRSH150Q to NMN synthesis for different reaction conditions, such as different MgCl 2 Concentration, different AMP concentration, different ATP-disodium concentration, different sodium polyphosphate concentration, under these conditions the mutant synthesizes NMN yield.
FIG. 6 is the effect of different metal ions on the enzyme activity of phosphoribosyl pyrophosphate synthetase and its mutant Ba-PRSH 150Q.
FIG. 7 shows the effect of metal ions on NMN production when NMN is prepared by catalyzing ribose-5-phosphate with nicotinamide using riboside pyrophosphatase mutant Ba-PRSH150Q, nicotinamide riboside transferase Cp-NAMPT, genetically engineered bacteria and genetically engineered bacteria containing pyrophosphatase Ch-PPK.
Detailed Description
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
example 1: synthesis of ribopyrophosphate synthase Gene, construction of mutant library and screening
The gene sequence of ATP fluorescence detection protein (ATPi) (GenBank: UKS 89445.1) is fully synthesized by the method of optimizing the codon of Escherichia coli by the company of Optimago, and the optimized sequence is shown as SEQ ID NO:11 and ligating the ATPi fragment between the linearized pETDuet-1 plasmids XbaI and EcoRI. Constructing a pETDuet-ATPi recombinant expression vector, and transferring the expression vector into E.coli BL21 (DE 3) to prepare E.coli BL21 (DE 3)/pETDuet-ATPi. The gene containing ribose pyrophosphoric acid synthetase Ba-PRS (GenBank: WP_ 013350761.1) is obtained by total gene synthesis of the Optimago company of the Optimago family through taking Escherichia coli as a host and carrying out codon optimization (the optimized nucleotide sequence is shown as SEQ ID NO: 1), and is inserted into pET28a (+) to construct a recombinant expression vector, and the CDNA segment of RK is linked to the position behind TATAACCAT (before NcoI restriction site) and the position behind CTCGAG (XhoI restriction site) in pET28a (+) and the expression vector is transferred into E.coli BL21 (DE 3) to prepare E.coli BL21 (DE 3)/pET 28a (+) -Ba-PRS. The preparation of the library of mutant ribose pyrophosphoric acid synthetase was carried out by 1 round of site-directed saturation mutation, the primer design is shown in Table 1, the carrier pET28a (+) -Ba-PRS in E.coli BL21 (DE 3)/pET 28a (+) -Ba-PRS is used as a template, and His150-F and His150-R in Table 1 are used as mutation primers of 150 amino acids. The 150 th histidine of the amino acid sequence of the mutant Ba-PRS of the ribopyrophosphate synthetase is mutated into the rest 19 amino acids by saturated mutation PCR, and transformed to obtain monoclonal mutants, and the mutant Ba-PRS-H150Q (marked as M150-Q) of the ribopyrophosphate synthetase, namely the 150 th histidine of the amino acid shown as SEQ ID NO.2 is mutated into glutamine) and the mutant Ba-PRS-H150L (marked as M150-L, namely the 150 th histidine of the amino acid shown as SEQ ID NO.2 is mutated into leucine) are obtained by dominant strain screening (Table 2).
PCR reaction System (50. Mu.L): 2. Mu.L of forward primer (10. Mu.M), 2. Mu.L of reverse primer (10. Mu.M), 25. Mu.L of 2 XPhanta buffer, 1. Mu.L of dNTP mix (10 mM each), 1. Mu.L of plasmid template, 1. Mu.L of DNA polymerase and 18. Mu.L of ultrapure water. The PCR procedure set up according to the Phanta Super-Fidelity DNA polymerase instructions was as follows: pre-denaturation at 95℃for 5min, followed by 29 cycles (denaturation at 95℃for 30s, annealing at 55℃for 30s, extension at 72℃for 5 min), final extension at 72℃for 10min, and incubation at 16 ℃. The recombinant plasmid obtained was transformed into E.coli BL21 (DE 3) competent cells by means of heat shock at 42℃and spread evenly on plates and incubated for 14h at 37 ℃. Then, the monoclonal cells were picked up and transferred to 1mL of LB liquid medium containing 50. Mu.g/mL kanamycin, and after 10 hours of incubation at 37℃and 180rpm in a 96-well plate, induced, cultured at 28℃for 12 hours, and then centrifuged to collect the cells. ATPi is used to enhance fluorescence uptake of ATP-disodium. The ATPi was subjected to the same culture method as that of ribopyrophosphate synthase to obtain bacterial cells E.coli BL21 (DE 3)/pETDuet-ATPi, and the bacterial cells were crushed at a wet bacterial cell concentration of 40g/L, centrifuged at 8000rpm for 10min, and the supernatant was collected. High throughput screening of the obtained mutants for dominant mutantsSimultaneously, E.coli BL21 (DE 3)/pET 28a (+) was selected in 96-well plates as negative and positive controls with the original strain. The screening conditions were as follows: the cell was resuspended in 100uL of phosphate buffer (50 mM) pH7.5, and 200uL of a conversion system composed of ATP-disodium and ribose-5-phosphate, which were used as substrates, was added to the reaction mixture at 37℃and 800rpm, wherein the amount of cell bodies in 1mL of the bacterial solution in the 96-well plate was the amount of the mutant Ba-PRS. After 10min of reaction, centrifuging a 96-well plate, taking 90uL of supernatant reaction liquid into a 96-well blackboard, adding 10uL of ATPi enzyme liquid, detecting a fluorescence value at an excitation wavelength of 485nm and an emission wavelength of 515nm by using a multifunctional enzyme-labeled instrument, and taking a mutant with a low fluorescence value as a dominant strain, sequencing the obtained dominant strain by using the mutant with the improved primary screening enzyme activity, and re-screening the mutant. The re-screening method was as follows, 30uL of the deposited bacterial liquid was transferred from a 96-well plate into 8mL of LB liquid medium containing 50. Mu.g/mL of kanamycin, cultured at 37℃for 10 hours at 180rpm, transferred into 100mL of LB liquid medium containing 50. Mu.g/mL of kanamycin at 1% (v/v) of inoculum size, cultured at 37℃for 2 hours at 180rpm, added with 0.1mM IPTG of final concentration, induced at 28℃for 12 hours, and centrifuged at 8000rpm for 10 minutes to collect the bacterial cells, thereby obtaining the corresponding wet bacterial cells. 1g of bacterial cells are weighed, 10mL of phosphate buffer solution is added, the mixture is uniformly mixed and then is subjected to ultrasonic crushing to obtain crude enzyme solution, the crude enzyme solution is reacted with ATP-disodium and ribose-5-phosphate with the final concentration of 10mM, the reaction is carried out at 37 ℃ and 800rpm for 10min, after the reaction, the reaction is stopped by 0.2mM phosphoric acid, and dd H is used 2 The liquid phase detection was performed after dilution of O by 20 times.
TABLE 1 site-directed saturation mutagenesis primer design for ribose pyrophosphate synthases
Example 2: control group ribose pyrophosphoric acid synthetase and mutant thereof, engineering bacteria containing nicotinamide ribosyl transferase Cp-NAMPT gene and engineering bacteria containing pyrophosphatase Ch-PPK gene are constructed and cultured.
Nicotinamide ribosyl transferase gene engineering bacteria: the gene of engineering bacteria (GenBank: CP 000383.1) of pyrophosphatase Ch-PPK gene is subjected to codon optimization by taking escherichia coli as a host (the optimized nucleotide sequence is shown as SEQ ID NO: 7), a recombinant expression vector is constructed by total gene synthesis and inserted into pET28a (+) by the Optimago, the cDNA segment of Ch-PPK is respectively linked to the position behind TATAACAT (before NcoI cleavage site) and the position behind CTCGAG (XhoI cleavage site) in pET-28a (+) by the same method, and the optimized nucleotide sequence is shown as SEQ ID NO:9, and respectively linking the CDNA fragment of Cp-NAMPT to TATACAT at the position in pET-28a (+) (before NcoI cleavage site) and before CTCGAG (XhoI cleavage site), and transferring the expression vector into E.coli BL21 (DE 3) to obtain E.coli BL21 (DE 3)/pET 28a (+) -Cp-NAMPT.
The ribopyrophosphate synthase and its mutant E.coli BL21 (DE 3)/pET 28a (+) -Ba-PRS and E.coli BL21 (DE 3)/pET 28a (+) -Ba-PRSH150Q of example 1, the starting strain E.coli BL21 (DE 3)/pET 28a (+) -Cp-NAMPT of example 2, E.coli BL21 (DE 3)/pET 28a (+) -Ch-PPK were inoculated into LB liquid medium containing kanamycin at a final concentration of 50. Mu.g/mL, cultured at 37℃for 10 hours, inoculated into fresh LB liquid medium containing kanamycin at a final concentration of 50. Mu.g/mL at 1.5% (v/v), cultured at 37℃for 2 hours at 180rpm, and then added with IPTG at a final concentration of 0.1mM, cultured at 28℃for 12 hours, and centrifuged at 4℃for 10 minutes at 8000rpm to obtain the corresponding wet cell. The obtained cells produce corresponding proteins, and can be used for preparing protein pure enzyme solution, and can also be used for synthesizing NMN by catalyzing substrate nicotinamide with crude enzyme solution, wherein the synthetic route is shown in figure 1.
Example 3: mutant library for catalyzing synthesis of NMN by ribose-5-phosphate
The enzyme solution prepared in example 2, enzyme solution comprising engineering bacteria containing phosphoribosyl pyrophosphate synthetase Ba-PRS, enzyme solution of nicotinamide ribosyl transferase Cp-NAMPT engineering bacteria and bacterial body crushing solution of engineering bacteria containing pyrophosphatase Ch-PPK genes are mixed according to the dry weight ratio of 1:1:1 into a mixed catalyst. Under the same conditions, the control strain E.coli BL21 (DE 3)/pET 28a (+) -Ba-PRSH150Q is used for replacing the mutant bacterial enzyme solution to prepare the control strain mixed catalyst.
Respectively taking mixed bacterial liquid of mutant strain and mixed bacterial liquid of control strain as catalysts, nicotinamide and ribose-5-phosphate as substrates, sodium polyphosphate, disodium adenosine triphosphate as auxiliary substrates and metal ion MgCl 2 . The reaction system is 10mL, the dosage of the three catalysts is 6g DCW/L, the final concentration of ribose-5-phosphate is 7.59g/L, the final concentration of nicotinamide is 2.44g/L, the final concentration of disodium adenosine triphosphate is 0.551g/L, and the final concentration of sodium polyphosphate is 8.047g/L based on the total dry weight of the mixed bacteria. The reaction medium of pH7.5 and 50mM phosphate buffer solution is used to construct a conversion system, the reaction is carried out at 37 ℃ and 800rpm for 30min, sampling is carried out, 50 mu L of reaction solution is taken, 50 mu L of 0.2mM phosphate precipitate protein is added, and 900 mu L of dd H is added 2 O, i.e., the reaction solution was diluted 20 times, centrifuged at 12000rpm for 3min, and the supernatant was passed through a 0.22 μm microfiltration membrane as a liquid phase sample, and NMN and nicotinamide were detected by HPLC to calculate the yield. Dominant mutants were selected using the product NMN as an index, and the experimental results are shown in Table 2.
Liquid phase detection conditions: chromatographic columnC18 (4.6X250 mm, acchrom, china) column, mobile phase: 20mM dipotassium hydrogen phosphate, 1.8g of potassium dihydrogen phosphate is added to adjust the pH to 7.0, the flow rate is 1.0mL/min, the detection wavelength is 254nm, the sample injection amount is 10 mu L, and the column temperature is 40 ℃. NMN and nicotinamide retention times were respectively: 2.8min,17.5min.
TABLE 2 catalytic Properties of Cp-NAMPT and mutants thereof
Example 4: purification of phosphoribosyl pyrophosphate synthetase Ba-PRS female parent and mutant thereof
Ba-PRS and dominant mutants obtained in example 3 (Ba-PRSH 150Q, ba-PRSH150L, ba-PRSH150V in Table 2), wet cells of phosphoribosyl pyrophosphate synthetase mutants were obtained according to the method described in example 2, and the cells were collected by centrifugation at 8000rpm,4℃for 10min, respectively, and washed twice with 0.9% (w/V) saline. Adding the mixture into phosphate buffer solution with pH of 7.5 and 50mM according to the total amount of 100g/L of wet thalli for resuspension, and carrying out ultrasonic crushing on an ice-water mixture for 6min under the condition of ultrasonic crushing: the power is 400W, the mutant strain crude enzyme liquid is obtained after crushing for 1s and suspending for 2 s. The supernatant was collected by centrifugation at 8000rpm at 4℃for 10min, and after microfiltration through a 0.45 μm membrane, the mutant protein was purified using a Ni affinity column.
The mutant proteins were purified using a nickel affinity column (1.6X10 cm, bio-Rad, USA) as follows: (1) the instrument line was flushed with buffer A (pH 7.5 containing 0.3M NaCl, 20mM PBS buffer) at a flow rate of 3.0 mL/min. (2) Mounting the nickel affinity column on an AKTA protein purifier, (3) washing impurities on the nickel column with a buffer A at a flow rate of 2.0mL/min until the target protein is loaded on the column after the conductivity is stable. The heteroprotein was eluted with buffer B (0.3M NaCl, 50mM imidazole in pH7.5, 20mM PBS buffer), followed by elution of the protein of interest with buffer C (0.3M NaCl, 500mM imidazole in pH7.5, 20mM PBS buffer). The collected eluate was dialyzed overnight against 20mM phosphate buffer (pH 7.5). All purification steps were carried out at 4 ℃. Protein size was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pure phosphoribosyl pyrophosphate synthetase of the control strain E.coli BL21 (DE 3)/pET 28a (+) -Ba-PRS was collected under the same conditions, and the electrophoresis result is shown in FIG. 2, and the target enzyme expression level of the mutant strain is not significantly changed compared with that of the E.coli BL21 (DE 3)/pET 28a (+) -phosphoribosyl pyrophosphate synthetase H150Q strain, so that the improvement of the enzyme activity of the mutant is not caused by the increase of the enzyme expression level and is related to the increase of the specific activity of the enzyme.
Example 5: determination of specific enzyme activity of female parent phosphoribosyl pyrophosphate synthetase and mutant enzyme thereof
The enzyme activity unit (U) is defined as: the amount of enzyme required per minute to consume 1. Mu. Mole of ATP at 37℃and pH7.5 is defined as one enzyme activity unit, U. Specific enzyme activity is defined as the number of units of activity, U/mg, per milligram of enzyme protein.
Standard conditions for enzyme activity detection: 10mM ATP-disodium, 10mM ribose-5-phosphate, 3uL of pure enzyme solution, 37 ℃,10 mL of pH7.5, 50mM dipotassium hydrogen phosphate buffer, 800rpm for 3min, sample treatment and HPLC detection analysis.
The protein concentration was determined using a biquinolinecarboxylic acid protein assay kit (Nanjing, nanjing Biotechnology development Co., ltd.).
TABLE 3 relative and specific enzyme activities of phosphoribosyl pyrophosphate synthases and mutants thereof
Example 6: selection of optimum pH for phosphoribosyl pyrophosphate synthetase Ba-PRSH150Q and optimum pH for three-enzyme co-catalytic synthesis of NMN.
Four buffers were prepared: 50mM citric acid-disodium hydrogen phosphate buffer, 50mM Tris-HCl buffer, 50mM glycine-sodium hydroxide buffer. The preparation method of the 50mM citric acid-disodium hydrogen phosphate buffer solution comprises the following steps: 50mM (0.06 g) disodium hydrogen phosphate was weighed into 8mL dd H 2 In O, the pH was adjusted to 5.0, 5.5, 6.0 with citric acid, respectively, and dd H was used, respectively 2 O was fixed to a volume of 10mL. The preparation method of the 50mM phosphate buffer solution comprises the following steps: 50mM (0.087 g) dipotassium hydrogen phosphate was weighed into 8mL dd H 2 In O, the pH was adjusted to 6.0, 6.5, 7.0, 7.5, 8.0 with potassium dihydrogen phosphate, respectively, and dd H was used, respectively 2 O was fixed to a volume of 10mL. The preparation method of the 50mM Tris-HCl buffer solution comprises the following steps: 50mM (0.061 g) Tris was weighed in 8mL dd H 2 In O, the pH was adjusted to 7.5, 8.0, 8.5, 9.0 with hydrochloric acid, respectively, and dd H was used, respectively 2 O was fixed to a volume of 10mL. The preparation method of the 50mM glycine-sodium hydroxide buffer solution comprises the following steps: 50mM (0.038 g) glycine was weighed into 8mL dd H 2 In O, the pH was adjusted to 9.0, 9.5, 10.0 with sodium hydroxide, respectively, and dd H was used, respectively 2 O was fixed to a volume of 10mL. Under the above pH conditions, ATP-disodium, ribose-5-phosphate and 3uLBa-PRSH150Q pure enzyme solution were added at a final concentration of 10mM, reacted at 37℃and 800rpm for 3min, and the samples were treated and subjected to HPLC detection analysis to compare the enzyme activities of Ba-PRSH 150Q. Under the condition of different pH values,the relative enzyme activities of phosphoribosyl pyrophosphate synthetase Ba-PRSH150Q are shown in FIG. 3. The results show that the enzyme activity of Ba-PRSH150Q is highest in Tris/HCl buffer with pH of 8.5, and Cp-NAMPT and Ch-PPK are considered simultaneously in the process of combining and catalyzing and synthesizing NMN by using Ba-PRSH150Q, so that the comparison of NMN yield is realized by co-catalyzing and synthesizing three enzymes under different pH conditions. By taking the pH buffer solution prepared in the above way as a buffer system, the addition amount of the mixed enzyme solution in the conversion system is reduced to 6g DCW/L, wherein the reduced dry weight of Cp-NAMPT is 2.66g DCW/L, the reduced dry weight of Ch-PPK is 1.67g DCW/L, and the reduced dry weight of Ba-PRSH150Q is 1.67g DCW/L. The feeding amount of the substrate nicotinamide is 2.90g/L (24 mM) respectively; ribose-5-phosphate concentration was 7.59g/L (33 mM) and ATP-disodium concentration was 0.551g/L (1 mM). Sodium polyphosphate concentration of 6.44g/L (20 mM), mgCl 2 The concentration was 0.952g/L (10 mM). The reaction medium was 10mL of a reaction system constructed with 50mM phosphate buffer solution at pH7.5, 37℃and 800rpm, and NMN production was examined for 1 hour. NMN yields at different pH conditions are shown in FIG. 3. As can be seen, NMN production is highest in phosphate buffer pH 7.5.
Example 7: NMN is catalyzed and synthesized by using phosphoribosyl pyrophosphate synthetase Ba-PRSH150Q under different substrate nicotinamide concentrations.
According to the cell culture conditions described in example 1, ba-PRSH 150-Q, cp-NAMPT and Ch-PPK wet cells were obtained by fermentation, and crude enzyme solutions corresponding to the three cells were obtained. And optimizing the substrate dosage in the reaction system, wherein the addition amount of the mixed enzyme liquid in the conversion system is reduced to 6g DCW/L, the reduced dry weight of Cp-NAMPT is 2.66g DCW/L, the reduced dry weight of Ch-PPK is 1.67g DCW/L, and the reduced dry weight of Ba-PRSH150Q is 1.67g DCW/L. Setting 9 substrate concentration gradients, wherein the feeding amount of substrate nicotinamide is 1.94g/L (16 mM) respectively; 2.44g/L (20 mM); 2.90g/L (24 mM); 3.39g/L (28 mM); 3.87g/L (32 mM); 4.36g/L (36 mM); 4.84g/L (40 mM); 5.32g/L (44 mM); 5.81g/L (48 mM), ribose-5-phosphate concentration 7.59g/L (33 mM), ATP-disodium concentration 0.551g/L (1 mM). Sodium polyphosphate concentration of 6.44g/L (20 mM), mgCl 2 The concentration was 0.952g/L (10 mM). pH7.5 and 50mM phosphate buffer solution as reaction mediumThe transformation system was set up to 10mL, reacted at 37℃and 800rpm, and NMN production was examined for 1 hour. And the optimal substrate amount of the bacterial cells was determined by comparing NMN yields, and NMN yields at each different nicotinamide amount are shown in FIG. 4. NMN production was maximized at a substrate nicotinamide concentration of 28 mM.
Example 8: three enzymes co-catalyze and synthesize NMN: different MgCl 2 Effects of concentration, different AMP concentrations, different ATP-disodium concentration, different sodium polyphosphate concentrations on NMN production.
The dosage ratio of the three enzymes of Ch-PPK, cp-NAMPT and Ba-PRSH150Q for co-catalytic synthesis of NMN is 1.6:1:1, wherein Cp-NAMPT has a reduced dry weight of 2.66g DCW/L, ch-PPK has a reduced dry weight of 1.67g DCW/L, and Ba-PRSH150Q has a reduced dry weight of 1.67g DCW/L. The dosage of substrate nicotinamide is 3.39g/L (28 mM), the ribose-5-phosphate concentration is 7.59g/L (33 mM), the dosage of ATP-disodium is optimized, and the concentration gradient is respectively set to be 0mM;0.25mM;0.5mM;0.75mM;1mM;1.5mM;2 mM), at which point the AMP concentration was 0mM, the sodium polyphosphate concentration was 20mM, mgCl 2 The concentration was 10mM. The reaction medium was 10mL of a reaction system constructed with 50mM phosphate buffer solution at pH7.5, 37℃and 800rpm, and NMN production was examined for 1 hour.
The AMP dosage is optimized, and the concentration gradient is respectively set to be 0mM;1mM;2mM;3mM;4mM, at which time the ATP-disodium concentration is 1mM, the sodium polyphosphate concentration is 20mM, mgCl 2 The concentration was 10mM. The reaction medium was 10mL of a reaction system constructed with 50mM phosphate buffer solution at pH7.5, 37℃and 800rpm, and NMN production was examined for 1 hour.
MgCl 2 Optimizing the dosage, and setting the concentration gradient to be 5mM respectively; 10mM;20mM;30mM;35mM;40mM, at which time the AMP concentration was 0mM, the ATP-disodium concentration was 1mM, the sodium polyphosphate concentration was 20mM, and MgCl 2 The concentration was 10mM. The reaction medium was 10mL of a reaction system constructed with 50mM phosphate buffer solution at pH7.5, 37℃and 800rpm, and NMN production was examined for 1 hour.
Optimizing the dosage of sodium polyphosphate, and setting the concentration gradient to be 5mM respectively; 10mM;15mM;20mM;30mM;35mM;40mM, at which time the AMP concentration was 0mM, the ATP-disodium concentration was 1mM, and MgCl 2 The concentration was 10mM. phosphate buffer at pH7.5, 50mMThe reaction medium was used as a reaction medium to construct a conversion system 10mL, the reaction was carried out at 37℃and 800rpm, and NMN production was detected for 1 h.
And determining ATP-disodium, AMP, mgCl by comparing NMN production 2 And optimal amounts of sodium polyphosphate, NMN yields at each of the different nicotinamide amounts are shown in FIG. 5. The substrate nicotinamide was dosed at 3.39g/L (28 mM), ATP-disodium was used at 1.5mM, at which time the AMP concentration was 0mM, sodium polyphosphate concentration was 20mM, mgCl 2 A concentration of 20mM is the optimal reaction system.
Example 9: influence of different metal ions on the enzymatic Activity of phosphoribosyl-pyrophosphate synthetase
The enzyme activity unit (U) of phosphoribosyl pyrophosphate synthetase is defined as: the amount of enzyme required per minute to consume 1. Mu. Mole of ATP at 37℃and pH7.5 is defined as one enzyme activity unit, U. Specific enzyme activity (U/mg) is defined as the number of units of activity per milligram of enzyme protein, U/mg.
Standard conditions for enzyme activity detection: 10mM ATP-disodium, 10mM ribose-5-phosphate, 3uL of pure enzyme solution, 37 ℃,10 mL of pH7.5, 50mM dipotassium hydrogen phosphate buffer, 800rpm for 10min, sample treatment and HPLC detection analysis.
The protein concentration was determined using a biquinolinecarboxylic acid protein assay kit (Nanjing, nanjing Biotechnology development Co., ltd.).
A pure enzyme solution containing phosphoribosyl pyrophosphate synthetase Ba-PRS engineering bacteria and mutant Ba-PRSH150Q thereof is obtained by the method of the example 4. Adding metal ions with the final concentration of 1mM, wherein the metal ions are respectively as follows: by CaCl 2 Formulated Ca 2+ The method comprises the steps of carrying out a first treatment on the surface of the K formulated with KCl + The method comprises the steps of carrying out a first treatment on the surface of the Na formulated with NaCl + The method comprises the steps of carrying out a first treatment on the surface of the By ZnCl 2 Formulated Zn 2+ The method comprises the steps of carrying out a first treatment on the surface of the By FeCl 2 Formulated Fe 2+ The method comprises the steps of carrying out a first treatment on the surface of the By FeCl 3 Formulated Fe 3 + The method comprises the steps of carrying out a first treatment on the surface of the With ZnBr 2 Formulated Zn 2+ The method comprises the steps of carrying out a first treatment on the surface of the With SnCl 2 Formulated Sn 2+ The method comprises the steps of carrying out a first treatment on the surface of the By MnCl 2 Formulated Mn 2+ The method comprises the steps of carrying out a first treatment on the surface of the With PdCl 2 Formulated Pd 2+ The method comprises the steps of carrying out a first treatment on the surface of the With RbCl 2 Formulated Rb 2+ The method comprises the steps of carrying out a first treatment on the surface of the By CoCl 2 Formulated Co 2+ The method comprises the steps of carrying out a first treatment on the surface of the With CrCl 3 Formulated Cr 3+ The method comprises the steps of carrying out a first treatment on the surface of the With NiCl 2 Formulated Ni 2+ 13 metal ions. And (3) measuring the enzyme activity of the original strain Ba-PRS engineering bacteria and the mutant Ba-PRSH150Q thereof under the standard condition of enzyme activity detection. The results are shown in FIG. 6. The results show Ca 2+ 、K + 、Fe 3+ Has obvious enzyme activity promoting effect on Ba-prs.
Example 10: influence of different metal ions on the yield of the three-enzyme co-catalytic synthesis NMN.
The dosage ratio of the three enzymes of Ch-PPK, cp-NAMPT and Ba-PRSH150Q for co-catalytic synthesis of NMN is 1.6:1:1, wherein Cp-NAMPT has a reduced dry weight of 2.66g DCW/L, ch-PPK has a reduced dry weight of 1.67g DCW/L, ba-PRSH150Q has a reduced dry weight of 1.67g DCW/L, and the total amount of three enzymes is 6g DCW/L. The substrate nicotinamide was dosed at 3.39g/L (28 mM), ribose-5-phosphate at 7.59g/L (33 mM), ATP-disodium at 1.5mM, AMP at 0mM, sodium polyphosphate at 20mM, mgCl 2 At a concentration of 20mM, ca was added to the reaction system 2+ ；K + ；Na + ；Zn 2+ ；Fe 2+ ；Fe 3+ ；Mn 2+ The final concentration was 1mM. The NMN yield after 2h of reaction was measured by constructing a conversion system 10mL in 50mM phosphate buffer, pH7.5, at 37℃and 800 rpm. The NMN yields are shown in FIG. 7 for each of the different metal ions. The results show Fe 3+ Has obvious promoting effect on NMN yield, and Fe is added 3+ After that, the NMN yield is increased by 0.51g/L, and finally the NMN yield reaches 7.4g/L.
Claims (10)
1. A phosphoribosyl-pyrophosphate synthetase mutant is characterized in that the phosphoribosyl-pyrophosphate synthetase mutant is obtained by mutating histidine at position 150 of an amino acid sequence shown in SEQ ID NO.2 into glutamine, leucine or valine.
2. The phosphoribosyl pyrophosphate synthetase mutant according to claim 1, wherein: the phosphoribosyl pyrophosphate synthetase mutant is obtained by mutating histidine at position 150 of an amino acid sequence shown in SEQ ID NO.2 into glutamine.
3. An expression plasmid for a phosphoribosyl pyrophosphate synthetase mutant according to claim 1.
4. A genetically engineered bacterium expressing the phosphoribosyl-pyrophosphate synthetase mutant according to claim 1.
5. Use of a phosphoribosyl pyrophosphate synthetase mutant according to claim 1 for the catalytic preparation of β -nicotinamide mononucleotide.
6. The application of claim 5, wherein the application is: the method comprises the steps of forming a conversion system by taking a phosphoribosyl pyrophosphate synthetase mutant, nicotinamide ribosyl transferase Cp-NAMPT and pyrophosphatase Ch-PPK as catalysts, ribose and nicotinamide as substrates, adenosine disodium triphosphate and sodium polyphosphate as auxiliary substrates and a phosphate buffer solution with the pH value of 7.5 and 50mM as a reaction medium, and reacting for 30min-2h at 37 ℃ and 800rpm to obtain a reaction solution containing beta-nicotinamide mononucleotide.
7. The use according to claim 6, wherein: the amino acid sequence of the nicotinamide ribosyl transferase Cp-NAMPT is shown in SEQ ID NO. 10; the amino acid sequence of the pyrophosphatase Ch-PPK is shown as SEQ ID NO. 8.
8. The use according to claim 6, wherein: in the conversion system, the final concentration of ribose is 7.5-30g/L, the final concentration of disodium adenosine triphosphate is 0.551-110.2g/L, the final concentration of nicotinamide is 1.94g/L-5.81g/L, and the final concentration of sodium polyphosphate is 1.61-12.88g/L.
9. The use according to claim 6, wherein: the phosphoribosyl pyrophosphate synthetase mutants, nicotinamide ribosyl transferase Cp-NAMPT and pyrophosphatase Ch-PPK are added in the form of pure enzyme liquid, wet bacterial cells of genetically engineered bacteria or cell disruption liquid.
10. The use according to claim 9, wherein: the catalyst is added in the form of cell disruption liquid of wet bacterial cells of genetic engineering bacteria of each enzyme, the dosage of the catalyst is 0.1-4 g DCW/L based on the dry weight of the total bacterial cells, and the mass ratio of the phosphoribosyl pyrophosphate synthetase mutant, nicotinamide ribosyl transferase Cp-NAMPT and pyrophosphatase Ch-PPK is1 based on the dry weight of cells: 1:1.6.
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