Enzyme composition and application thereof in preparation of lactose-N-trisaccharide

Abstract

The invention relates to the field of bioengineering, in particular to an enzyme composition and application thereof in preparation of lactose-N-trisaccharide. In particular, the present invention provides an enzyme composition comprising: N-acetylaminohexosyl-1-kinase, N-acetylglucosamine-1-phosphate uridine transferase, beta-1, 3-N-acetylglucosaminotransferase, and a polyphosphate kinase. A four-enzyme system is screened through an enzyme screening modification technology, the four enzymes are co-expressed, 100-500 mM of a substrate is successfully converted within 5 h through a one-pot method, and the conversion rate reaches up to 95% or above. The polyphosphokinase screened by the invention can simultaneously carry out ATP (adenosine triphosphate) and UTP (uridine triphosphate) circulation, has the characteristics of strong affinity, high conversion speed and good stability, can utilize cheap sodium hexametaphosphate as a substrate, successfully reduces the reaction cost, and has the strength of production amplification.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, in particular to an enzyme composition and application thereof in preparing lactose-N-triose. Background Art

[0002] Lactose-N-triose (LNT II, ​​GlcNAcβ1-3Galβ1-4Glc) is an important structural unit of human milk oligosaccharides (HMOs), and can be used as a synthetic precursor for lactose-N-neotetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4Glc) and lactose-N-tetraose (LNT, Galβ1-3GlcNAcβ1-3Galβ1-4Glc). These two tetrasaccharides have been certified as Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration (FDA). Lactose-N-neotetraose was approved twice by the National Health Commission in September 2023 and September 2024 as a new type of food nutrient fortifier, which can be added to infant formula, modified milk powder (for children) and special medical infant food. At the same time, the application for lactose-N-tetraose as a new type of food additive was accepted by the National Health Commission in January 2024.

[0003] In 2002, researchers at the French National Center for Scientific Research used modified Escherichia coli JM109 (lacY + Z - ) plasmid expressing β-1,3-N-acetylglucosamine transferase (NmLgtA) was used for high-density fermentation to synthesize 6g / L of lactose-N-trisaccharide. The research group of Christoph Albermann at the University of Stuttgart in Germany integrated the gene encoding NmLgtA into the chromosome of Escherichia coli, and after optimizing the fermentation carbon source, the lactose-N-trisaccharide exceeded 15g / L. The research group of Mu Wanmeng at Jiangnan University optimized the plasmid copy number and ribosome binding site sequence, and further achieved an E. coli fermentation level of 46.2g / L of lactose-N-trisaccharide. In addition to using E. coli as the chassis cell, the research group of Liu Long at Jiangnan University used the modified Bacillus subtilis BY01 (lacY + ) plasmid expresses NmLgtA and strengthens the supply of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) to achieve the synthesis of lactose-N-triose 2.98 g / L. The prior art discloses that the fermentation yield of the modified Kluyveromyces StrainKL-2-08 (∆LAC4::HPLgtA) and StrainKM-2-08 (∆LAC4::HPLgtA) exceeds 20 g / L.

[0004] Compared with microbial fermentation, biocatalytic synthesis of human milk oligosaccharides has the advantages of less impurities, controllable reaction, and high product concentration. Xi Chen's research group at the University of California, Davis, used N-acetylglucosamine-1-kinase NahK, N-acetylglucosamine uridyltransferase PmGlmU, inorganic pyrophosphatase PmPpA, and β1-3-N-acetylglucosamine transferase NmLgtA to catalyze the synthesis of lactose-N-trisaccharide in one step.

[0005] However, the existing biocatalytic method requires the exogenous addition of expensive cofactors adenosine triphosphate (ATP) and uridine triphosphate (UTP). The present invention aims to introduce a low-cost ATP / UTP mixed regeneration system that relies on polyphosphate and polyphosphate kinase, greatly reducing the amount of cofactors, thereby reducing the material cost of biocatalytic synthesis of lactose-N-triose. Summary of the invention

[0006] In view of this, the present invention provides an enzyme composition and its application in the preparation of lactose-N-triose. The present invention aims at the shortcomings of the existing fermentation method for synthesizing LNT II, ​​such as complex engineering bacteria construction, difficulty of microorganisms to tolerate extreme environments, slow reaction speed, and low product yield. Through enzyme screening and transformation technology, a 4-enzyme system including N-acetylglucosamine-1-kinase, N-acetylglucosamine-1-phosphate uridyltransferase, β-1,3-N-acetylglucosamine transfer and polyphosphate kinase is screened, and N-acetylglucosamine-1-kinase and polyphosphate kinase, N-acetylglucosamine-1-phosphate uridyltransferase and β-1,3-N-acetylglucosamine transferase are co-expressed, and 100~500mM substrate is successfully converted in one pot within 5h, with a conversion rate of more than 95%. And the polyphosphate kinase screened by the present invention can simultaneously cycle ATP and UTP, has the characteristics of strong affinity, fast conversion speed and good stability, can use cheap sodium hexametaphosphate as a substrate, successfully reduces the cost of the reaction, and has the strength of production amplification.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides an enzyme composition, comprising: N-acetylglucosamine-1-kinase, N-acetylglucosamine-1-phosphate uridyltransferase, β-1,3-N-acetylglucosaminyltransferase and polyphosphate kinase.

[0009] In some embodiments of the present invention, in the above enzyme composition, the N-acetylglucosamine-1-kinase is derived from Clostridiales bacterium, the N-acetylglucosamine-1-phosphate uridyltransferase is derived from Mycobacterium tuberculosis, the β-1,3-N-acetylglucosaminyltransferase is derived from Haemophilusinfluenzae and the polyphosphate kinase is derived from Dysgonomonas sp.

[0010] The present invention also provides application of the enzyme composition in preparing lactose-N-triose.

[0011] The present invention also provides a method for preparing lactose-N-triose, which uses N-acetylglucosamine, adenosine triphosphate, uridine triphosphate, lactose, magnesium chloride and phosphate as raw materials to obtain the lactose-N-triose under the catalytic action of the above enzyme composition.

[0012] In some embodiments of the present invention, in the above preparation method, the enzyme composition exists in the form of a host expressing each enzyme, an enzyme solution or an immobilized enzyme.

[0013] In some embodiments of the present invention, in the above preparation method, the host contains a vector for expressing each enzyme or a vector for co-expressing two enzymes.

[0014] In some embodiments of the present invention, in the above-mentioned preparation method, the co-expression vector includes: co-expression vector 1 and co-expression vector 2; the co-expression vector 1 includes: a nucleic acid molecule expressing the N-acetylglucosamine-1-kinase and a nucleic acid molecule expressing the polyphosphate kinase; the co-expression vector 2 includes: a nucleic acid molecule expressing the N-acetylglucosamine-1-phosphate uridyltransferase and a nucleic acid molecule expressing the β-1,3-N-acetylglucosaminyltransferase.

[0015] In some embodiments of the present invention, in the above-mentioned preparation method, the chassis strain of the host includes: Escherichia coli.

[0016] In some embodiments of the present invention, in the above-mentioned preparation method, the added amount of the N-acetylglucosamine-1-kinase is 1100~3600 U, the added amount of the N-acetylglucosamine-1-phosphate uridyltransferase is 2800~5400 U, the added amount of the β-1,3-N-acetylglucosamine transferase is 5000~7200 U and the added amount of the polyphosphate kinase is 4200~6000 U.

[0017] In some embodiments of the present invention, in the above preparation method, the amount of N-acetylhexosamine-1-kinase added is 1960 U, 2430 U or 3560 U; and / or

[0018] The added amount of the N-acetylglucosamine-1-phosphate uridyltransferase is 2900 U, 3350 U or 4250 U; and / or

[0019] The added amount of the β-1,3-N-acetylglucosamine transferase is 4150U, 5100U or 6790U; and / or

[0020] The added amount of the polyphosphokinase is 2760U, 3980U or 5420U.

[0021] In some embodiments of the present invention, in the above preparation method, the catalytic temperature is 33-40° C., and the pH value is 7.5-8.0.

[0022] In some embodiments of the present invention, in the above preparation method, the catalytic temperature is 33°C, 35°C or 40°C.

[0023] In some embodiments of the present invention, in the above preparation method, the catalytic pH value is 7.5, 7.8 or 8.0.

[0024] In some embodiments of the present invention, in the above preparation method, the molar ratio of the N-acetylglucosamine, the adenosine triphosphate, the uridine triphosphate, the lactose, the magnesium chloride and the phosphate is: (100~500): (2~9): (2~10): (100~580): 60: (50~240).

[0025] In some embodiments of the present invention, in the above preparation method, the concentration of N-acetylglucosamine is 100-500 mM; and / or

[0026] The concentration of the adenosine triphosphate is 2-9 mM; and / or

[0027] The concentration of the uridine triphosphate is 2-10 mM; and / or

[0028] The concentration of the lactose is 100-580 mM; and / or

[0029] The concentration of magnesium chloride is 60 mM; and / or

[0030] The concentration of the phosphate radical is 50-240 mM.

[0031] The present invention provides an enzyme composition, comprising: N-acetylglucosamine-1-kinase, N-acetylglucosamine-1-phosphate uridyltransferase, β-1,3-N-acetylglucosaminyltransferase and polyphosphate kinase.

[0032] The present invention aims at the disadvantages of the existing fermentation method for synthesizing LNT II, ​​such as the complex construction of engineering bacteria, the difficulty of microorganisms to tolerate extreme environments, the slow reaction speed, and the low product yield. Through enzyme screening and transformation technology, a 4-enzyme system including N-acetylglucosamine-1-kinase, N-acetylglucosamine-1-phosphate uridyl transferase, β-1,3-N-acetylglucosamine transfer and polyphosphate kinase is screened, and N-acetylglucosamine-1-kinase and polyphosphate kinase, N-acetylglucosamine-1-phosphate uridyl transferase and β-1,3-N-acetylglucosamine transferase are co-expressed, and 100-500mM substrate is successfully converted in one pot within 5 hours, with a conversion rate of more than 95%. In addition, the polyphosphate kinase screened by the present invention can simultaneously circulate ATP and UTP, has the characteristics of strong affinity, fast conversion speed and good stability, can use cheap sodium hexametaphosphate as a substrate, successfully reduces the cost of the reaction, and has the strength of production amplification. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0034] Figure 1 The co-expression plasmid pETDuet-CbNahK-DsPPK is shown;

[0035] Figure 2 The co-expression plasmid pETDuet-MtGlmU-HiLgtA is shown;

[0036] Figure 3 Shown is the synthesis route of GlcNAc to GlcNAc-1-P;

[0037] Figure 4 Shows the liquid phase spectrum of GlcNAc-1-P synthesized from GlcNAc;

[0038] Figure 5 Shown is the synthetic route of GlcNAc-1-P to UDP-GlcNAc;

[0039] Figure 6 Shows the liquid phase spectrum of GlcNAc-1-P synthesizing UDP-GlcNAc;

[0040] Figure 7 The synthetic route of LNT II synthesized by UDP-GlcNAc is shown;

[0041] Figure 8 Shows the liquid phase spectrum of LNT II synthesized by UDP-GlcNAc;

[0042] Fig. 9 The complete synthetic route of GlcNAc synthesis of LNT II is shown;

[0043] Fig.10 Shown is SDS-PAGE picture;

[0044] Fig.11 Example 3 shows the one-pot synthesis of LNT II by four single enzymes;

[0045] Fig.12 Example 4 shows the one-pot synthesis of LNT II by co-expression of enzymes;

[0046] Fig.13 Example 5 shows the one-pot synthesis of LNT II by co-expression of enzymes;

[0047] Fig.14 Example 6 shows the one-pot synthesis of LNT II by co-expression of enzymes;

[0048] Fig.15 The catalytic reaction of the polyphosphate kinase DsPPK isozyme DsPPK-11 in Comparative Example 1 is shown;

[0049] Fig.16 The comparative example 2 β-1,3-N-acetylglucosaminyltransferase HiLgtA isozyme HiLgtA-05 catalyzes the reaction. DETAILED DESCRIPTION

[0050] The invention discloses an enzyme composition and application thereof in preparing lactose-N-triose.

[0051] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0052] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0053] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0054] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0055] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0056] The technical scheme provided by the present invention is: LNT II is synthesized using N-acetylglucosamine (GlcNAc) as a substrate, firstly, GlcNAc and ATP are catalyzed by N-acetylglucosamine-1-kinase (EC: 2.7.1.162) to generate GlcNAc-1-P and adenosine diphosphate (ADP), then GlcNAc-1-P and UTP are catalyzed by N-acetylglucosamine-1-phosphate uridyltransferase (EC: 2.7.7.23) to generate UDP-GlcNAc and pyrophosphate, and then lactose is added to generate LNT II and uridine diphosphate (UDP) under the catalysis of β-1,3-N-acetylglucosamine transferase (EC: 2.4.1.149). ADP and UDP generated by the conversion of ATP and UTP in the reaction are catalyzed by polyphosphate kinase (EC: 2.7.4.1), sodium hexametaphosphate is used to provide phosphate, and magnesium chloride is added as an enzyme activator to cyclically regenerate ATP and UTP. In order to reduce the fermentation pressure of enzymes, N-acetylglucosamine-1-kinase and polyphosphate kinase, β-1,3-N-acetylglucosamine transferase and N-acetylglucosamine-1-phosphate uridyltransferase were co-expressed.

[0057] According to this route, the 1 L reaction solution is prepared as follows: 22.1-110.5 g N-acetylglucosamine (100-500 mM), 1.1-5.5 g adenosine triphosphate disodium ATP-2Na (2-10 mM), 1.2-6.1 g 5'-uridine triphosphate trisodium UTP-3Na (2-10 mM), 34.2 g-205.2 g lactose (100-600 mM), 12.2 g magnesium chloride hexahydrate (60 mM) and 30.6-152.9 g sodium hexametaphosphate (50-250 mM) are weighed and added to a beaker, and water is added and stirred to dissolve at 40-60°C. After the solution is stirred until clear, 4 M NaOH is added to adjust the solution to pH 7.0-8.5 and the volume is fixed to 1000 mL.

[0058] Prepare the reaction solution according to the above steps, transfer it to a three-necked flask, control the temperature of the water bath at 30~40℃, add enzyme solution to the reaction bottle to start the reaction, and titrate the reaction conditions with NaOH solution to control the pH between 7.0~8.5. The amount of enzyme solution added is as follows: 1100~3600 U of N-acetylglucosamine-1-kinase, 2800~5400 U of N-acetylglucosamine-1-phosphate uridyltransferase, 5000~7200 U of β-1,3-N-acetylglucosamine transferase, and 4200~6000 U of polyphosphate kinase.

[0059] Abbreviations and English meanings in this invention

[0060]

[0061] In Examples 1 to 6 of the present invention and Comparative Examples 1 to 2, all the raw materials and reagents used can be purchased from the market.

[0062] The present invention will be further described below in conjunction with embodiments:

[0063] Example 1 Construction of co-expression enzyme system

[0064] Through enzyme screening, we obtained N-acetylglucosamine-1-kinase CbNahK (from Clostridiales bacterium), N-acetylglucosamine-1-phosphate uridyltransferase MtGlmU (from Mycobacterium tuberculosis), β-1,3-N-acetylglucosaminyltransferase HiLgtA (from Haemophilus influenzae) and polyphosphate kinase DsPPK (from Dysgonomonas sp.).

[0065] The gene sequences of each enzyme are shown below:

[0066] N-acetylhexaminohexose-1-kinase CbNahK (Clostridiales bacterium) protein sequence>A0A3D5ZJ04: MGDFTKEYFTAAKRFLHDCEIVDVSPYGEGHINKTLLVTTPDKRYIMQKMNTGIFPDSDGLMSNILYVTQSLRSRGVETLDVVFTDDGKPYLKAADGNYRMYKFIENTVSYQTTTDKKVLYNSGKAFGKFQNYLADFDASVLVEVIPHFHDTPKRIADFAAAVKADKYDLVKTCREEVDFVLSRAETVGKIVDGLACGSIPLRVTHNDTKLNNVLMDAVTKEARAVIDLDTIMPGSMLYDFGDSIRFGASTAAADERDTSKVHFDIGLFEAYARGYVGEVKSSITATEAELLPYSAYLMTLECGSRFLADYLEGNVYFATRYAEHNLVRARTQFALVAEHERNFDQMSAIIGKILAE. (As shown in SEQ ID NO:1)

[0067]

[0068] N-acetylglucosamine-1-phosphate uridyltransferase MtGlmU (Mycobacterium tuberculosis) protein sequence> A5U161: MTFPGDTAVLVLAAGPGTRMRSDTPKVLHTLAGRSMLSHVLHAIAKLAPQRLIVVLGHDHQRIAPLVGELADTLGRTIDVALQDRPLGTGHAVLCGLSALPDDYAGNVVVTSGDTPLLDADTLADLIATHRAVSAAVTVLTTTLDDPFGYGRILRTQDHEVMAIVEQTDATPSQREIREVNAGVYAFDIAALRSALSRLSSNNAQQELYLTDV IAILRSDGQTVHASHVDDSALVAGVNNRVQLAELASELNRRVVAAHQLAGVTVVDPATTWIDVDVTIGRDTVIHPGTQLLGRTQIGGRCVVGPDTTLTDVAVGDGASVVRTHGSSSSIGDGAAVGPFTYLRPGTALGADGK LGAFVEVKNSTIGTGTKVPHLTYVGDADIGEYSNIGASSVFVNYDGTSKRRTTVGSHVRTGSDTMFVAPVTIGDGAYTGAGTVVREDVPPGALAVSAGPQRNIENWVQRKRPGSPAAQASKRASEMACQQPTQPPDADQTP. (As shown in SEQID NO:3)

[0069]

[0070] β-1,3-N-acetylglucosaminyltransferase HiLgtA (Haemophilus influenzae) protein sequence>Q57287: MENCPLVSVIVCAYNAEQYIDESISSIINQTYENLEIIVINDGSTDLTLSHLEEISKLDKRIKIISNKYNLGFINSLNIGLGCFSGKYFARMDADDIAKPSWIEKIVTYLEKNDHITAMGSYLEIIVEKECGIIGSQYKTGDIWKNPLLHNDICEAMLFYNPIHNNTMIMRANVYREHKLIFNKDYPYAEDYKFWSEVSRLGCLANYPEALVKYRLHGNQTSSVYNHEQNETAKKIKRENITYYLNKIGIDIKVINSVSLLEIYHVDKSNKVLKSILYEMYMSLDKYTITSLLHFIKYHLELFDLKQNLKIIKKFIRKINVIF. (as shown in SEQ ID NO:5)

[0071] β-1,3-N-acetylglucosaminyltransferase HiLgtADNA sequence: ATGGAAAACTGCCCGCTGGTGAGTGTTATTGTGTGCGCATATAATGCCGAACAGTATATTGATGAAAGTATTAGTAGCATCATCAACCAGACCTATGAAAATCTGGAAATTATTGTTATCAACGACGGCAGTACCGATCTGACCCTGAGTCATCTGGAAGAAATTAGTAAACTGGATAAACGCATTAAGATCATTAGCAATAAGTACAATCTGGGTTTTATCAATAGCCTGAATATTGGTCTGGGTTGTTTTAGCGGTAAATATTTTGCACGTATGGATGCCGATGATATTGCAAAACCGAGCTGGATTGAAAAAATTGTGACCTATCTGGAAAAGAATGATCATATTACCGCAATGGGTAGCTATCTGGAAATTATCGTTGAAAAAGAATGCGGTATTATCGGCAGTCAGTATAAAACCGGTGATATTTGGAAAAATCCGCTGCTGCATAATGATATTTGTGAAGCAATGCTGTTTTACAATCCGATTCATAATAACACCATGATTATGCGTGCCAATGTGTATCGCGAACATAAACTGATTTTTAATAAGGATTACCCGTACGCAGAAGATTATAAATTTTGGAGCGAAGTTAGCCGTCTGGGCTGCCTGGCCAATTATCCGGAAGCACTGGTGAAATATCGTCTGCATGGCAATCAGACCAGCAGTGTTTATAATCATGAACAGAATGAAACCGCCAAAAAAATTAAACGTGAAAATATCACCTACTACCTGAATAAAATCGGCATTGATATTAAGGTTATCAACAGCGTTAGCCTGCTGGAAATTTATCATGTTGATAAAAGTAACAAGGTGCTGAAAAGCATTCTGTATGAAATGTATATGAGCCTGGATAAATACACCATTACCAGTCTGCTGCATTTTATTAAATACCATCTGGAACTGTTCGATCTGAAACAGAATCTGAAAATTATCAAGAAGTTCATCCGTAAAATCAACGTTATTTTCTAA. (as shown in SEQ ID NO:6)

[0072] Polyphosphate kinase DsPPK (Dysgonomonas sp.) protein sequence>A0A3D3TIS3:MDKEVLKKLLAEPGKKHKVSGFSTDYTAGMNKKEAKKELEDNTLKLRELQNVLYAHDRYAVLLIFQAMDAAGKDGTIKHVMSGVNPQGCQVFSFKQPSDEELDHDYLWRIYKSLPERGRIGIFNRSHYEDVLIAKVHPNIVLGTKLPHVKKLKDINGKFWEKRYRQINNFEEHLVENGTIVIKFFLNLSKDEQEKRFLARLEDDSKNWKFSASDLKERPYWDDYMKAYSDMLTNTSTKYAPWYVIPADNKWFMRYAVGQIICDRIAELDLHYPTLSQAAKEEIKKAKEELSSKMKNEDKKQKDEEAVRK. (as shown in SEQ ID NO:7)

[0073] DNA sequence of diphosphokinase DsPPK: ATGGATAAGGAAGTTCTGAAAAAGCTGCTGGCAGAACCGGGTAAAAAACATAAAGTGAGCGGCTTTAGTACCGATTATACCGCCGGTATGAATAAAAAAGAAGCAAAAAAAGAGCTGGAGGATAATACCCTGAAACTGCGCGAACTGCAGAATGTTCTGTATGCACATGATCGCTATGCAGTTCTGCTGATTTTTCAGGCAATGGATGCAGCAGGCAAAGATGGTACCATTAAACATGTGATGAGTGGCGTGAATCCGCAGGGCTGCCAGGTTTTTAGTTTTAAACAGCCGAGTGATGAAGAACTGGATCATGATTATCTGTGGCGTATTTATAAAAGCCTGCCGGAACGCGGCCGCATTGGCATTTTTAATCGCAGTCATTATGAAGATGTGCTGATTGCCAAAGTGCATCCGAATATTGTTCTGGGCACCAAACTGCCGCATGTTAAAAAACTGAAAGATATTAACGGCAAGTTCTGGGAAAAACGCTATCGCCAGATTAATAATTTTGAAGAACACCTGGTTGAGAATGGCACCATTGTGATTAAATTTTTTCTGAATCTGAGCAAGGACGAACAGGAAAAACGCTTTCTGGCCCGTCTGGAAGATGATAGTAAAAATTGGAAATTCAGCGCAAGTGATCTGAAAGAACGTCCGTATTGGGATGATTATATGAAAGCATATAGCGATATGCTGACCAATACCAGCACCAAATATGCACCTTGGTATGTTATTCCGGCCGATAATAAATGGTTTATGCGTTATGCAGTGGGTCAGATTATTTGTGATCGCATTGCAGAACTGGATCTGCATTATCCGACCCTGAGTCAGGCCGCCAAAGAAGAAATTAAAAAAGCCAAAGAAGAGCTGAGCAGTAAAATGAAAAATGAAGATAAGAAGCAGAAGGATGAAGAAGCAGTTCGTAAATAA. (as shown in SEQ ID NO:8)

[0074] Co-expression:

[0075] The DNA sequences of N-acetylglucosamine-1-kinase CbNahK and polyphosphate kinase DsPPK were respectively inserted into two multiple cloning sites of the vector pETDuet-1 to form the co-expression plasmid pETDuet-CbNahK-DsPPK.

[0076] The DNA sequences of N-acetylglucosamine-1-phosphate uridyltransferase MtGlmU and β-1,3-N-acetylglucosaminyltransferase HiLgtA were respectively inserted into the two multiple cloning sites of the vector pETDuet-1 to form the co-expression plasmid pETDuet-MtGlmU-HiLgtA.

[0077] The correct plasmid was verified by gene sequencing and then transferred into the E. coli BL21 (DE3) strain. A small amount of culture was carried out at 37°C in 5 mL of LB culture medium containing 50μM kanamycin. When the cells grew to OD 0.5-0.8, 0.4mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and protein expression was induced at 37°C for 4 hours. Finally, the cells were collected, the cells were broken by freeze-thaw method, and high-speed centrifugation was performed. The collected supernatant was then used to confirm protein expression by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The strain with correct protein expression was cultured step by step to a 5-liter fermenter, induced to express at 37°C for 6 hours under 0.5 mM IPTG conditions, and the wet cells of each enzyme were collected. The LB medium consists of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate, and 5% glycerol.

[0078] As shown in Table 1, through activity measurement, the enzyme activity of N-acetylglucosamine-1-kinase CbNahK was 300-592 U / mL, the enzyme activity of N-acetylglucosamine-1-phosphate uridyltransferase MtGlmU was 130-210 U / mL, the enzyme activity of β-1,3-N-acetylglucosaminyltransferase HiLgtA was 60-95 U / mL, and the enzyme activity of polyphosphate kinase DsPPK was 110-180 U / mL.

[0079] Table 1 Enzyme activities in the co-expression system

[0080]

[0081] Example 2 Stepwise synthesis of LNT II using N-acetylglucosamine as substrate

[0082] (1) GlcNAc is used as a substrate to synthesize GlcNAc-1-P.

[0083] like Figure 3As shown, weigh 22.1 g GlcNAc (100 mM), 55 g ATP-2Na (100 mM), and 12.2 g magnesium chloride hexahydrate (60 mM) into a beaker, and add water to dissolve by stirring at room temperature. After the solution is stirred until clear, add 4 M NaOH to adjust the solution to pH 7.0 and dilute to 1000 mL.

[0084] The reaction solution was transferred to a three-necked flask, the temperature was controlled at 35°C in a water bath, the enzyme solution was added to the reaction bottle to start the reaction, the NaOH solution was titrated to control the reaction conditions to pH 7.5, and finally 2200 U N-acetylhexosamine-1-kinase CbNahK was added to start the reaction. The reaction was completed after 2.0 h, and the main component of the obtained reaction solution was GlcNAc-1-P, and some magnesium chloride, GlcNAc, ATP, ADP, etc. were retained. The yield was greater than 95%, and the output was greater than 30 g / L (such as Figure 4 as shown).

[0085] (2) GlcNAc-1-P is used as a substrate to synthesize UDP-GlcNAc.

[0086] like Figure 5 As shown, after the first step of the reaction, 61 g of UTP-3Na (100 mM) was added to the reaction bottle and dissolved in the reaction solution by stirring.

[0087] The temperature of the water bath was controlled at 35°C, and the enzyme solution was added to the reaction bottle to start the reaction. The NaOH solution was titrated to control the reaction conditions between pH 7.5, and finally 3500 U of N-acetylglucosamine-1-phosphate uridyltransferase MtGlmU was added to start the reaction. The reaction was completed after 1.0 h, and the main component of the reaction solution was UDP-GlcNAc, with a yield of >90% and a yield of more than 50 g / L (such as Figure 6 as shown).

[0088] (3) UDP-GlcNAc is used as a substrate to synthesize LNT II.

[0089] like Figure 7 As shown, after the second step reaction, 34.3 g lactose (100 mM) was added to the reaction bottle and stirred to dissolve in the reaction solution.

[0090] The temperature of the water bath was controlled at 35°C, and the enzyme solution was added to the reaction bottle to start the reaction. The reaction conditions were controlled to pH 7.5 by titration with NaOH solution, and finally 6000 U of β-1,3-N-acetylglucosaminyltransferase HiLgtA was added to start the reaction. The reaction was completed after 2 h, and the main component of the reaction solution was LNT II, ​​with a yield greater than 95% and a yield of >40 g / L (such as Figure 8 as shown).

[0091] Combining the results of the three-step reaction, the total yield is >81%. The step-by-step reaction is complicated, requires a lot of input materials, and cannot achieve the recycling of ATP and UTP.

[0092] Example 3 One-pot synthesis of LNT II using four single enzymes

[0093] like Fig. 9 As shown, the 1 L reaction solution is configured as follows: 66.3 g GlcNAc (300 mM), 3.3 g ATP-2Na (6 mM), 3.6 g UTP-3Na (6 mM), 102.6 g lactose (300 mM), 12.2 g magnesium chloride hexahydrate (60 mM) and 91.8 g sodium hexametaphosphate (150 mM) are weighed and added to a beaker, and water is added and stirred to dissolve at room temperature. After the solution is stirred until clear, 4 M NaOH is added to adjust the solution to pH 7.5 and the volume is fixed to 1000 mL.

[0094] According to the above steps, several reaction solutions were prepared and transferred to different three-necked flasks. The temperature of the water bath was controlled at 35°C. The enzyme solution was added to the reaction bottle to start the reaction. The reaction conditions were controlled at pH 7.5 by titration of NaOH solution. The enzyme activity of each enzyme was added as follows: N-acetylglucosamine-1-kinase CbNahK 2800 U, N-acetylglucosamine-1-phosphate uridyltransferase MtGlmU4500 U, β-1,3-N-acetylglucosamine transferase HiLgtA 5900 U, and polyphosphate kinase DsPPK 4600 U.

[0095] like Fig.11 As shown, the yield of LNT II after 4 h of reaction was 160.1 g / L, the total yield was >96%, and the conversion rate was >99%.

[0096] Example 4 One-pot synthesis of LNT II by co-expression of enzymes

[0097] The 1 L reaction solution was prepared as follows: 22.1 g GlcNAc (100 mM), 1.1 g ATP-2Na (2 mM), 1.2 g UTP-3Na (2 mM), 35 g lactose (100 mM), 12.2 g magnesium chloride hexahydrate (60 mM) and 30.6 g sodium hexametaphosphate (50 mM) were weighed and added to a beaker, and water was added and stirred to dissolve at room temperature. After the solution was stirred until clear, 4 M NaOH was added to adjust the solution to pH 7.6, and the volume was fixed to 1000 mL.

[0098] The reaction solution was prepared according to the above steps, transferred to a three-necked flask, the water bath temperature was controlled at 33°C, the enzyme solution obtained in Example 1 was added to the reaction flask to start the reaction, and the reaction conditions were controlled between pH 7.8 by titration of NaOH solution. The enzyme activity input was as follows: N-acetylglucosamine-1-kinase CbNahK 1960 U, N-acetylglucosamine-1-phosphate uridyltransferase MtGlmU2900 U, β-1,3-N-acetylglucosamine transferase HiLgtA 4150 U, polyphosphate kinase DsPPK 2760 U.

[0099] like Fig.12 As shown, the yield of LNT II was 54.9 g / L after 2 h of reaction, the total yield was >97%, and the conversion rate was >99%.

[0100] Example 5 One-pot synthesis of LNT II by co-expression of enzymes

[0101] The preparation of 1 L of reaction solution is as follows: weigh 110.6 g GlcNAc (500 mM), 1.1 g ATP-2Na (9 mM), 5.5 g UTP-3Na (10 mM), 198.4 g lactose (580 mM), 12.2 g magnesium chloride hexahydrate (60 mM) and 146.8 g sodium hexametaphosphate (240 mM) into a beaker, heat in a water bath at 60°C to dissolve, add 4 M NaOH to adjust the solution to pH 7.0, and make up to 1000 mL.

[0102] The reaction solution was prepared according to the above steps and transferred to a three-necked flask. The temperature of the water bath was controlled at 35° C. The enzyme solution obtained in Example 1 was added to the reaction flask to start the reaction. The reaction conditions were controlled to pH 7.5 by titration with NaOH solution. The enzyme activity input was as follows: N-acetylglucosamine-1-kinase CbNahK 3560 U, N-acetylglucosamine-1-phosphate uridyltransferase MtGlmU4250 U, β-1,3-N-acetylglucosaminyltransferase HiLgtA 6790 U, and polyphosphate kinase DsPPK 5420 U.

[0103] like Fig.13 As shown, the yield of LNT II after 4 h of reaction was 268.7 g / L, the total yield was >97%, and the conversion rate was >98.6%.

[0104] Example 6 One-pot synthesis of LNT II by co-expression of enzymes

[0105] The preparation of 1 L of reaction solution is as follows: weigh 55.3 g GlcNAc (250 mM), 2.8 g ATP-2Na (5 mM), 3.3 g UTP-3Na (6 mM), 102.6 g lactose (300 mM), 12.2 g magnesium chloride hexahydrate (60 mM) and 73.4 g sodium hexametaphosphate (120 mM) into a beaker, heat in a water bath at 50°C to dissolve, add 4 M NaOH to adjust the solution to pH 7.0, and make up to 1000 mL.

[0106] The reaction solution was prepared according to the above steps and transferred to a three-necked flask. The temperature of the water bath was controlled at 40° C. The enzyme solution obtained in Example 1 was added to the reaction flask to start the reaction. The reaction conditions were controlled to pH 8.0 by titration with NaOH solution. The enzyme activity input was as follows: N-acetylglucosamine-1-kinase CbNahK 2430 U, N-acetylglucosamine-1-phosphate uridyltransferase MtGlmU3350 U, β-1,3-N-acetylglucosamine transferase HiLgtA 5100 U, and polyphosphate kinase DsPPK 3980 U.

[0107] like Fig.14 As shown, the yield of LNT II after 3 h of reaction was 135.1 g / L, the total yield was >96%, and the conversion rate was >99.1%.

[0108] Comparative Example 1 Catalytic reaction of polyphosphate kinase DsPPK isozyme DsPPK-11

[0109] The preparation of 1 L of reaction solution is as follows: weigh 61.9 g GlcNAc (280 mM), 3.3 g ATP-2Na (6 mM), 3.3 g UTP-3Na (6 mM), 102.6 g lactose (300 mM), 12.2 g magnesium chloride hexahydrate (60 mM) and 73.4 g sodium hexametaphosphate (120 mM) into a beaker, heat in a water bath at 50°C to dissolve, add 4 M NaOH to adjust the solution to pH 7.0, and make up to 1000 mL.

[0110] The reaction solution was prepared according to the above steps and transferred to a three-necked flask. The temperature was controlled at 37° C. in a water bath. The enzyme solution was added to the reaction flask to start the reaction. The reaction condition was controlled to pH 7.6 by titration with NaOH solution. The enzyme activity input was as follows: 2430 U of N-acetylglucosamine-1-kinase CbNahK, 3350 U of N-acetylglucosamine-1-phosphate uridyltransferase MtGlmU, 5100 U of β-1,3-N-acetylglucosaminyltransferase HiLgtA, and 4100 U of polyphosphate kinase DsPPK isozyme DsPPK-11 (the co-expression system of polyphosphate kinase DsPPK isozyme DsPPK-11 is the same as the co-expression plasmid pETDuet-CbNahK-DsPPK in Example 1, except that polyphosphate kinase DsPPK was replaced by polyphosphate kinase DsPPK isozyme DsPPK-11).

[0111] like Fig.15 As shown, the yield of LNT II was 123 g / L after 6 h of reaction, the total yield was >75%, and the conversion rate was >80.6%.

[0112] Comparative Example 2 β-1,3-N-acetylglucosaminyltransferase HiLgtA isozyme HiLgtA-05

[0113] The preparation of 1 L of reaction solution is as follows: weigh 44.2 g GlcNAc (200 mM), 2.2 g ATP-2Na (4 mM), 3.3 g UTP-3Na (6 mM), 102.6 g lactose (300 mM), 12.2 g magnesium chloride hexahydrate (60 mM) and 61.2 g sodium hexametaphosphate (100 mM) into a beaker, heat in a water bath at 50°C to dissolve, add 4 M NaOH to adjust the solution to pH 7.0, and make up to 1000 mL.

[0114] The reaction solution was prepared according to the above steps and transferred to a three-necked flask. The temperature was controlled at 40°C in a water bath. The enzyme solution was added to the reaction flask to start the reaction. The reaction conditions were controlled to pH 8.0 by titration with NaOH solution. The enzyme activity input was as follows: N-acetylglucosamine-1-kinase CbNahK 2430 U, N-acetylglucosamine-1-phosphate uridyltransferase MtGlmU 3350 U, β-1,3-N-acetylglucosamine transferase HiLgtA isozyme HiLgtA-05 5230 U (the co-expression system of β-1,3-N-acetylglucosaminyltransferase HiLgtA isozyme HiLgtA-05 is the same as the co-expression plasmid pETDuet-MtGlmU-HiLgtA in Example 1, except that β-1,3-N-acetylglucosaminyltransferase HiLgtA is replaced by β-1,3-N-acetylglucosaminyltransferase HiLgtA isozyme HiLgtA-05), polyphosphate kinase DsPPK 3980 U.

[0115] like Fig.16 As shown, the yield of LNT II was 76.7 g / L after 6 h of reaction, the total yield was >75.1%, and the conversion rate was >70.4%.

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made 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. An enzyme composition, characterized in that include: N-acetylglucosamine-1-phosphate uridyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase, β-1,3-N-acetylglucosaminyltransferase, and polyphosphate kinase.

2. The enzyme composition according to claim 1, characterized in that The N-acetylglucosamine-1-kinase is derived from Clostridiales bacterium, the N-acetylglucosamine-1-phosphate uridyltransferase is derived from Mycobacterium tuberculosis, the β-1,3-N-acetylglucosaminyltransferase is derived from Haemophilusinfluenzae, and the polyphosphate kinase is derived from Dysgonomonas sp.

3. Use of the enzyme composition according to claim 1 or 2 in the preparation of lactose-N-triose.

4. A method for preparing lactose-N-triose, characterized in that: The lactose-N-triose is obtained by using N-acetylglucosamine, adenosine triphosphate, uridine triphosphate, lactose, magnesium chloride and phosphate as raw materials under the catalytic action of the enzyme composition according to claim 1 or 2.

5. The preparation method according to claim 4, characterized in that: The enzyme composition exists in the form of a host expressing each enzyme, an enzyme solution or an immobilized enzyme.

6. The preparation method according to claim 5, characterized in that: The host contains a vector for expressing each enzyme or a vector for co-expressing two enzymes.

7. The preparation method according to any one of claims 4 to 6, wherein the amount of N-acetylglucosamine-1-kinase added is 1100-3600 U, the amount of N-acetylglucosamine-1-phosphate uridyltransferase added is 2800-5400 U, the amount of β-1,3-N-acetylglucosamine transferase added is 5000-7200 U, and the amount of polyphosphate kinase added is 4200-6000 U.

8. The preparation method according to any one of claims 4 to 7, characterized in that: The catalytic temperature is 33-40° C., and the pH value is 7.5-8.

0.

9. The preparation method according to any one of claims 4 to 8, characterized in that: The molar ratio of the N-acetylglucosamine, the adenosine triphosphate, the uridine triphosphate, the lactose, the magnesium chloride and the phosphate is: (100-500): (2-9): (2-10): (100-580): 60: (50-240).

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