Polyphosphate kinase and its use in increasing atp production

Abstract

The application discloses a polyphosphate kinase and application thereof in improving ATP production, and belongs to the technical field of genetic engineering.The polyphosphate kinase disclosed by the application is PePPK, PsPPK, RbPPK or RrPPK, and the amino acid sequence is shown as SEQ ID NO.5-8.The polyphosphate kinase disclosed by the application can improve ATP production, and can synthesize glutathione and 3'-phosphoadenosine-5'-phosphosulfate.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to polyphosphokinases and their application in increasing ATP production. Background Technology

[0002] In vitro biotransformation is an innovative biomanufacturing platform, but energy supply remains a critical challenge. Directly supplementing with stoichiometric amounts of ATP is extremely costly and can lead to problems such as enzyme inhibition. The ATP regeneration process involving highly efficient PPKs (polyphosphokinases) is an important way to solve the energy supply problem in in vitro biotransformation. PPKs can catalyze the conversion of inorganic polyphosphate (Poly P) and ADP into ATP, constructing a highly efficient ATP regeneration module that avoids dependence on large amounts of exogenous ATP, providing continuous energy support for in vitro enzymatic reactions. Secondly, compared with high-valent phosphate compounds such as phosphopyruvate used in substrate-level phosphorylation, Poly P has significant advantages in terms of low cost and high stability. PPKs utilize Poly P as a phosphate donor to regenerate ATP, significantly reducing the economic cost of in vitro biotransformation and overcoming the practicality barrier caused by the excessively high cost of phosphate donors in traditional substrate-level phosphorylation.

[0003] Some studies selected from Corynebacterium glutamicum PPK2B cg Catalyze the conversion of ADP to ATP and introduce it into the body. Streptomyces albulus This increased intracellular ATP levels and ultimately increased ε-polylysine production. Other studies will draw from... Meiothermus ruber PPK enzymes are used to provide ATP production in glutathione synthesis, enabling a glutathione conversion rate of up to 80%. Further studies have analyzed the activities of various PPK enzymes on ADP and polyphosphate substrates of different degrees of polymerization, finding that they originate from... Pseudomonas aeruginosa PPK2C pa It exhibits the highest specific activity for polyP6, enabling a continuous supply of ATP from 3'-adenosine-5'-phosphosulfate.

[0004] Therefore, providing polyphosphokinases and their applications in increasing ATP production is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides polyphosphokinase and its application in increasing ATP production.

[0006] This invention innovatively provides four polyphosphokinases: PePPK, PsPPK, RbPPK, and RrPPK, which respectively interact with PPK2C, which has excellent ATP regeneration effects. paThey exhibited homology of 30.45%, 31.65%, 30.74%, and 32.81%, respectively. PePPK, PsPPK, RbPPK, and RrPPK achieved significantly superior results compared to the aforementioned derivatives in the extracellular synthesis of glutathione and the intracellular synthesis of 3'-phosphoadenosine-5'-phosphate, respectively. P. aeruginosa The comparison PPK2C pa Its effects are advanced and have practical value.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Polyphosphate kinases, namely PePPK, PsPPK, RbPPK, or RrPPK, have amino acid sequences as shown in SEQ ID NO. 6-9.

[0009] Furthermore, the application of the aforementioned polyphosphokinase in increasing ATP production.

[0010] Furthermore, the application of the aforementioned polyphosphate kinase in the synthesis of intracellular and extracellular products.

[0011] Furthermore, the application of the polyphosphate kinase in the extracellular synthesis of glutathione or the intracellular synthesis of 3'-adenosine-5'-phosphosulfate.

[0012] The recombinant vectors contain the genes for the four wild-type polyphosphate kinases mentioned above.

[0013] The recombinant strains each contain the genes for the four wild-type polyphosphate kinases mentioned above.

[0014] A method for preparing polyphosphokinase includes the following steps: ligating a polyphosphokinase encoding gene to an original expression vector to obtain a recombinant vector; transforming the recombinant vector into host cells to obtain PePPK, PsPPK, RbPPK, and RrPPK recombinant strains, respectively. The original expression vector is pET-28a(+), and the host cell is *Escherichia coli* BL21(DE3).

[0015] The polyphosphate kinases PePPK, PsPPK, RbPPK, and RrPPK were used to construct an ATP regeneration system.

[0016] The purified polyphosphokinase solution was used to synthesize glutathione or 3'-adenosine-5'-phosphosulfate.

[0017] As can be seen from the above technical solutions, compared with the prior art, this invention discloses polyphosphate kinases and their application in increasing ATP production. The polyphosphate kinases obtained through screening using the EITLEM algorithm, an enzyme catalytic kinetic parameter prediction model, aim to provide a variety of highly active wild-type polyphosphate kinases. Among them, PePPK predicts...k cat / K m It is 6.92s -1 ·mM -1 PsPPK predictions k cat / K m It is 8.05s -1 ·mM -1 RbPPK prediction k cat / K m It is 7.24s -1 ·mM -1 RrPPK prediction k cat / K m It is 10.18s -1 ·mM -1 In practical applications, these wild-type polyphosphokinases exhibit higher ATP generation efficiency than other wild-type polyphosphokinases, significantly improving reaction efficiency and reducing ATP consumption, thus offering economic advantages. The four wild-type polyphosphokinases PePPK, PsPPK, RbPPK, and RrPPK provided in this invention possess high activity compared to the control polyphosphokinase PPK2C. pa At 30°C, the enzyme activities were 36.3 times, 24.3 times, 24.4 times, and 36.5 times higher, respectively. The application of wild-type polyphosphokinases provided by this invention utilizes highly active wild-type polyphosphokinases to construct an ATP regeneration system, catalyzing the synthesis of ATP from the ATP byproduct ADP during the reaction, thus achieving ATP recycling. Simultaneously, these wild-type polyphosphokinases can serve as mutant templates to construct polyphosphokinase mutants with even higher activity. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 SDS-PAGE for PePPK, PsPPK, RbPPK, and RrPPK.

[0020] Figure 2The amount of ATP produced by PePPK, PsPPK, RbPPK, and RrPPK at 30 °C, 40 °C, and 50 °C, respectively.

[0021] Figure 3 For PePPK, PsPPK, RbPPK, RrPPK and PPK2C pa Application of regenerated ATP in glutathione production.

[0022] Figure 4 For PePPK, PsPPK, RbPPK, RrPPK and PPK2C pa Application of regenerated ATP in the production of 3'-adenosine-5'-phosphosulfate. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Direct synthesis from Paenirhodobacter enshiensis, Pararhodobacter sp., Rhodobacteraceae bacterium and Rugamonas rubra The PPK gene of ATCC 43154, which contains both NcoI and XhoI restriction sites, with the base GC added after the NcoI site, is synthesized into a PPK gene sequence with restriction sites and protective bases as shown in SEQ ID NO.1-4.

[0025] From Paenirhodobacter enshiensis The PPK (PePPK) gene:

[0026] ccatggctcgag ;SEQ ID NO.1.

[0027] From Pararhodobacter sp. The PPK (PsPPK) gene:

[0028] ccatggctcgag ;SEQ ID NO.2.

[0029] From Rhodobacteraceae bacterium The PPK (RbPPK) gene:

[0030] ccatggctcgag ; SEQ ID NO.3.

[0031] From Rugamonas rubra The PPK (RrPPK) gene of ATCC 43154:

[0032] ccatggGCATGAACCTGCAAGATCAAGAAATCTTGTCTCGTATCCATCGTGACATCGCTGACTCTTACGATGAAGAGCTGGAACTGGAACTGGAAGATCACCTGGAAGATGCGTACGCTGATCCGGCTGGTGGTCGTTCTGAAGCGGATAAAGCTGCACGTCGTGCTTACTTCCGTGAACTGTTCCGTCTGCAAGCTGAACTGGTTAAACTGCAAGACTGGGTTGTTGCGACCGGTCACAAAGTTGTTATCTTGTTCGAAGGTCGTGATGCAGCAGGTAAAGGCGGTGTTATCAAACGTATCACTCAGCGTCTGAATCCACGTGTTTGCCGTGTTGCAGCACTGCCGGCTCCGAACGATCGTGAACGCACTCAGTGGTACTTCCAGCGTTACGTTAGCCACCTGCCGGCTGCTGGTGAAATCGTATTGTTCGATCGTAGCTGGTACAACCGTGCTGGTGTTGAACGTGTTATGAACTTCTGCACCGATGAACAGTACGAAGAGTTCTTCCGTACCGTTCCGGAATTCGAACGTATGCTGGCTCGTTCCGGTATCCAGCTGGTTAAATACTGGTTCTCCATCTCTGACGAAGAACAGCACCTGCGTTTCTTGGGCCGTATCCACGATCCGCTGAAACAGTGGAAACTGTCTCCGATGGATCTGGAAAGCCGTCGTCGTTGGGAAGAATACACCAAAGCGAAAGAAATCATGCTGGAACGTACTCACATCCCGGAAGCGCCGTGGTGGGTTGTTCAAGCAGTTGACAAGAAACGTGCTCGTCTGAACTGCATCCACCACCTGTTGCAGCAGATGCCGTACGAAGAAGTAGCTCGTAGCTCTATCGATCTGCCGCAGCGTGAACGTCACGACGACTACGAACGTCAGCCGGTACCGCAAGAGATCTTGGTTCCGGAAATCTAC ctcgag ; SEQ ID NO.4.

[0033] from Pseudomonas aeruginosa PPK (PPK2Cpa )Gene:

[0034] ccatggctcgag ;SEQ ID NO.5.

[0035] The gene synthesis was completed by Beijing Qingke Biotechnology Co., Ltd.

[0036] PePPK amino acid sequence:

[0037] MEDLNQSAVPAREPAVTAVAPQSGPTGAETVPLSGEQPDTEPGEGRLEGPKVFPTVDPNAIRLAFESGKYPYPRRMGRPAYEKEKALLQAELLKVQIWAQETGQKFVILFEGRDAAGKGGTIKRFMEHLNPRFARVVALNKPTEVEKGQWFFQRYIQHLPTAGEMVFYDRSWYN RAGVERVMGFCTPSEYLEFMRQTPELERMLVRSGVRLYKYWFSVTRDEQRRRFAERGTDPLKRWKLSPIDIASLDKWDDYTEAKEAMFFYTDTADSPWVIVKSNDKKRARLNCMKHFLSTLDYPDKDPAIATLPDPLIVGHASHVVRASEHILGAALHPAQAAARTAAAKA; SEQ ID NO.6.

[0038] The amino acid sequence of PsPPK:

[0039] MTDTAAASPTESATPEPVETGSQPVATDVASGADGDALQRQTAPSGAASPRDAVRLAAEKGGPISPEVLKTAFESAHYPYPHKMARKPYEAEKARLQAELLKVQIWAQDTGQKFVMLFEGRDAAGKGGTIKRFTEHLNPRFARVVALNKPTESERGQWYFQRYIEHLP TAGEMVFYDRSWYNRAGVERVMGFCTPEEYLEFMRQAPEFERMLVRSGIRLYKFWFSVTPDEQRRRFAERETDPLKRWKLSPIDKASLDLWDEYTAAKEAMFFYTDTADAPWTIIKSKDKKRARLNAMKHFLSTLDYPGKDPEVVGKPDPLIVGHARHVMRHADF; SEQ ID NO.7.

[0040] The amino acid sequence of RbPPK:

[0041] MPQDAAAKPNGSVASQPVPQAAAQGPAGMPAGMPAGMPAGMPAGAAVQATALPGAPAQPRSYPRADPAEIARGFETGAYPYRDRMGVRAYEAEKMRLQAELLKVQIWTQETGQRFVLLFEGRDAAGKGGTIKRFMEHLNPRYARTVALTKPSDVEKGQWFFQRYLAHLPTAGEMVFYDRSWYNRAGVERVMGFCTPNEYLEFMREAPEIERMLVRSGIRLYKFWFSVTREEQRRRFLARETDPLKRWKLSPIDKASLDRWDDYTEAKEAMFFHTDTADAPWVIVKSDDKKRARLNVMKYFLASVDYPDKDLSVVGQPDPLIVGRASQVIHMPEGILTKATPEAMRRVGRARG; SEQ ID NO.8.

[0042] Amino acid sequence of RrPPK:

[0043] MNLQDQEILSRIHRDIADSYDEELELELEDHLEDAYADPAGGRSEADKAARRAYFRELFRLQAELVKLQDWVVATGHKVVILFEGRDAAGKGGVIKRITQRLNPRVCRVAALPAPNDRERTQWYFQRYVSHLPAAGEIVLFDRSWYNRAGVERVMNFCTDEQYEEFFRTVPEFERMLARSGIQLVKYWFSISDEEQHLRFLGRIHDPLKQWKLSPMDLESRRRWEEYTKAKEIMLERTHIPEAPWWVVQAVDKKRARLNCIHHLLQQMPYEEVARSSIDLPQRERHDDYERQPVPQEILVPEIY; SEQ ID NO.9.

[0044] PPK2C pa Amino acid sequence of

[0045] MFESAEVGHSIDKDTYEKAVIELREALLEAQFELKQQARFPVIILINGIEGAGKGETVKLLNEWMDPRLIEVQSFLRPSDEELERPPQWRFWRRLPPKGRTGIFFGNWYSQMLYARVEGHIKEAK LDQAIDAAERFERMLCDEGALLFKFWFHLSKKQLKERLKALEKDPQHSWKLSPLDWKQSEVYDRFVHYGERVLRRTSRDYAPWYVVEGADERYRALTVGRILLEGLQAALATKERAKRQPHAAPL VSSLDNRGLLDSLDLGQYLDKDAYKEQLAAEQARLAGLIRDKRFRQHSLVAVFEGNDAAGKGGAIRRVTDALDPRQYHIVPIAAPTEEERAQPYLWRFWRHIPARRQFTIFDRSWYGRVLVERIE SEQ ID NO.10.

[0046] Example 1: Construction of recombinant Escherichia coli BL21(DE3) expressing the PPK gene

[0047] PPK genes come from... Paenirhodobacter enshiensis, Pararhodobacter sp., Rhodobacteraceae bacterium and Rugamonas rubra ATCC 43154 was cloned into pET28a(+) via the NcoI and XhoI restriction sites. GC was added after the NcoI site to prevent frameshift mutations. The total volume of the restriction enzyme digestion system was typically 100 μL: 5000 ng plasmid or 2000 ng DNA fragment (as shown in SEQ ID NO. 1-4), 10 μL 10× Buffer, restriction endonucleases calculated at 1 μL corresponding to 1000 ng template, and sterile ddH2O added to a final volume of 100 μL. The reaction was carried out at 37 ℃ in a metal bath for 40 min. The corresponding enzymes were named PePPK, PsPPK, RbPPK, and RrPPK, with their corresponding amino acid sequences shown in SEQ ID NO. 6-9.

[0048] The total volume of the ligation system was 20 μL: 1 μL T4 DNA ligase, 2 μL 10×T4 DNA Ligase Buffer, 100 ng of the digested plasmid, and the fragment was added in a specific volume based on 5 times the number of plasmid moles. Deionized water was added to bring the total volume to 20 μL, and ligation was carried out at 4 ℃ for at least 12 h.

[0049] The constructed recombinant plasmid pET28a-PPK was transformed into [a specific plasmid] via thermal shock method. Escherichia coli BL21(DE3), the specific procedure is as follows: Take 5 μL of recombinant plasmid and add it to 100 μL of... Escherichia coli BL21(DE3) competent cells were placed in broth on ice for 30 min, then heat-shocked at 42 ℃ for 90 s, and immediately placed on ice for 2 min. 900 μL of LB liquid medium (10 g / L Tryptone, 5 g / L Yeast extract, 10 g / L NaCl) was added, and the cells were incubated at 37 ℃ and 200 rpm for 1 h. 200 μL of the culture was then spread onto LB solid medium containing kanamycin and incubated at 37 ℃ for 12 h. The resulting single colony was the recombinant BL21(DE3) (pET28a-PPK). Single colonies were picked and incubated overnight in LB liquid medium. The samples were then sent to Qingke Biotechnology for sequencing. After verification, the bacterial culture was stored in glycerol cryovials at -80 ℃.

[0050] Example 2: Expression, purification, and SDS-PAGE of PPK

[0051] Seed culture: The strain preserved in glycerol at -80 ℃ was inoculated into LB liquid medium containing 0.05 mg / mL kanamycin in test tubes and placed in a shaking incubator and cultured at 37 ℃ and 180 rpm for 12 h to form seed culture.

[0052] Shake flask culture and induction: The seed culture was inoculated into a shake flask containing 100 mL LB liquid medium (containing 0.05 mg / mL kanamycin) (350 μL of seed culture was added to every 50 mL LB liquid medium), and placed in a shaker. The culture was incubated at 37 ℃ and 180 rpm for three hours, and the OD of the bacterial culture was monitored. 600 When the expression level reached 0.8, 100 μL of isopropyl thiogalactoside (IPTG) at a concentration of 50 mg / mL was added to the bacterial culture for induction, and the culture was carried out at 16 °C for 12 h to induce PPK expression.

[0053] Protein purification: After induction culture, transfer the bacterial culture to a 50 mL centrifuge tube. Centrifuge at 4 ℃ and 4500 rpm for 12 minutes, discard the supernatant, and collect the bacterial cells (after discarding the supernatant, gently shake to remove as much culture medium as possible). Add 10 mL of Tris-HCl buffer solution (50 mM, pH=8.5) to the centrifuge tube using a 10 mL pipette, and repeatedly pipette to fully resuspend the bacterial cells. After resuspending, place the bacterial culture in an ice-water bath and sonicate to lyse the cells (2 s operation, 2 s interval, 40% power) for 30 minutes. After lysing the cells, centrifuge the lysate at 4 ℃ and 4500 rpm for 20 minutes and collect the supernatant (i.e., crude enzyme solution). Open both ends of the Ni-NTA affinity chromatography column, and after the protective buffer has drained naturally, add 30 mL of pre-chilled Tris-HCl (50 mM, pH=8.0) buffer solution. After the buffer solution has drained, slowly transfer the crude enzyme solution into the column. Once drained, add 30 mL of pre-cooled Tris-HCl solution containing 10 mM imidazole to elute any remaining proteins, and allow it to drain completely. Add 15 mL of pre-cooled Tris-HCl solution containing 350 mM imidazole to elute the target enzyme, and collect all the eluent using a 30 kDa ultrafiltration tube. Wash the Ni-NTA affinity chromatography column sequentially with 30 mL of Tris-HCl solution containing 350 mM imidazole and then with 30 mL of Tris-HCl solution containing 10 mM imidazole, and seal with 20% ethanol at 4 °C. The eluent collected in the ultrafiltration tube was centrifuged at 4000 rpm for 30 minutes at 4 °C. 15 mL of Tris-HCl buffer solution was added to the concentrated enzyme solution after centrifugation and ultrafiltration was performed again to dilute the imidazole in the enzyme solution. The resulting enzyme solution was transferred to a 1.5 mL centrifuge tube, and the protein content was determined using an Implen NanoPhotometer and temporarily stored at 4 °C.

[0054] Mix the crude enzyme solution and loading buffer at a ratio of 3:1 (v / v) and heat-treat at 95 °C for 5-10 min. Add an appropriate amount of electrophoresis buffer to the electrophoresis tank, then add 20 μL of sample to each well, and add 10 μL of Marker (M) to a single well. Set the voltage to a constant 120 volts and electrophoresis for approximately 50 min. Transfer the gel to a culture dish, wash with deionized water, and then pour in Coomassie Brilliant Blue G-250 staining solution to cover the gel. Stain on an 80 rpm shaker for 60 min. Discard the staining solution, wash 1-3 times with deionized water, and repeat the destaining process with shaking until the protein bands are clear.

[0055] The molecular weights of PePPK, PsPPK, RbPPK, and RrPPK were 40.14 kDa, 38.92 kDa, 40.67 kDa, and 37.23 kDa, respectively, which matched the size of the darkest band in the SDS-PAGE results, proving that PePPK, PsPPK, RbPPK, and RrPPK are all soluble and expressed at high levels. Figure 1 ).

[0056] Example 3 Determination of PPK activity

[0057] The enzyme reaction system and conditions are as follows: PBS buffer (2.67 mM KCl, 137.07 mM NaCl, 10 mM Na₂HPO₄, 1.84 mM KH₂PO₄), 5 mM MgCl₂, 5 mM ADP, 5 mM sodium hexametaphosphate, adjusted to pH 7.0, and the amount of enzyme added is 0.1 mg / mL. -1 Each system was reacted at 30 °C, 40 °C, and 50 °C for 15 min, respectively. The reaction was terminated by adding 4% (m / v) trichloroacetic acid, centrifuged, and the supernatant was filtered through a membrane. As a control of the recombinant polyphosphate kinase of this invention, [the following was used]... Pseudomonas aeruginosa The PPK gene PK22_PSEAE was cloned into the pET28a vector according to the method in Example 1 to construct an expression strain, and the enzyme was named PPK2C. pa (The gene sequence is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.10). Pure enzyme solution was obtained by inducing expression according to the method in Example 2, and the reaction was carried out according to the above method.

[0058] ATP content was determined by HPLC using a Waters-C18 column (25 cm × 4.6 mm, 5 µm). The mobile phase consisted of 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid methanol solution, followed by ultrasonic degassing for 15 min. Detection conditions were: UV 254 nm, column temperature 30 ℃, and flow rate 0.8 mL / min. –1 The injection volume was 2 µL, and the mobile phase ratio was aqueous phase:organic phase (v / v) = 1:9.

[0059] At 30 °C, PePPK, PsPPK, RbPPK, and RrPPK produced 2.72 mM, 1.82 mM, 1.83 mM, and 2.74 mM of ATP, respectively, which were significantly higher than the control PPK2C. pa 0.07 mM ATP produced Figure 2At 40 °C, PePPK, PsPPK, RbPPK, and RrPPK produced 3.39 mM, 2.42 mM, 2.2 mM, and 0.96 mM of ATP, respectively, which were significantly higher than the control PPK2C. pa 0.08 mM ATP was produced. At 50 °C, PePPK, PsPPK, RbPPK, and RrPPK produced 4.18 mM, 2.95 mM, 3 mM, and 0.54 mM ATP, respectively, compared to control PPK2C. pa It produces only 0.08 mM ATP.

[0060] Example 4: Application of PePPK, PsPPK, RbPPK, and RrPPK in Glutathione (GSH) Synthesis

[0061] The GSH synthesis system consists of 130 mM monosodium glutamate, 130 mM cysteine, 130 mM glycine, 50 mM magnesium chloride, 50 mM sodium hexametaphosphate, and 5 mM ATP, dissolved in 200 mM Tris-HCl, with the pH adjusted to 8.5. GshFHp originates from... Haemophilus parahaemolyticus GeneBank: WP_311418989.1. The amount of GshFHp pure enzyme added was 0.2 mg / mL. -1 The amount of PPK pure enzyme added was 0.2 mg / mL. -1 The reaction was carried out at 30 ℃ and 500 rpm, and samples were taken after 1–6 h. The reaction was terminated with 2% trichloroacetic acid solution. GSH content was determined by liquid chromatography (HPLC) using a Waters-C18 column (25 cm × 4.6 mm, 5 µm). The mobile phase consisted of 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid methanol solution, with ultrasonic degassing for 15 min. Detection conditions were: UV 220 nm, column temperature 30 ℃, and flow rate 1 mL / min. -1 The injection volume was 2 µL, and the mobile phase ratio was aqueous phase:organic phase (v / v) = 1:9.

[0062] In two-enzyme systems composed of PePPK, PsPPK, RbPPK, and RrPPK and GshFHp, respectively, 10.72 g·L⁻¹ were produced after 1 h of reaction. -1 8.86 g·L -1 4.46 g·L -1 and 2.91 g·L -1 GSH was collected, and the reaction continued, accumulating to 31.12 g·L⁻¹ at 6 h. -1 29.97 g·L -1 24.11 g·L -1 and 5.00 g·L-1 GSH, compared to PPK2C pa 1.46 g·L was produced at 1 h and 6 h, respectively. -1 and 2.13 g·L -1 GSH ( Figure 3 This demonstrates that PePPK, PsPPK, RbPPK, and RrPPK are effective in regenerating ATP in a two-enzyme system.

[0063] Example 5: Application of PePPK, PsPPK, RbPPK, and RrPPK in the synthesis of 3'-adenosine-5'-phosphosulfate (PAPS)

[0064] The 3'-adenosine-5'-phosphate sulfate synthase expression gene was ligated to pET-19b using seamless cloning ligation technology.

[0065] The gene for expressing 3'-adenosine-5'-phosphosulfate synthase with a homologous arm is shown in SEQ ID NO.11.

[0066] agatataccATGGCCATGGGCcaccaccaccaccaccactga; SEQ ID NO.11.

[0067] In SEQ ID NO.11, 1-21bp is the 5' homologous arm sequence, 10-2247bp is the 3'-adenosine-5'-phosphosulfate synthase expression gene, and 2248-2268bp is the 3' homologous arm sequence.

[0068] The total volume of the ligation system was 10 μL: After detecting the concentrations of the gene fragment and the vector fragment, they were mixed according to the Gibson assembly molar ratio of 1:3. The total volume of the gene and vector fragments was 5 μL. The 2×Basic Mix enzyme was mixed with the gene fragment in equal volume and incubated at 50 ℃ for 15 min, and then temporarily stored at 4 ℃.

[0069] The Gibson assembly product was introduced into Trans 10 competent cells via chemical transformation. The specific procedure was as follows: 10 μL of the recombinant plasmid was added to 100 μL of Trans 10 competent cell culture medium. The mixture was placed on ice for 30 min, heat-shocked at 42 ℃ for 30 s, and immediately placed on ice for 2 min. 500 μL of LB liquid medium (10 g / L Lyptone, 5 g / L Yeast extract, 10 g / L NaCl) was added, and the mixture was incubated at 37 ℃ and 200 rpm for 1 h. 100 μL of the culture was then spread onto LB solid medium containing ampicillin and incubated at 37 ℃ for 12 h. The resulting single colony was the recombinant strain Trans10 (pET19b-PAPS). Single colonies were picked and cultured overnight in LB liquid medium. The samples were sequenced, and after verification, the plasmid DNA was purified using the EasyPure Plasmid MiniPrep Kit.

[0070] The empty pET-28a plasmid and the four pET28a-PPK plasmids constructed above, along with the pET19b-PAPS plasmid, were introduced into the system via chemical transformation. Escherichia coli BL21(DE3) competent cells, with the pET-28a empty plasmid introduced as the control strain. The specific procedure is as follows: 200 ng of pET28a-PPK plasmid and pET19b-PAPS plasmid were added to 100 μL, respectively. Escherichia coliBL21(DE3) competent cells were placed in saturated broth on ice for 30 min, then heat-shocked at 42 °C for 90 s, and immediately placed on ice for 2 min. 900 μL of LB liquid medium (10 g / L Tryptone, 5 g / L Yeastextract, 10 g / L NaCl) was added, and the cells were incubated at 37 °C for 200 rpm for 1 h. 200 μL of the culture was then spread onto LB solid medium containing kanamycin and ampicillin, and incubated at 37 °C for 12 h. The resulting single colony was the recombinant BL21(DE3)-PPK-PAPS. Single colonies were picked, incubated overnight in LB liquid medium, and then the bacterial culture was stored in glycerol cryovials at -80 °C.

[0071] Seed culture: The strain preserved in glycerol at -80 ℃ was inoculated into LB liquid medium containing 0.05 mg / mL kanamycin and 0.10 mg / mL ampicillin in test tubes, and placed in a shaking incubator and cultured at 37 ℃ and 180 rpm for 12 h to form seed culture.

[0072] Shake flask culture and induction: The seed culture was inoculated into a shake flask containing 50 mL of fermentation medium (10 g / L NaCl, 10 g / L Lyptone, 5 g / L Yeast extract, 0.05 mg / mL kanamycin, 0.10 mg / mL ampicillin, 20 mM magnesium sulfate, 10 mM sodium hexametaphosphate) (1 mL seed culture was added to every 50 mL LB liquid medium). The flask was placed in a shaker and cultured at 37 ℃ and 200 rpm for 2.5 h. The OD of the bacterial culture was then monitored. 600 When the concentration reached 0.8, 0.5 mM isopropyl thiogalactoside (IPTG) was added to the bacterial culture for induction, and then the culture was carried out in a shaker at 30 °C for 24 h.

[0073] Cell disruption: After centrifugation at 4000 rpm for 10 min, the supernatant was discarded and the bacterial cells were collected. The cells were resuspended in Tris-HCl buffer (50 mM Tris, 300 mM NaCl, 20% glycerol, pH=7.4). The cells were then disrupted by high-pressure homogenizer at 800-1000 bar and centrifuged at 4000 rpm for 20 min to remove cell debris. The supernatant was filtered through a 0.22 μm filter and the PAPS content was determined by HPLC.

[0074] PAPS content was determined by HPLC using a Polyamine II column (4.6 × 250 mm, 12 nm). The mobile phase consisted of 50 mM KH₂PO₄ and 0.1% triethylamine solution, followed by ultrasonic degassing for 15 min. Detection conditions were: UV 254 nm, column temperature 25℃, and flow rate 0.6 mL / min. –1 The injection volume was 5 µL, and the detection time was 35 min.

[0075] In an intracellular system of *E. coli* composed of RrPPK, PsPPK, RbPPK, PePPK, and PAPS synthase, respectively, 25.71 mg·L⁻¹ of enzymes were synthesized after 24 h of fermentation. -1 32.98 mg·L -1 28.11 mg·L -1 and 37.96 mg·L - 1 PAPS was superior to 16.48 mg·L⁻¹ produced by the control strain (empty pET-28a plasmid). -1 and PPK2C pa The produced 21.23 mg·L -1 ( Figure 4 This demonstrates that RrPPK, PsPPK, RbPPK, and PePPK are effective in regenerating ATP in intracellular synthesis systems.

[0076] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of polyphosphate kinase PePPK in increasing ATP production, characterized in that, The amino acid sequence of the polyphosphate kinase PePPK is shown in SEQ ID NO.

6.

2. The application of polyphosphate kinase PePPK in the extracellular synthesis of glutathione or the intracellular synthesis of 3'-adenosine-5'-phosphosulfate, characterized in that, The amino acid sequence of the polyphosphate kinase PePPK is shown in SEQ ID NO.6.

Images