Multi-enzyme catalysis system and method for synthesizing functional glycan

Abstract

The invention belongs to the technical field of biosynthesis, and relates to a multi-enzyme catalysis system and method for synthesizing functional glycans. The system comprises a substrate module comprising nucleoside monophosphate, monosaccharide, polyphosphate and acceptor molecules; the nucleoside monophosphoric acid is one or more of uridine monophosphoric acid, adenosine monophosphoric acid, guanosine monophosphoric acid or cytidine monophosphoric acid; an enzyme module comprising a polyphosphate kinase, at least one remedial pathway synthetase, and at least one glycosyltransferase; the cofactor module comprises magnesium ions and optional inorganic pyrophosphatase; the system can realize in-situ regeneration of sugar nucleotide with nucleoside monophosphate as an initial cofactor and one-pot synthesis of target glycan. The invention systematically proposes that cheap NMP is used as a unique or main starting cofactor to replace expensive NTP or NDP for in-situ regeneration of various sugar nucleotides for the first time. Cofactor cost can be reduced by dozens of times, and a foundation is laid for large-scale production of glycans.

Description

Technical Field

[0001] This invention belongs to the field of biosynthesis technology and relates to a multi-enzyme catalytic system and method for synthesizing functional polysaccharides. Background Technology

[0002] Glycans (including oligosaccharides and polysaccharides) play a vital role in living organisms, participating extensively in numerous biological processes such as cell recognition, immune responses, signal transduction, pathogen infection, embryonic development, and tumor metastasis. However, these structurally complex and sequence-uniform functional glycans are present in extremely low amounts in nature, making direct extraction insufficient to meet research and clinical needs. Therefore, developing efficient, controllable, and large-scale preparation technologies has become one of the core tasks in the field of glycoscience.

[0003] Leloir-type glycosyltransferases (GTs) are key enzymes in the synthesis of complex polysaccharides in organisms. They efficiently, regio- and stereoselectively catalyze the formation of glycosidic bonds using activated sugar nucleotides (SNs) as glycosyl donors. Common SNs include uridine 5'-diphosphate galactose (UDP-Gal), UDP- N -Acetylgalactosamine (UDP- N -acetylgalactosamine, abbreviated as UDP-GalNAc), UDP- N -acetylglucosamine (UDP- NUDP-glucosamine (UDP-GlcNAc), UDP-glucuronic acid (UDP-GlcA), guanosine 5'-diphosphate L-fucose (GDP-Fuc), and cytidine 5'-monophosphate sialic acids (CMP-Sia) are among the enzymes used. GTs (glucosamine esters) can be used to reconstruct multi-enzyme cascades in vitro, mimicking the in vivo glycan synthesis pathway and achieving precise assembly of complex glycan chains. Compared to traditional chemical synthesis methods, this enzymatic strategy offers advantages such as simple steps, environmental friendliness, high selectivity, and no need for protection or deprotection, making it particularly suitable for preparing oligosaccharides or polysaccharides with well-defined structures and homogeneous functions. However, the widespread application of GTs, especially in large-scale preparation, is limited by two main factors: firstly, commercially available SNs (semiconductor sessiles) are extremely expensive. Secondly, the nucleoside diphosphates (NDP) or nucleoside monophosphates (NMP) released during the glycosylation reaction have a product-inhibiting effect on GTs, significantly reducing the reaction rate and yield.

[0004] To overcome the aforementioned limitations, early developments focused on in-situ regeneration systems for SNs (specimens and glycosaminoglycans) centered around phosphoenolpyruvate (PEP) and pyruvate kinase (PK). While these systems achieved gram-scale synthesis of various important glycans, PEP itself is unstable and costly, and its degradation product, pyruvate, inhibits PK activity. These inherent drawbacks severely limit the industrial application of this system. Therefore, developing an enzyme catalytic platform that can directly utilize the inexpensive NMP as a starting cofactor, efficiently and universally regenerate various SNs, and is suitable for the large-scale synthesis of various functional glycans has significant scientific and industrial application value. Summary of the Invention

[0005] To address the problems of high cost of glyconucleotides and insufficient efficiency or narrow applicability of cofactor regeneration systems in existing technologies, this invention provides a multi-enzyme catalytic system and method for synthesizing functional polysaccharides based on in-situ regeneration of glyconucleotides using nucleoside monophosphate. The core of this invention lies in the coupling of a highly efficient, broad-spectrum polyphosphokinase with salvage pathway synthases and glycosyltransferases. Only inexpensive nucleoside monophosphates and polyphosphates are needed to achieve continuous in-situ regeneration of various glyconucleotides, thereby driving the efficient synthesis of a series of important functional polysaccharides.

[0006] The technical solution adopted by this invention to solve its technical problem is: A multi-enzyme catalytic system for synthesizing functional polysaccharides, the system comprising: Substrate module: contains nucleoside monophosphate, monosaccharide, polyphosphate and receptor molecule; Enzyme module: contains polyphosphoric acid kinase, at least one salvage pathway synthase, and at least one glycosyltransferase; The system can achieve in-situ regeneration of glyconucleotides using the nucleoside monophosphate as a starting cofactor, and synthesize the target polysaccharide through a one-pot catalytic reaction.

[0007] Preferably, the nucleoside monophosphate is one or more of adenosine monophosphate (AMP), guanosine monophosphate (GMP), cytidine monophosphate (CMP), and uridine monophosphate (UMP).

[0008] Preferably, the polyphosphate is a linear polyphosphate with a degree of polymerization ≥3; the final concentration of the polyphosphate in the reaction system is 10 mM to 300 mM; more preferably, the polyphosphate is sodium hexametaphosphate.

[0009] Preferably, the polyphosphokinase is type III polyphosphokinase 2 (PPK2-III), which can catalyze the phosphorylation of AMP, GMP, CMP and UMP to generate the corresponding nucleoside triphosphates (NTPs); more preferably, the polyphosphokinase is derived from Erysipelotrichaceae bacterium (EbPPK).

[0010] Preferably, the salvage pathway synthase comprises a monosaccharide kinase and a glyconucleotide pyrophosphorylase; the monosaccharide kinase is selected from at least one of galactokinase (GalK), N-acetylhexosamine kinase (NahK), fucokinase (FKP), and glucuronide kinase (GlcAK); the glyconucleotide pyrophosphorylase is selected from at least one of UDP-glycopyrophosphorylase (USP), UDP-N-acetylgalactosamine pyrophosphorylase (AGX1), GDP-fucopyrophosphorylase (FKP), and CMP-sialic acid synthase (CSS).

[0011] Preferably, the glycosyltransferase is a Leloir-type glycosyltransferase capable of catalyzing glycosyltransfer reactions from glyconucleotide donors; more preferably, the glycosyltransferase is selected from at least one of β1,4-galactosyltransferase (NmLgtB), α1,4-galactosyltransferase (NmLgtC), β1,3-N-acetylgalactosamine transferase (HiLgtD), β1,3-galactosyltransferase (HiLgtD, Cvβ3GalT), β1,3-N-acetylglucosamine transferase (NmLgtA), α1,2-fucosyltransferase (GE2FT), α2,3-sialic acid transferase (PmST3), and hyaluronic acid synthase (PmHAS).

[0012] Preferably, the receptor molecule is a sugar or glycoside compound containing a free hydroxyl group; more preferably, the receptor molecule is salidroside, lactose, or its glycosylated derivatives (such as Tyrosol-Lac, Tyrosol-Gb3, Tyrosol-Gb4, Tyrosol-Gb5, LNT II, ​​LNT).

[0013] Preferably, the system further comprises magnesium ions and / or inorganic pyrophosphatase; more preferably, the inorganic pyrophosphatase is derived from Pasteurella multocida (PmPPA).

[0014] The present invention also provides a multi-enzyme catalytic method for synthesizing functional polysaccharides, which utilizes the above-mentioned multi-enzyme catalytic system to carry out a one-pot catalytic reaction.

[0015] Preferably, the reaction is carried out in a buffer system with a pH of 5.5-9.0, at a reaction temperature of 25-45°C, and for a reaction time of 1-72 hours.

[0016] Preferably, the concentrations of each component in the reaction system satisfy one or more of the following: a. The concentration of the nucleoside monophosphate is from 0.01 mM to 100 mM; b. The concentration of the monosaccharide is from 10 mM to 100 mM; c. The concentration of the receptor molecule is from 10 mM to 100 mM; d. The concentration of the polyphosphate is from 10 mM to 300 mM; e. The concentration of magnesium ions is from 10 mM to 300 mM.

[0017] More preferably, the concentration of the nucleoside monophosphate is selected according to its type: AMP concentration is 0.01-1.5 mM, GMP concentration is 4-30 mM, CMP concentration is 8-60 mM, and UMP concentration is 0.5-20 mM.

[0018] Preferably, the target glycans synthesized by the method include, but are not limited to, tumor-associated glycoantigens (such as Tyrosol-Globo H, Tyrosol-SSEA4), human milk oligosaccharides (such as LNT II, ​​LNT, LNnT, LNFP I, 3'-SL), glycosaminoglycans, and other polysaccharides (such as hyaluronic acid, HA).

[0019] Compared with the prior art, the beneficial effects of the present invention are at least reflected in the following aspects: 1. Significantly Reduced Costs: This invention is the first to systematically propose using inexpensive NMPs (such as UMP, CMP) as the sole or primary starting cofactor to replace expensive NTPs or NDPs for the in-situ regeneration of various SNs (UDP-Gal, UDP-GlcNAc, UDP-GalNAc, CMP-Sia, GDP-Fuc, etc.). The cost of cofactors can be reduced by tens of times, laying an economic foundation for the large-scale production of glycans. 2. Simplified reaction system: Since the PPK used (such as EbPPK) has a broad spectrum of NMP phosphorylation ability, especially effective in treating pyrimidine nucleotides (CMP, UMP), the regeneration of SNs such as CMP-Sia does not require the addition of cytidine kinase, which simplifies the type of enzyme and the reaction process. 3. High Efficiency and Versatility: The three-module (NTP regeneration module, SNs synthesis module, and glycosylation module) coupling system constructed in this invention achieves efficient recycling of cofactors and a high total conversion number. This system has been successfully applied to the multi-gram-level high-efficiency synthesis of more than ten important functional polysaccharides in three major categories (tumor-associated glycoantigens, human milk oligosaccharides, and hyaluronic acid), with yields all exceeding 71%, and the highest exceeding 99%, demonstrating the platform's powerful versatility and high efficiency. 4. Overcoming product inhibition: The PPK / PolyPn system continuously rephosphorylates the NDP / NMP generated in the reaction into NTP, maintaining the concentration of high-energy phosphate donors in the system. This effectively alleviates the GTs product inhibition caused by the accumulation of NDP / NMP and promotes the reaction towards product formation. 5. Simple operation and easy to scale up: The one-pot reaction reduces intermediate purification steps and cofactor additions, making the operation simple and easier to scale up and produce on a large scale. Attached Figure Description

[0020] Figure 1These are the thin-layer chromatography results of the synthesis of corresponding NTPs by phosphorylation of four NMPs with EbPPK; where 1 is AMP standard, 2 is ADP standard, 3 is GMP standard, 4 is GDP standard, 5 is UMP standard, 6 is UDP standard, 7 is CMP standard, 8 is CDP standard, and a is the reaction solution catalyzed by EbPPK. Figure 2 These are the thin-layer chromatography results of the Tyrosol-Lac synthesis reaction; where 1 is Gal standard, 2 is Salidroside standard, 3 is UMP standard, 4 is Gal-1-P standard, 5 is UDP-Gal standard, 6 is Tyrosol-Lac standard, and 7 is the reaction solution. Figure 3 It is the preparation of Tyrosol-Lac 1 H and 13 C NMR spectrum; where, (a) 1 (b) H nuclear magnetic resonance spectrum; 13 C nuclear magnetic resonance spectrum; Figure 4 These are the thin-layer chromatography results of the Tyrosol-Gb3 synthesis reaction; where 1 is Gal standard, 2 is Tyrosol-Lac standard, 3 is UDP-Gal standard, 4 is control group, and 5 is reaction solution; Figure 5 It is the prepared Tyrosol-Gb3 1 H and 13 C NMR spectrum; where, (a) 1 (b) H nuclear magnetic resonance spectrum; 13 C nuclear magnetic resonance spectrum; Figure 6 These are the thin-layer chromatography results of the Tyrosol-Gb4 synthesis reaction; where 1 is Tyrosol-Gb3 standard, 2 is UDP-GalNAc standard, 3 is control group, and 4 is reaction solution; Figure 7 It is the prepared Tyrosol-Gb4 1 H and 13 C NMR spectrum; where, (a) 1 (b) H nuclear magnetic resonance spectrum; 13 C nuclear magnetic resonance spectrum; Figure 8 These are the thin-layer chromatography results of the Tyrosol-Gb5 synthesis reaction; where 1 is Gal-1-P standard, 2 is UDP-Gal standard, 3 is Tyrosol-Gb4 standard, 4 is Tyrosol-Gb5 standard, 5 is control group, and 6 is reaction solution; Figure 9It is the prepared Tyrosol-Gb5 1 H and 13 C NMR spectrum; where, (a) 1 (b) H nuclear magnetic resonance spectrum; 13 C nuclear magnetic resonance spectrum; Figure 10 These are the thin-layer chromatography results of the Tyrosol-Globo H synthesis reaction; among them, 1 is AMP standard, 2 is GMP standard, 3 is Fuc standard, 4 is GDP-Fuc standard, 5 is Tyrosol-Gb5 standard, 6 is Tyrosol-Globo H standard, 7 is control group, and 8 is reaction solution; Figure 11 It is the prepared Tyrosol-Globo H 1 H and 13 C NMR spectrum; where, (a) 1 (b) H nuclear magnetic resonance spectrum; 13 C nuclear magnetic resonance spectrum; Figure 12 These are the thin-layer chromatography results of the Tyrosol-SSEA4 synthesis reaction; where 1 is CMP standard, 2 is CTP standard, 3 is Sia standard, 4 is CMP-Sia standard, 5 is Tyrosol-Gb5 standard, and 6 is the reaction solution. Figure 13 It is the prepared Tyrosol-SSEA4 1 H and 13 C NMR spectrum; where, (a) 1 (b) H nuclear magnetic resonance spectrum; 13 C nuclear magnetic resonance spectrum; Figure 14 These are the thin-layer chromatography results of the LNT II synthesis reaction; where 1 is UMP standard, 2 is UDP-GlcNAc standard, 3 is GlcNAc standard, 4 is Lactose standard, and 5 is the reaction solution. Figure 15 It is the prepared LNT II 1 H and 13 C NMR spectrum; where, (a) 1 (b) H nuclear magnetic resonance spectrum; 13 C nuclear magnetic resonance spectrum; Figure 16 These are the thin-layer chromatography results of the LNnT synthesis reaction; among them, 1 is UMP standard, 2 is Gal standard, 3 is Gal-1-P standard, 4 is UDP-Gal standard, 5 is LNT II standard, 6 is LNnT standard, 7 is control group, and 8 is reaction solution; Figure 17 It is the prepared LNnT 1 H and 13 C NMR spectrum; where, (a) 1 (b) H nuclear magnetic resonance spectrum; 13 C nuclear magnetic resonance spectrum; Figure 18 These are the thin-layer chromatography results of the LNT synthesis reaction; among them, 1 is UMP standard, 2 is Gal standard, 3 is Gal-1-P standard, 4 is UDP-Gal standard, 5 is LNT II standard, 6 is control group, and 7 is reaction solution; Figure 19 It is the prepared LNT. 1 H and 13 C NMR spectrum; where, (a) 1 (b) H nuclear magnetic resonance spectrum; 13 C nuclear magnetic resonance spectrum; Figure 20 These are the thin-layer chromatography results of the LNFP I synthesis reaction; among them, 1 is AMP standard, 2 is ATP standard, 3 is GMP standard, 4 is GTP standard, 5 is Fuc standard, 6 is Fuc-1-P standard, 7 is GDP-Fuc standard, 8 is LNT standard, and 9 is the reaction solution. Figure 21 It is the prepared LNFP I 1 H and 13 C NMR spectrum; where, (a) 1 (b) H nuclear magnetic resonance spectrum; 13 C nuclear magnetic resonance spectrum; Figure 22 These are the thin-layer chromatography results of the 3'-SL synthesis reaction; where 1 is CMP standard, 2 is CTP standard, 3 is Sia standard, 4 is CMP-Sia standard, 5 is Lactose standard, and 6 is the reaction solution. Figure 23 It is the prepared 3'-SL 1 H and 13 C NMR spectrum; where, (a) 1 (b) H nuclear magnetic resonance spectrum; 13 C nuclear magnetic resonance spectrum; Figure 24 These are the thin-layer chromatography results of the HA synthesis reaction; among them, 1 is GlcNAc standard, 2 is UDP-GlcNAc standard, 3 is GlcA standard, 4 is UDP-GlcA standard, 5 is HA standard, and 6 is reaction solution. Detailed Implementation

[0021] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to the following embodiments. In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the reagents and materials used are commercially available.

[0022] Example 1: Enzyme Expression and Purification Recombinant expression vectors for the enzymes NahK, GalK, USP, FKP, AGX1, CSS, GlcAK, PmPPA, EbPPK, NmLgtA, NmLgtB, NmLgtC, HiLgtD, PmST3, PmHAS, and Cvβ3GalT were obtained according to literature methods. All of these enzymes are known enzymes with publicly disclosed nucleotide and amino acid sequences.

[0023] The amino acid sequence of α-1,2-fucosyltransferase (GE2FT) is shown in WP_143414822.1, and the encoding nucleotide sequence is shown in NZ_CP041632.1. The artificially synthesized GE2FT gene was inserted into the pET30a vector between the NdeI and XhoI restriction sites via enzyme digestion, resulting in a recombinant vector of α-1,2-fucosyltransferase GE2FT.

[0024] The above recombinant expression vector was transformed into... E. coli BL21(DE3) competent cells. Single colonies were picked and cultured in LB medium containing the appropriate antibiotic until the OD600 reached approximately 0.6-0.8. 0.2 mM IPTG was added, and expression was induced at 16-25 °C for 20 hours. Bacterial cells were collected using a vertical centrifuge (8000 rpm, 10 min, 4 °C). The bacterial resuspended in buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl). The bacterial resuspended was then subjected to sonication in an ice-water mixture (40 W, 3 s sonication, 2 s interval, 30 min). The disrupted bacterial solution was centrifuged (4 °C, 12000 rpm, 30 min), and the supernatant was collected.

[0025] The proteins encoded by the above-mentioned enzyme genes all contain six histidine residues and can be purified using immobilized metal ion affinity chromatography. Therefore, the collected supernatant was purified using nickel ion affinity chromatography, following the instructions of the relevant chromatography materials. The main steps were as follows: mixing the supernatant with the chromatography medium or passing it through a chromatography column; washing away impurities with a buffer containing 30 mM imidazole; eluting the target protein with a buffer containing 500 mM imidazole; ultrafiltration, desalting, and concentration to obtain a recombinant protein solution with a purity exceeding 90%. This solution was then aliquoted and stored at -20 °C for later use. The activity of all enzymes was verified using their corresponding substrate transformation experiments.

[0026] Example 2: Substrate Adaptability Study of EbPPK Ideally, a polyphosphokinase should efficiently phosphorylate all four common nucleoside monophosphates (NMPs) and catalyze the formation of their corresponding nucleoside triphosphates (NTPs). Using 10 mM AMP, GMP, UMP, or CMP as substrates, 20 mM sodium hexametaphosphate as the phosphate donor, and 20 mM MgCl2 as the divalent metal ion, the pH of the mixed reaction solution was adjusted to 7.5. 1 mg / mL EbPPK was added, and the reaction was carried out overnight at 37 °C. Thin-layer chromatography (TLC) detected the formation of the four nucleoside triphosphates. Figure 1 ).

[0027] The above results demonstrate that the EbPPK selected in this invention can efficiently identify four common NMPs and catalyze the generation of corresponding NTPs.

[0028] Example 3: Synthesis of Tyrosol-Lac (tyrosol lactoside) using UMP as the sole cofactor The reaction system (200 mL) contained: 100 mM Tris-HCl (pH 5.5), 50 mM rhodioloside (receptor sugar), 60 mM galactose, 2 mM UMP, 96 mM MgCl2, 96 mM sodium hexametaphosphate, EbPPK (220 mg), BiGalK (220 mg), BiUSP (220 mg), PmPPA (110 mg), and NmLgtB (220 mg). The system was reacted at 37 ℃ and 220 rpm for 9 hours. TLC monitoring showed that the receptor was completely consumed. Figure 2 The reaction solution was terminated with ethanol, centrifuged, purified by C18 column chromatography and BioGel P-2 gel filtration to obtain a white solid, Tyrosol-Lac, in 96% yield. The structure of the product was verified by nuclear magnetic resonance spectroscopy. Figure 3 ).

[0029] The above results demonstrate that Tyrosol-Lac was successfully prepared using the method provided in this invention.

[0030] Example 4: Synthesis of Tyrosol-Gb3 (tyrosol-Gb3 or tyrosol-trihexosylceramide) using UMP as the sole cofactor The reaction system (180 mL) contained: 100 mM Tris-HCl (pH 7.5), 50 mM Tyrosol-Lac, 60 mM galactose, 2 mM UMP, 96 mM MgCl2, 96 mM sodium hexametaphosphate, EbPPK (180 mg), BiGalK (180 mg), BiUSP (180 mg), PmPPA (90 mg), and NmLgtC (180 mg). The reaction was carried out at 37 °C for 6 hours. The reaction was monitored by TLC until completion. Figure 4 The product was purified by C18 column chromatography to obtain Tyrosol-Gb3 powder in 98% yield. The structure of the product was verified by nuclear magnetic resonance spectroscopy. Figure 5 ).

[0031] The above results demonstrate that Tyrosol-Gb3 was successfully prepared using the method provided in this invention.

[0032] Example 5: Synthesis of Tyrosol-Gb4 (tyrosol-Gb4 or tyrosol-tetrahexosylceramide) using UMP as the sole cofactor The reaction system (700 mL) contained: 100 mM Tris-HCl (pH 7.5), 10 mM Tyrosol-Gb3, 12 mM MGaNAc, 1.5 mM UMP, 19.2 mM MgCl2, 19.2 mM sodium hexametaphosphate, 1 mM DTT, EbPPK (700 mg), BiNahK (700 mg), AGX1 (700 mg), PmPPA (350 mg), and LgtD (700 mg). The reaction was carried out at 37 °C for 4.5 hours. TLC monitoring showed substrate depletion (…). Figure 6 The product was purified by C18 column chromatography to obtain a white solid, Tyrosol-Gb4, in 94% yield. The structure of the product was verified by nuclear magnetic resonance spectroscopy. Figure 7 ).

[0033] The above results demonstrate that Tyrosol-Gb4 was successfully prepared using the method provided in this invention.

[0034] Example 6: Synthesis of Tyrosol-Gb5 (tyrosol-Gb5 or tyrosol-pentahexose ceramide) using UMP as the sole cofactor The reaction system (250 mL) contained: 100 mM Tris-HCl (pH 7.0), 20 mM Tyrosol-Gb4, 40 mM galactose, 20 mM UMP, 64 mM MgCl2, 64 mM sodium hexametaphosphate, 10 mM DTT, EbPPK (250 mg), BiGalK (250 mg), BiUSP (250 mg), PmPPA (125 mg), and LgtD (250 mg). The reaction was carried out at 30 °C for 48 hours. The reaction progress was confirmed by TLC. Figure 8 Tyrosol-Gb5 was obtained after purification using a C18 column and BioGel P-2, with a yield of 71%. The product structure was verified by NMR spectroscopy. Figure 9 ).

[0035] The above results demonstrate that Tyrosol-Gb5 was successfully prepared using the method provided in this invention.

[0036] Example 7: Synthesis of Tyrosol-Globo H using AMP and GMP as cofactors The reaction system (100 mL) contained: 100 mM Tris-HCl (pH 8.0), 1.5 mM AMP, 4 mM GMP, 24 mM MgCl2, 24 mM sodium hexametaphosphate, EbPPK (150 mg), BiFKP (200 mg), PmPPA (50 mg), GE2FT (100 mg), 15 mM fucose, and 10 mM Tyrosol-Gb5. The reaction was carried out at 37 °C until TLC showed substrate depletion. Figure 10 The reaction solution was purified by C18 column chromatography and BioGel P-2 to obtain Tyrosol-Globo H in 99% yield. The product structure was verified by nuclear magnetic resonance spectroscopy. Figure 11 ).

[0037] The above results demonstrate that Tyrosol-Globo H was successfully prepared using the method provided in this invention.

[0038] Example 8: Synthesis of Tyrosol-SSEA4 using CMP as a cofactor The reaction system (100 mL) contained: 100 mM Tris-HCl (pH 8.5), 10 mM Tyrosol-Gb5, 20 mM acetylneuraminic acid, 8 mM CMP, 192 mM MgCl2, 192 mM sodium hexametaphosphate, EbPPK (200 mg), NmCSS (100 mg), PmPPA (50 mg), and PmST3 (100 mg). The reaction was carried out at 37 °C for 24 hours. The reaction was monitored by TLC until completion. Figure 12 Tyrosol-SSEA4 was obtained by purification using a C-18 column and BioGel P-2, with a yield of 87%. The structure of the product was verified by nuclear magnetic resonance spectroscopy. Figure 13 ).

[0039] The above results demonstrate that Tyrosol-SSEA4 was successfully prepared using the method provided in this invention.

[0040] Example 9: Synthesis of LNT II (lactose-N-neotetrasaccharide a) using UMP as the sole cofactor The reaction system (350 mL) contained: 20 mM lactose, 24 mM GlcNAc, 2 mM UMP, 38.4 mM MgCl2, 38.4 mM sodium hexametaphosphate, EbPPK (350 mg), BiNahK (350 mg), AGX1 (350 mg), PmPPA (175 mg), LgtA (700 mg), pH 7.5. The reaction was carried out at 37 °C for 18 hours. The reaction was monitored by TLC until completion. (See attached image) Figure 14 The reaction solution was purified by activated carbon column chromatography and BioGel P-2 to obtain LNT II in 77% yield. The product structure was verified by nuclear magnetic resonance spectroscopy. Figure 15 ).

[0041] The above results demonstrate that LNT II was successfully prepared using the method provided in this invention.

[0042] Example 10: Synthesis of LNnT (lacto-N-neotetrasaccharide) using UMP as the sole cofactor The reaction system (100 mL) contained: 100 mM Tris-HCl (pH 5.5), 20 mM LNT II, ​​24 mM galactose, 10 mM UMP, 52.8 mM MgCl2, 52.8 mM sodium hexametaphosphate, EbPPK (100 mg), BiGalK (100 mg), BiUSP (100 mg), PmPPA (50 mg), and NmLgtB (100 mg). The reaction was carried out at 37 °C for 19 hours. The reaction was monitored by TLC until completion. Figure 16LNnT was obtained by purification using activated carbon column chromatography and BioGel P-2, with a yield of 86%. The product structure was verified by nuclear magnetic resonance spectroscopy. Figure 17 ).

[0043] The above results demonstrate that LNnT was successfully prepared using the method provided in this invention.

[0044] Example 11: Synthesis of LNT (lactose-N-neotetrasaccharide) using UMP as the sole cofactor The reaction system (100 mL) contained: 100 mM Tris-HCl (pH 7.5), 20 mM LNT II, ​​24 mM galactose, 0.5 mM UMP, 96 mM MgCl2, 96 mM sodium hexametaphosphate, EbPPK (100 mg), BiGalK (100 mg), BiUSP (100 mg), PmPPA (50 mg), and Cvβ3GalT (100 mg). The reaction was carried out at 37 °C for 20 hours. TLC monitoring showed complete conversion of LNT II. Figure 18 LNTs were obtained by purification using activated carbon column chromatography and BioGel P-2, with a yield of 84%. The product structure was verified by nuclear magnetic resonance spectroscopy. Figure 19 ).

[0045] The above results demonstrate that LNTs were successfully prepared using the method provided by this invention.

[0046] Example 12: Synthesis of LNFP I (lactose-N-fucopentose I) using AMP and GMP as cofactors The reaction system (30 mL) contained: 100 mM Tris-HCl (pH 8.0), 6 mM AMP, 30 mM GMP, 96 mM MgCl2, 96 mM sodium hexametaphosphate, EbPPK (45 mg), BiFKP (60 mg), PmPPA (15 mg), GE2FT (60 mg), 60 mM fucose, and 50 mM LNT. The reaction was carried out at 37 °C for approximately 47 hours. Complete consumption of LNT was confirmed by TLC. Figure 20 LNFP I was obtained by purification using activated carbon column chromatography and BioGel P-2, with a yield of 84%. The product structure was verified by nuclear magnetic resonance spectroscopy. Figure 21 ).

[0047] The above results demonstrate that LNFP I was successfully prepared using the method provided in this invention.

[0048] Example 13: Synthesis of 3'-SL (3'-sialyl lactose) using CMP as a cofactor The reaction system (40 mL) contained: 100 mM Tris-HCl (pH 8.0), 50 mM lactose, 60 mM N-acetylneuraminic acid, 30 mM CMP, 78 mM MgCl2, 78 mM sodium hexametaphosphate, EbPPK (80 mg), NmCSS (40 mg), PmPPA (20 mg), and PmST3 (40 mg). The reaction was carried out at 37 °C for 18 hours. The reaction progress was monitored by TLC. Figure 22 The product was purified by activated carbon column chromatography and BioGel P-2 to obtain 3'-SL in 82% yield. The structure of the product was verified by nuclear magnetic resonance spectroscopy. Figure 23 ).

[0049] The above results demonstrate that 3'-SL was successfully prepared using the method provided in this invention.

[0050] Example 14: Synthesis of hyaluronic acid (HA) using UMP and AMP as cofactors The reaction system (200 mL) contains: 100 mM Tris-HCl (pH 8.0), 20 mM glucuronic acid, and 20 mM... N α-acetylglucosamine, 0.1 mM AMP, 20 mM UMP, 64 mM MgCl2, 64 mM sodium hexametaphosphate, 10 mM KCl, 1.5 mM MnCl2, EbPPK (200 mg), BiNahK (100 mg), AGX1 (100 mg), AtGlcAK (100 mg), AtUSP (100 mg), PmPPA (50 mg), PmHAS (800 mg). The reaction was carried out at 30 °C for 48 hours. HA formation was monitored by TLC. Figure 24 The reaction solution was boiled, centrifuged, dialyzed (7 kDa MWCO), and freeze-dried to obtain white flocculent HA.

[0051] The above results demonstrate that HA was successfully prepared using the method provided by this invention.

[0052] The above examples demonstrate that the multi-enzyme catalytic method using NMP as an initiating cofactor provided by the present invention can efficiently and economically synthesize a variety of complex and functionally important polysaccharides, and has broad prospects for industrial application.

Claims

1. A multi-enzyme catalytic system for synthesizing functional polysaccharides, characterized in that, The system includes: Substrate module: contains nucleoside monophosphate, monosaccharide, polyphosphate and receptor molecule; Enzyme module: contains polyphosphoric acid kinase, at least one salvage pathway synthase, and at least one glycosyltransferase; The system is capable of in-situ regeneration of glyconucleotides starting with the nucleoside monophosphate as a cofactor, and catalyzes the glycosylation of the receptor molecule to synthesize the target glycan.

2. The multi-enzyme catalytic system according to claim 1, characterized in that, The nucleoside monophosphate is one or more of adenosine monophosphate, guanosine monophosphate, cytidine monophosphate, and uridine monophosphate.

3. The multi-enzyme catalytic system according to claim 1, characterized in that, The polyphosphokinase is a type III polyphosphokinase, which can catalyze the phosphorylation of at least one of adenosine monophosphate, guanosine monophosphate, cytidine monophosphate and uridine monophosphate to generate the corresponding nucleoside triphosphate.

4. The multi-enzyme catalytic system according to claim 1, characterized in that, The salvage pathway synthases include monosaccharide kinases and glyconucleotide pyrophosphorylases; the monosaccharide kinases are selected from at least one of galactokinase, N-acetylhexosamine kinase, fucokinase, and glucuronide kinase; the glyconucleotide pyrophosphorylases are selected from at least one of UDP-glycopyrophosphorylase, UDP-N-acetylgalactosamine pyrophosphorylase, GDP-fucopyrophosphorylase, and CMP-sialic acid synthase.

5. The multi-enzyme catalytic system according to claim 1, characterized in that, The glycosyltransferase is a Leloir-type glycosyltransferase that can catalyze glycosylation reactions in which glycosyl groups are provided by UDP-galactose, UDP-N-acetylgalactosamine, UDP-N-acetylglucosamine, GDP-fucose, or CMP-sialic acid.

6. The multi-enzyme catalytic system according to claim 1, characterized in that, The receptor molecule is a sugar or glycoside compound containing a free hydroxyl group.

7. The multi-enzyme catalytic system according to claim 1, characterized in that, The target polysaccharide is an oligosaccharide or polysaccharide containing a specific glycosidic bond structure determined by the glyconucleotide and glycosyltransferase.

8. The multi-enzyme catalytic system according to claim 1, characterized in that, The system also includes an inorganic pyrophosphatase for hydrolyzing the pyrophosphate generated in the reaction.

9. A multi-enzyme catalytic method for synthesizing functional polysaccharides, characterized in that, Using the multi-enzyme catalytic system of any one of claims 1-8, the substrate module and the enzyme module are mixed to perform a one-pot catalytic reaction to generate the target polysaccharide.

10. The multi-enzyme catalysis method according to claim 9, characterized in that, The reaction conditions must meet one or more of the following: a. The initial concentration of the nucleoside monophosphate is from 0.01 mM to 100 mM; b. The initial concentration of the monosaccharide is from 10 mM to 100 mM; c. The initial concentration of the receptor molecule is from 10 mM to 100 mM; d. The initial concentration of the polyphosphate is from 10 mM to 300 mM; e. The concentration of magnesium ions in the reaction system is from 10 mM to 300 mM.

Images