UDP (User Datagram Protocol) preference type polyphosphate kinase mutant and application thereof

Abstract

The invention provides a UDP (User Datagram Protocol) preference type polyphosphate kinase mutant and application thereof, compared with wild type polyphosphate kinase from Rhodobacter sp.140A, the amino acid sequence of the UDP preference type polyphosphate kinase mutant has the advantage that single-point mutation or combined mutation is carried out on amino acid at the 73rd site, the 114 site, the 133rd site, the 210 site, the 225th site or the 298th site. The thermal stability of the constructed polyphosphate kinase mutant is obviously improved compared with that of a wild type, the polyphosphate kinase mutant is used for preparing adenosine-3 '-O-glucose, the sustainability of UTP regeneration is obviously improved, the yield of adenosine-3'-O-glucose can reach 55 mM after a reaction is carried out for 3 hours, the conversion rate exceeds 90%, and the polyphosphate kinase mutant has a good industrial application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology and bioengineering, and in particular to a UDP-preferring polyphosphate kinase mutant with improved thermal stability. Background Art

[0002] In vitro multi-enzyme catalysis technology, which uses the assembly of multiple enzymes or enzyme complexes to construct existing or non-existent metabolic pathways in vitro to produce target compounds, has been widely used in recent years to manufacture various biological products. In vitro multi-enzyme catalysis systems have the advantages of easy control and optimization, fast reaction speed, high yield, easy product separation, and almost no by-products. They can even create metabolic pathways that do not exist in microorganisms. To date, the manufacturing potential of in vitro multi-enzyme catalysis has been widely used (Cai et al. Cell-free chemoenzymatic starch synthesis from carbondioxide. 2021).

[0003] Glycosylation reaction has always been a research hotspot in the field of in vitro multi-enzyme catalysis. Glycosylation reaction can use biomass such as straw cellulose, starch, dextrin as substrates to synthesize various oligosaccharides, polysaccharides, glycosides, glycoproteins, etc., which is an ideal "zero-carbon" new development model (Frohnmeyer and Elling. Enzyme cascades for the synthesis of nucleotide sugars: Updates to recent production strategies. 2023). The glycosylation activation donor is mainly UDP-sugar. The synthesis of all kinds of UDP-sugar requires expensive high-energy compound UTP for energy supply. How to regenerate UDP, a byproduct after UDP-sugar is utilized, into UTP is a difficult problem that needs to be solved urgently in the field of in vitro glycosylation.

[0004] The energy regeneration system based on polyphosphate kinase-inorganic polyphosphate has been widely used in in vitro energy-consuming enzyme catalysis reactions. Inorganic polyphosphate is a cheap phosphate donor compound that can be generated by the polymerization of phosphate at high temperature, or produced by microorganisms through the biopolymerization of phosphate ions. It has the advantages of low price and good stability, and shows attractive prospects in biocatalytic systems. In recent years, UDP-preferring polyphosphate kinase has been newly discovered. It has the new function of catalyzing the reaction of UDP and inorganic polyphosphate to generate UTP. It has the potential to be used as a tool enzyme for efficient regeneration of UTP and applied to in vitro glycosylation reactions.

[0005] So far, the research and application of UDP-preferring polyphosphate kinases have achieved certain success, but they still face some common problems. The most prominent one is that UDP-preferring polyphosphate kinases have very poor thermal stability, with a half-life of only 3 minutes at 50°C, which leads to very limited application of this energy regeneration system. Therefore, UDP-preferring polyphosphate kinases must be engineered to improve their thermal stability. Summary of the invention

[0006] The first object of the present invention is to provide a UDP-preferring polyphosphate kinase mutant with a long half-life and improved thermal stability.

[0007] The second object of the present invention is to provide a use of a UDP-preferring polyphosphate kinase mutant with improved thermal stability in catalyzing the preparation of adenosine-3'-O-glucose.

[0008] In order to achieve the first object of the present invention, the present invention provides a UDP-preferring polyphosphate kinase mutant, wherein the amino acid sequence of the mutant is compared with the wild-type polyphosphate kinase from Rhodobacter sp.140A (NCBI No. RBP84891.1), and a single point mutation or a combined mutation is performed at the amino acid at position 73, position 114, position 133, position 210, position 225 or position 298.

[0009] As a preferred embodiment, the single point mutation is: glutamic acid (E) at position 73 mutates to valine (V), and the mutation is conventionally named E73V; aspartic acid (D) at position 114 mutates to lysine (K), and the mutation is conventionally named D114K; alanine (A) at position 133 mutates to proline (P), and the mutation is conventionally named A133P; aspartic acid (D) at position 210 mutates to serine (S), and the mutation is conventionally named D210S; arginine (R) at position 225 mutates to alanine (A), and the mutation is conventionally named R225A; glutamic acid (E) at position 298 mutates to proline (P), and the mutation is conventionally named E298P; the combined mutation is two or more mutations in the single point mutation.

[0010] As a preferred embodiment, the combined mutations are: multi-point combined mutations at positions 73, 210 and 298, E73V, D210S and E298P, and the mutant is named M1;

[0011] The multiple combined mutations at positions 114, 133, and 225, D114K, A133P, and R225A, were named M2;

[0012] The mutant was named M3 with multiple combined mutations at positions 114, 210, 225 and 298, D114K, D210S, R225A and E298P.

[0013] In order to achieve the second object of the present invention, the present invention provides the use of the UDP-preferring polyphosphate kinase mutant in catalyzing the preparation of adenosine-3'-O-glucose, using the mutant as a catalyst and adenosine as a substrate to prepare adenosine-3'-O-glucose.

[0014] As a preferred solution, UDP, MgCl 2 , sodium hexametaphosphate, glucose-1-phosphate, and adenosine are used as substrates, UTP glucose-1-phosphotransferase and glycosyltransferase are used as biocatalysts, and the UDP-preferring polyphosphate kinase mutant is added as a UTP regenerator to catalyze the preparation of adenosine-3'-O-glucose.

[0015] As a further preferred embodiment, the reaction system pH is 6-8, the temperature is 30-50°C, the amount of UDP added is 1-3 mM, MgCl 2 The added dosage is 40-60mM, the added dosage of sodium hexametaphosphate is 10-40mM, the added dosage of glucose-1-phosphate is 10-60mM, and the added dosage of adenosine is 10-60mM.

[0016] The invention also provides a coding gene of a UDP-preferring polyphosphate kinase mutant and a recombinant genetic engineering bacterium containing the coding gene.

[0017] The advantage of the present invention is that the UDP-preferring polyphosphate kinase mutant with improved thermal stability provided by the present invention has significantly improved stability compared with the wild-type enzyme. The polyphosphate kinase mutant with improved thermal stability constructed by the present invention is used to prepare adenosine-3'-O-glucose, which significantly improves the sustainability of UTP regeneration. The mutant obtained by the present invention has an adenosine-3'-O-glucose yield of 37.6-55mM after 3 hours of reaction, and the highest conversion rate exceeds 90%. The production rate and yield of adenosine-3'-O-glucose obtained by catalyzing adenosine at 50°C are greatly improved, and it has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 . Expression of polyphosphate kinase, 1: cell lysis solution before induction of polyphosphate kinase expression; 2: cell lysis solution after induction of polyphosphate kinase expression; 3 and 4 are BL21 (DE3) blank controls without plasmid.

[0019] Figure 2 . Schematic diagram of the preparation of adenosine-3'-O-glucose.

[0020] Figure 3 .High performance liquid chromatography detection chart. DETAILED DESCRIPTION

[0021] The technical scheme in the embodiments of the present invention is clearly and completely described below in conjunction with specific embodiments. The test methods used in the following embodiments are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0022] Example 1. Screening of mutants

[0023] Molecular dynamics simulation, structural analysis and folding free energy calculation were used to screen single point mutations that may improve stability.

[0024] The basis for judgment includes:

[0025] (1) The mutation point should be far away from the active center site;

[0026] (2) Mutations should introduce new forms of forces that are beneficial to thermal stability, such as hydrogen bonds, salt bridges, and hydrophobic interactions.

[0027] (3) The overall folding free energy of the protein should decrease after mutation.

[0028] After the above analysis, the mutants were selected in which the glutamic acid (E) at position 73 was replaced by valine (V); the aspartic acid (D) at position 114 was replaced by lysine (K); the alanine (A) at position 133 was replaced by proline (P); the valine (V) at position 147 was replaced by tyrosine (Y); the glutamic acid (E) at position 199 was replaced by lysine (K); the valine (V) at position 200 was replaced by isoleucine (I); the aspartic acid (D) at position 210 was replaced by serine (S); the arginine (R) at position 225 was replaced by alanine (A); and the glutamic acid (E) at position 298 was replaced by proline (P).

[0029] Example 2. Construction of mutants

[0030] The pET28a recombinant plasmid containing the wild-type polyphosphate kinase gene (from Rhodobacter sp. 140A, NCBI No. RBP84891.1) was used as a template to perform site-directed mutagenesis on the above sites. PCR site-directed mutagenesis was performed after primers were designed using oligo7 software, and the primers are shown in Table 1.

[0031] Table 1. Primer sequences (SEQ ID NO.1 to SEQ ID NO.18)

[0032]

[0033]

[0034] Table 2. Reaction system

[0035] template 10μL DNA polymerase 25μL F Primer 3μL R Primer 3μL <![CDATA[ddH 2 The]]> 9μL Total volume 50μL

[0036] Table 3. PCR cycle process

[0037] Preheat 98℃5min transsexual 98℃10s annealing 55℃30s extend 72℃1min Final extension 72℃10min

[0038] The denaturation-annealing-extension cycle was repeated 30 times. DNA polymerase was purchased from Nanjing Novozyme Biotechnology Co., Ltd. The PCR product was recovered and detected by agarose (1%) gel electrophoresis, then mixed evenly with the vector, and the two PCR products were connected at 37°C for 15 minutes using homologous recombinase (Shanghai Tolo Biotechnology Co., Ltd.) to obtain a circularized mutant plasmid. The ligation product was transformed into Escherichia coli, and after culture on kanamycin-resistant plates, a single clone was selected for sequencing to verify that the sequence was correct, and a mutant plasmid was obtained. The construction of the combined mutation is based on the pET28a recombinant plasmid containing a single mutation or multiple mutation polyphosphate kinase gene as a template, and is obtained through the same operation as above.

[0039] Example 3. Preparation of pure enzyme

[0040] The plasmid carrying the polyphosphate kinase mutant was transformed into BL21 (DE3) to obtain recombinant bacteria.

[0041] The recombinant bacteria were inoculated into 50 mL of LB containing 50 mg / L kanamycin and cultured at 37°C, 220 rpm for 6-8 h. Subsequently, 2% inoculum was inoculated into 100 mL of LB and cultured at 37°C. 600 When the pH value reaches 0.6-0.8, add IPTG with a final concentration of 0.2mM and induce at 18°C ​​and 220rpm for 16-18h. Collect the cells by centrifugation, discard the supernatant, and add an appropriate amount of 50mM NaH containing 10mM imidazole and 500mM NaCl. 2 PO 4 The resuspended cells were added to a buffer (pH 8.0). The resuspended cells were added to a high-pressure homogenizer for crushing at a pressure of 700 bar until the bacterial solution became clear. The crushed cells were collected and centrifuged to obtain the supernatant, which was poured into a nickel column and homogenized with NaH containing different concentrations of imidazole. 2 PO 4 Buffer was used for elution, and the eluate at 200 mM imidazole concentration was collected. The eluate was concentrated by ultrafiltration until the residual volume reached about 1 mL, and 50% molecular grade glycerol was added at a volume ratio of 1:1, mixed, and then divided and stored at -80°C for future use. Figure 1 . is the expression of polyphosphate kinase.

[0042] Example 4, polyphosphate kinase activity and thermal stability test

[0043] Thermostability was calculated by incubating the enzyme at 50° C. for different time intervals in 100 mM Tris-HCl buffer, pH 7.0, and measuring the residual activity of the enzyme at 50° C. The inactivation rate constant of the enzyme was estimated using a first-order kinetic reaction model.

[0044] lnA=lnA 0 -kt

[0045] A is the relative enzyme activity after heat treatment for t hours; A 0 is the initial relative enzyme activity; k is the inactivation rate constant. The time when the enzyme activity is reduced to half of the initial enzyme activity is recorded as t 1 / 2 .

[0046] The reaction system contained 100 mM Tris-HCl buffer at pH 7.0, 10 mM UDP, 20 mM MgCl 2 , 5mM sodium hexametaphosphate, pure enzyme 0.001g / L, and water was used to make up the system to 1mL. The reaction solution was reacted at 50℃ for 2min, and a sample was taken. 1M hydrochloric acid was added at a volume ratio of 1:1 to terminate the reaction. The sample was placed on ice for 10min, then centrifuged at 12000 rpm for 10 minutes, the supernatant was collected, and the UTP content was detected by high performance liquid chromatography. The enzyme activity was calculated based on the measured UTP concentration, and the enzyme activity was defined as the amount of enzyme (g) that produces 1μmol of product per minute.

[0047] Table 4. Enzyme activity results

[0048]

[0049]

[0050] Conclusion: The enzyme activities and half-lives of wild-type and mutants were measured and it was found that the thermal stability of E73V, D114K, A133P, D210S, R225A, E298P, M1, M2 and M3 mutants was improved compared with the wild-type. The stability of V147Y, E199K and V200I mutants was worse than that of the wild-type, excluding these three single-point mutations.

[0051] Example 5. Preparation of Adenosine-3'-O-Glucose

[0052] The recombinant bacteria expressing UTP glucose-1-phosphotransferase (NCBI No. WP_028847555) and glycosyltransferase (NCBI No. WP_142191293.1) preserved in this laboratory were used to prepare pure enzymes according to the method described in Example 3. Figure 2 It is a reaction diagram.

[0053] The reaction system contained 100 mM Tris-HCl buffer at pH 7.0, 2 mM UDP, 60 mM MgCl 2 , 30mM sodium hexametaphosphate, 60mM glucose-1-phosphate, 60mM adenosine. Then add 0.2g / L of UTP glucose-1-phosphotransferase and glycosyltransferase, 0.1g / L of polyphosphate kinase, and the total volume is 1mL. After reacting at 50℃ for 3 hours, the adenosine-3'-O-glucose content ( Figure 3 ).

[0054] Table 5. Adenosine-3'-O-glucose production levels of different polyphosphate kinase mutants

[0055]

[0056]

[0057] in conclusion:

[0058] The polyphosphate kinase mutants provided by the present invention include single-point mutants and combined mutants. Compared with the wild-type polyphosphate kinase, the three mutants of V147Y, E199K and V200I have a reduced yield of the final adenosine-3'-O-glucose due to poor stability. The single-point mutants and combined mutants of E73V, D114K, A133P, D210S, R225A and E298P have a longer half-life at 50°C; especially the combined mutants, which show the superposition effect of the thermal stability of the single-point mutants, which increases the final yield of adenosine-3'-O-glucose. Based on this, the polyphosphate kinase mutants provided by the present invention have better thermal stability, are suitable for catalytic reactions at higher temperatures for a longer time, and significantly improve the sustainability of UTP regeneration.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A UDP-preferring polyphosphate kinase mutant, It is characterized in that Compared with the wild-type polyphosphate kinase from Rhodobacter sp.140A, the amino acid sequence of the mutant undergoes single point mutation or combined mutation at the 73rd, 114th, 133rd, 210th, 225th or 298th amino acid.

2. A UDP-preferring polyphosphate kinase mutant according to claim 1, It is characterized in that The single point mutations are: glutamic acid at position 73 mutates to valine; aspartic acid at position 114 mutates to lysine; glutamic acid at position 133 mutates to arginine; aspartic acid at position 210 mutates to serine; arginine at position 225 mutates to alanine; glutamic acid at position 298 mutates to proline; and the combined mutations are two or more mutations among the single point mutations.

3. A UDP-preferring polyphosphate kinase mutant according to claim 2, It is characterized in that The combined mutations are: combined mutations at positions 73, 210 and 298; combined mutations at positions 114, 133 and 225; combined mutations at positions 114, 210, 225 and 298.

4. A gene encoding the UDP-preferring polyphosphate kinase mutant according to any one of claims 1 to 3.

5. A recombinant genetically engineered bacterium comprising the coding gene according to claim 4.

6. Use of the UDP-preferring polyphosphate kinase mutant according to any one of claims 1 to 3 in catalyzing the preparation of adenosine-3'-O-glucose.

7. The use according to claim 6, It is characterized in that UDP, MgCl 2 , sodium hexametaphosphate, glucose-1-phosphate, and adenosine are used as substrates, UTP glucose-1-phosphotransferase and glycosyltransferase are used as biocatalysts, and the UDP-preferring polyphosphate kinase mutant described in any one of claims 1 to 3 is added as a UTP regeneration agent to catalyze the preparation of adenosine-3'-O-glucose.

8. The use according to claim 7, It is characterized in that The reaction system pH is 6-8, the temperature is 30-50°C, the amount of UDP added is 1-3 mM, and the MgCl 2 The added dosage is 40-60mM, the added dosage of sodium hexametaphosphate is 10-40mM, the added dosage of glucose-1-phosphate is 10-60mM, and the added dosage of adenosine is 10-60mM.

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