ENZYMATIC METHOD FOR PREPARATION OF CMP-NEU5AC
20240352497 ยท 2024-10-24
Inventors
Cpc classification
C12N9/1205
CHEMISTRY; METALLURGY
C12N9/1229
CHEMISTRY; METALLURGY
C12Y207/07043
CHEMISTRY; METALLURGY
C12P19/46
CHEMISTRY; METALLURGY
C12Y207/04014
CHEMISTRY; METALLURGY
C12P19/26
CHEMISTRY; METALLURGY
C12P19/44
CHEMISTRY; METALLURGY
International classification
C12P19/46
CHEMISTRY; METALLURGY
C12N9/12
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a method for producing cytidine 5-monophospho-N-acetyl-neuraminic acid (CMP-Neu5Ac, 1) from low-cost substrates N-acetyl-D-glucosamine (GlcNAc), pyruvate, cytidine and polyphosphate in a single reaction mixture with a set of optionally immobilized or optionally co-immobilized enzymes comprising N-acylglucoamine 2-epimerase (AGE), an N-acetylneuraminate lyase (NAL), an N-acylneuraminate cytidylyltransferase (CSS), a uridine kinase (UDK), a uridine monophosphate kinase and a polyphosphate kinase 3 (PPK3). Further, said process may be adapted to produce Neu5Acylated i.e. sialylated biomolecules and biomolecules including a saccharide, a peptide, a protein, a glycopeptide, a glycoprotein, a glycolipid, a glycan, an antibody, and a glycoconjugate, in particular, an antibody drug conjugate, and a carbohydrate conjugate vaccine, or a flavonoid.
Claims
1. A method for producing cytidine 5-monophospho-N-acetyl-neuraminic acid (CMP-Neu5Ac, 1) ##STR00030## comprising: A) providing a solution comprising N-acetyl-
2. The method according to claim 1, wherein the set of enzymes further comprising an inorganic diphosphatase (PPA, EC 3.6.1.1).
3. The method according to claim 1, wherein the set of enzymes further comprises a one-domain polyphosphate kinase 2 (1D-PPK2, EC 2.7.4.1) and/or a two-domain polyphosphate kinase 2 (2D-PPK2, EC 2.7.4.1).
4. The method according to claim 1, wherein the resulting solution has a pH value in the range of 7.0-9.0.
5. The method according to claim 1, wherein a concentration of N-acetyl-
6. The method according to claim 1, wherein the ratio of the N-acetyl-
7. The method according to claim 1, wherein the resulting solution further comprises Mg.sup.2+ with a concentration in the range of 0.1 mM to 200 mM.
8. The method according to claim 1, wherein each of the enzymes has the following amino acid sequence: the N-acylglucosamine 2-epimerase (AGE) comprises at least 80% of an amino acid sequence as set forth in SEQ ID NO: 1; the N-acetylneuraminate lyase (NAL) comprises at least 80% of an amino acid sequence as set forth in SEQ ID NO: 2; the N-acylneuraminate cytidylyltransferase (CSS) comprises at least 80% of an amino acid sequence as set forth in SEQ ID NO: 3; the uridine kinase (UDK) comprises at least 80% of an amino acid sequence as set forth in SEQ ID NO: 4; the uridine monophosphate kinase (URA6) comprises at least 80% of an amino acid sequence as set forth in SEQ ID NO: 5; the polyphosphate kinase 3 (PPK3) comprises at least 80% of an amino acid sequence as set forth in SEQ ID NO: 6; the inorganic diphosphatase (PPA) comprises at least 80% of an amino acid sequence as set forth in SEQ ID NO: 7; the one-domain polyphosphate kinase 2 (1D-PPK2) comprises at least 80% of an amino acid sequence as set forth in SEQ ID NO: 9,
9. The method according to claim 11, wherein the solid support is composed of beads or resins comprising a polymer with epoxide functional groups, with amino epoxide functional groups, with ethylenediamine functional groups, with amino C2 functional groups, with amino C6 functional groups, with anionic/amino C6 spacer functional groups.
10. The method according to claim 1, wherein the ratio of N-acetyl-
11. The method according to claim 1, wherein the set of enzymes is directly co-immobilized on a solid support from cell lysate or cell homogenate.
12. The method according to claim 1, wherein the N-acetyl-
13. A method for producing a Neu5Acylated biomolecule comprising i) performing the method according to claim 1 to obtain a CMP-Neu5Ac, ii) reacting the CMP-Neu5Ac with a biomolecule, wherein the biomolecule is a saccharide, a glycopeptide, a glycoprotein, a glycolipid, a glycan, a peptide, a protein, an antibody, an antibody drug conjugate, a carbohydrate conjugate vaccine, virus, virus like particles, virus vaccine, or a flavonoid by forming an O-glycosidic bond with an hydroxyl group of the biomolecule with removal of CMP group in the presence of a sialyltransferase.
14. The method according to claim 13, wherein the biomolecule contains any of galactoside (Gal), galactosamininde (GalN), N-acetylgalactosaminide (GalNAc), neuraminide (Neu), N-acetyl neuraminide (Neu5Ac), N-glycolylneuraminide, 3-Deoxy-D-glycero-D-galacto-2-nonulosonic Acid (KDN), and N-acetyllacosaminide (Gal-3-1-3-GlcNAc) moiety as terminal end group.
15. A set of enzymes comprising a set of enzymes comprising an N-acylglucosamine 2-epimerase (AGE, EC 5.1.3.8), an N-acetylneuraminate lyase (NAL, EC 4.1.3.3), an N-acylneuraminate cytidylyltransferase (CSS, EC 2.7.7.43), a uridine kinase (UDK, EC 2.7.1.48), a uridine monophosphate kinase (URA6, EC 2.7.4.22) and a polyphosphate kinase 3 (PPK3, EC 2.7.4.1), wherein the set of enzymes is co-immobilized on a polymer by covalent bonds.
16. The set of enzymes according to claim 15 further comprising an inorganic diphosphatase (PPA), a one-domain polyphosphate kinase 2 (1D-PPK2, EC 2.7.4.1) and/or a two-domain polyphosphate kinase 2 (2D-PPK2, EC 2.7.4.1).
17. The set of enzymes according to claim 15, further comprising an N-acetyl-glucosamine deacetylase (EC 3.5.1.33).
18. The set of enzymes according to claim 16, further comprising an N-acetyl-glucosamine deacetylase (EC 3.5.1.33).
Description
DESCRIPTION OF THE FIGURES
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[0764] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
[0765] Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.
EXAMPLES
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TABLE-US-00002 Abbreviations and Acronyms AGE N-acetyl-D-glucosamine epimerase/isomerase ADP adenosine 5-diphosphate AMP adenosine 5-monophosphate ATP adenosine 5-triphosphate dH.sub.2O deionized water CMP cytidine 5-monophosphate CDP cytidine 5-diphosphate CTP cytidine 5-triphosphate CSS N-acylneuraminate cytidylyltransferase NmCSS N-acylneuraminate cytidylyltransferase of Neisseria meningitidis serogroup B (strain MC58) NAL N-acetylneuraminate lyase, or N-acetylneuraminate pyruvate lyase GlcN D-glucosamine GlcNAc N-acetyl-D-glucosamine ManNAc N-acetyl-D-mannosamine Neu5Ac N-acetyl-D-neuraminic acid, or 5-(acetylamino)-3,5-dideoxy-D- glycero--D-galacto-non-2-ulopyranosonic acid CMP-Neu5Ac cytidine 5-monophosphate N-acetyl-D-neuraminic acid PolyP polyphosphate PPi pyrophosphate Pi phosphate PPK2 polyphosphate kinase 2 PPK3 polyphosphate kinase 3 1D-PPK2 1-domain polyphosphate kinase 2, polyphosphate:ADP phosphotransferase 2D-PPK2 2-domain polyphosphate kinase 2, polyphosphate:AMP phosphotransferase URA6 uridine monophosphate kinase PPA inorganic pyrophosphatase PmPpA Pasteurella multocida inorganic pyrophosphatase
Chemicals & Reagents
[0767] Unless otherwise stated, all chemicals and reagents were acquired from Sigma-Aldrich and CarboSynth, and were of the highest purity available. Solid supports were obtained from Resindion, ChiralVision, Rohm GmbH & Co. KG and micromod GmbH.
Example 1: Preparation of Enzymes
[0768] Each plasmid was individually transformed into E. coli BL21 and followed by cultivation on LB agar plates with selection markers. For each enzyme expression the following protocol was followed:
[0769] A single colony from agar plate was incubated in LB media and a selection marker at 37 C. overnight. The main culture was prepared by applying a seeding factor of 100 from the overnight culture and incubation in TB media with 1 mM MgSO.sub.4 and a selection marker at 37 C. up to OD.sub.600 0.8. The gene expression was induced by addition of 0.4 mM IPTG and cultivation at 16 C. for 20 hrs. Cells were harvested by centrifugation at 7,000g for 30 min. Afterwards, the cell pellet was resuspended in lysis buffer. Cells lysis was conducted by high pressure homogenization (800-1000 psi). Cell lysates were centrifuged at 7,000g for 30 min and filtered through a 0.8 m filter. Enzymes were purified through nickel affinity chromatography (see following slides).
TABLE-US-00003 TABLE 1 Enzymes used in this example Enzyme Abbreviation EC class Origin SEQ ID AGE family AGE 5.1.3.8 Trichormus SEQ ID 1 epimerase/isomerase variabilis N-acetylneuraminate NAL 4.1.3.3 Pasteurella SEQ ID 2 lyase multocida (strain Pm70) N-acylneuraminate CSS 2.7.7.43 Neisseria SEQ ID 3 cytidylyltransferase meningitidis MC58 (serogroup B) Uridine kinase UDK 2.7.1.48 Escherichia coli SEQ ID 4 (strain K12) Uridine monophosphate URA6 2.7.4.47 Arabidopsis SEQ ID 5 kinase thaliana Polyphosphate kinase 3 PPK3 2.7.4.1 Ruegeria pomeroyi SEQ ID 6 (strain ATCC 700808/DSM 15171/DSS-3) Inorganic PPA 3.6.1.1 Pasteurella SEQ ID 7 diphosphatase multocida (strain Pm70) 2-domain polyphosphate 2D-PPK2 2.7.4.1 Pseudomonas SEQ ID 8 kinase 2 aeruginosa 1-domain polyphosphate 1D-PPK2 2.7.4.1 Pseudomonas SEQ ID 9 kinase 2 aeruginosa
Enzyme Purification
[0770] The clear cell lysate was loaded on a Ni-NTA affinity column on an AKTA system. The column was washed with 20% of elution buffer. Enzymes were eluted using elution buffer. Enzyme solutions were concentrated and dialysed (to remove imidazole) with 3 kDa Amicon filters and then stored in storage buffer at 20 C.
Buffer Composition of Lysis/Binding Buffer (A), Elution Buffer (B) and Storage Buffer (C)
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TABLE-US-00004 TABLE 2 Lysis/binding buffer (A) Lysis/Binding buffer Conc. (mM) MOPS 50 mM NaCl 300 mM MgCl.sub.2 10 mM glycerol 5% imidazole 10 mM pH 7.4
TABLE-US-00005 TABLE 3 Elution buffer (B) Elution buffer Conc. (mM) MOPS 50 mM NaCl 300 mM MgCl.sub.2 10 mM glycerol 5% imidazole 250 mM pH 7.4
TABLE-US-00006 TABLE 4 Storage buffer (C) Enzyme Storage buffer Conc. (mM) MOPS 25 mM NaCl 150 mM MgCl.sub.2 5 mM glycerol 5% pH 7.4
Plasmids and Stock Cultures
[0772] Stock solutions of all E. coli cultures carrying the plasmids (pET28a with kanamycin resistance) with the gene sequences were available from earlier studies [1,2]. The stock solutions contained 50% glycerol and were kept at 20 C.
[0773] The gene and corresponding protein sequences were obtained from the UniProt database: AGE (WP_011320279.1), NAL (Q9CKB0), CSS (P0A0Z7), UDK (P0A8F4, URA6 (004905), PPA (P57918), PPK3 (Q5LSN8), 2DPPK2 (Q9HYF1), and 1 DPPK2 (Q92SA6). The plasmids were ordered from commercial suppliers (BioCat GmbH):
TABLE-US-00007 TABLE 5 Enzymes and plasmids used in the experiments Enzyme Vector Selection marker AGE pET-28a(+) Kanamycin NANA pET-22b(+) Ampicilin UDK pET-28a(+) Kanamycin CSS pET-100/D-TOPO Ampicilin URA6 pET-28a(+) Kanamycin PPK3 pET-28a(+) Kanamycin PmPPA pET-28a(+) Kanamycin 1D-PPK2 pET-28a(+) Kanamycin 2D-PPK2 pET-28a(+) Kanamycin
[0774] PPA (PmPpA; enzymes carrying a C-terminal hexahistidin-tag (His-tag)), PPK3 and URA6 (for an N-terminal His-tag). After transformation of the plasmids into E. coli, the DNA was isolated and the accuracy of the constructs was checked by gene sequencing (Eurofins Genomics, Ebersberg, Germany). [0775] 1. Mahour, R., et al., Establishment of a five-enzyme cell-free cascade for the synthesis of uridine diphosphate N-acetylglucosamine. Journal of Biotechnology, 2018. 283: p. 120-129. [0776] 2. Rexer, T. F. T., et al., One pot synthesis of GDP-mannose by a multi-enzyme cascade for enzymatic assembly of lipid-linked oligosaccharides. Biotechnology and Bioengineering, 2018. 115(1): p. 192-205.
One-Pot Cascade Reactions
[0777] Immobilized enzymes can often be separated from solutions and reused. Moreover, they may exhibit higher activity and can be used for a wide range of processes, such as continuous synthesis in packed bed reactors. A wide range of commercially available solid supports were tested for the co-immobilization of the CMP-Neu5Ac multi-enzyme cascade.
Reactions
[0778] Cascade reactions were conducted in 1.5 mL safe-lock Eppendorf vials in volumes of 150 L at 35 C. in a thermomixer at 450 rpm shaking. [0779] All enzymes were mixed into one vial [0780] Reactions were started by mixing enzymes, buffers and reactants [0781] For profiling the time course of the reaction, at each time point 3 L sample were aliquoted and quenched by adding 297 L cold (4 C.) dH2O and measured by anion exchange chromatography without delay.
Measurements
[0782] High-performance anion exchange chromatography (HPAEC) with UV (260 nm) and pulsed amperometric detection (PAD) was utilized to measure concentrations of reactants. For analyte separation and quantification a step gradient elution method was developed and validated chromatographic separation was performed at a system flow of 0.5 mL/min using a non-porous pellicular column CarboPac PA200 (2502 mm). The HPAEC system (ICS5000) as well as all columns, components and software were purchased from Thermo Scientific (Waltham, USA).
Enzyme Immobilization
[0783] It should be noted that finding the optimal solid support is always down to experimental trial and error as insufficient knowledge about the immobilization of enzymes exist to predict the optimal solid support.
[0784] The surprising finding was that the multi-enzyme cascade showed activity when co-immobilized on a wide range of epoxy supports. The epoxy supports that were tested and showed activity varied in support matrix, particle size, pore size and oxiran content. Other solid supports where enzymes are immobilized by hydrophobic adsorption, ionic interaction or covalent crosslinking with glutaraldehyde showed very little to no activity implying that at least one of the five key enzymes are little active to inactive. Moreover, the multi-enzyme cascade was active on epoxy supports when a large range of different rations of proteins to solid supports where used. For the synthesis of CMP-Neu5Ac, many of the epoxy supports loaded with the enzymes could be used in more than 20 reaction cycles without re-immobilizing the enzymes on the supports. Tested Epoxy supports are summarized in Table 7.
TABLE-US-00008 TABLE 7 Selection of tested epoxy (including amino-epoxy) supports Resin Mass (mg) EC-EP 109 EP403/M 91 IB-COV1 95 IB-COV2 111 IB-COV3 91 Eupergit CM 100 ECR8215F 92 ECR8204F 84 ECR8209F 84 ECR8285 84 EP403/S 82 EP400/SS 102 EC-HFA/M 111 HFA403/M 98 HFA403/S 94 EC-HFA/S 104
Experiment A
[0785] A wide range of commercially available solid supports (see Table 7) were tested for the co-immobilization of the enzymes, NmCSS, PPK3 and URA6, used in the CMP-Neu5Ac synthesis (see
[0786] To test the multi-enzyme cascade on various enzyme loaded beads, a given mass (see Table 1) of each resin was added.
[0787] 100 L of reaction buffer (see below) was added to the beads and incubated at 30 C. and 550 rpm for 20 h. Afterwards, the supernatant was analyzed for CMP-Neu5Ac. The CMP-Neu5Ac concentrations were then measured by HPAEC-UV/PAD.
Results:
[0788] Surprisingly it has been found that co-immobilization of the set of enzymes results in a higher productivity in the production of cytidine 5-monophospho-N-acetyl-neuraminic acid (CMP-Neu5Ac) compared to non-immobilized or separately immobilization of the enzymes. Thus, preferably the enzymes used in the inventive methods described herein are co-immobilized on a solid support. Surprisingly, the beads with the enzyme can be used in more than 11 cycles (see
TABLE-US-00009 TABLE 8 Concentration of reactants in the feed solution of Experiment A Substrate Conc. (mM) Neu5NAc 5 CMP 5 ATP 5 PolyP.sub.25 30 MgCl.sub.2 HEPES
Experiment CProof of Concept
[0789] To check whether CMP-Neu5Ac can be produced in a one-pot reaction using the designed pathway, a reaction with the concentrations as detailed in the table below was conducted. The concentrations of reactants were measured over the time (see chromatograms after 4 min and 30 hours below). As shown in the chromatogram below, CMP-Neu5Ac was produced. The concentration time course of CMP-Neu5Ac is shown below.
TABLE-US-00010 TABLE 9 Concentration of reactants in the feed solution. Enzyme/Compound Conc./Mass AGE 2.57 g NANA 3 g CSS 3 g UDK 8.8 g URA6 8 g PPK3 21 g GlcNAc 5 mM Cytidine 2 mM Pyruvate 5 mM ATP 4 mM PolyP.sub.25 4 mM MOPS 50 mM MgCl2 20 mM pH 7.4 Volume 150 L
Results
[0790] It was shown that with the set of enzyme, CMP-Neu5Ac could successfully be produced from GlcNAc, pyruvate and cytidine.
Experiment DIncreased Substrate Concentrations
[0791] In experiment D, two cascade reaction (D1 with and D2 without PPA) were conducted with higher substrate concentrations (see below for initial concentrations). After overnight incubation the reactant concentrations were measured by HPAEC-UV detection.
[0792] CMP-Neu5Ac was successfully synthesized through the cascades. However, considerable concentration of CMP, CDP and CTP were detected implying low yields.
TABLE-US-00011 TABLE 10A Initial concentration of reactants for reaction D1 - cascade reaction without PPA (see FIG. 6) Enzyme Conc. (g/L) UDK 95 URA6/PPK3 185 CSS 1225 AGE 28 NANA 841 Reactants Conc. (mM) Cytidine 36 GlcNAc 39.6 Pyruvate 39.6 ATP 3.6 PolyP.sub.25 14.5 Buffer Conc. (mM) Tris (8.5) 145 Co-factor Conc. (mM) MgCl.sub.2 54 Total volume (L) 227.5
TABLE-US-00012 TABLE 10B Initial concentration of reactants for reaction D2 - cascade reaction with PPA (see FIG. 7) Enzyme Conc. (g/L) UDK 93 URA6/PPK3 155 CSS 1203 AGE 27 NANA 826 PPA 56 Reactants Conc. (mM) Cytidine 35.4 GlcNAc 39 Pyruvate 39 ATP 3.5 PolyP.sub.25 14.1 Buffer Conc. (mM) Tris (8.5) 140 Co-factor Conc. (mM) MgCl.sub.2 53 Total volume (L) 282.5
Experiment EInhibition of AGE by CTP
[0793] As know from the literature, CTP inhibits AGE. We independently verified this in a reaction starting the synthesis of CMP-Neu5Ac from GlcNAc and CTP (see below). The initial substrate and enzyme concentrations are shown in the table below.
[0794] In an overnight reaction very little CMP-Neu5Ac was detected, verifying the inhibition of AGE by CTP (see chromatogram below).
##STR00027##
TABLE-US-00013 TABLE 11 Initial substrate and enzyme concentration of experiment E (see also FIG. 8) Enzyme Conc. (g/L) UDK 26 URA6/PPK3 778 CSS 1275 PPA 47 Reactants Conc. (mM) GlcNAc 10 Pyruvate 10 CTP 10 Buffer Conc. (mM) Tris (8.5) 150 Co-factor Conc. (mM) MgCl.sub.2 50 Total volume (L) 200
Experiment FIncreasing the Yield
[0795] It is known that CTP inhibits AGE and ATP activates AGE. However, by adjusting the initial substrate and enzyme concentrations the yield and product concentration can be optimised. Decreasing cytidine is increasing the yield but decreases the CMP-Neu5Ac end concentration. In Experiment D, the ratio of cytidine-ATP-PolyP was kept constant (1-0.3-0.8) while the cytidine concentrations were increased (see below for initial concentrations).
[0796] After 40 hours of incubation reactions F1 and F2 resulted in almost full conversion of cytidine to CMP-Neu5Ac as shown in
TABLE-US-00014 TABLE 12 Initial concentration of reactions F1-F4 (see also FIG. 9) Reaction F1 F2 F3 F4 Enzyme Conc. (g/L) UDK 65 61 56 53 URA6/PPK3 82 76 71 66 CSS 1262 1173 1094 1024 AGE 38 35 33 31 NANA 1155 1075 1002 937 PPA 31 29 27 25 Reactants Conc. (mM) Cytidine 10 18.4 25.7 32 GlcNAc 75 69 64.2 60.2 Pyruvate 80 73.5 68.5 64.2 ATP 3 5.3 7.8 9.6 PolyP.sub.25 8 14.5 20.5 26 Buffer Conc. (mM) Tris (8.5) 150 140 130 120 Cofactor Conc. (mM) MgCl.sub.2 75 70 65 60 Total volume (L) 202 217 233 249
Experiment G
[0797] An additional reaction was carried out measuring concentrations over time (see below for initial concentrations). The reaction shows the conversion of cytidine, GlcNAc and pyruvate to CMP-Neu5Ac with a yield of about 75% with respect to cytidine and CMP-Neu5Ac titers of about 25 mM (16 g/L).
TABLE-US-00015 TABLE 13 Initial concentration of experiment G (see also FIG. 10) Enzyme Conc. (g/L) UDK 56 URA6/PPK3 71 CSS 1094 AGE 33 NANA 1002 PPA 27 Reactants Conc. (mM) Cytidine 33 GlcNAc 75 Pyruvate 80 ATP 9 PolyP.sub.25 24 Buffer Conc. (mM) Tris (8.5) 150 Co-factor Conc. (mM) MgCl.sub.2 75 Total volume (L) 233.5
Example H: Simultaneous Production of Enzymes for the Synthesis of CMP-Neu5Ac at 100 mL Scale
[0798] For the production of CMP-Neu5Ac from CMP, Neu5Ac, PolyP.sub.n, and catalytic amounts of ATP one single strain was generated from which all three enzymes are overexpressed simultaneously. The genes and vectors used in this work are shown below:
TABLE-US-00016 TABLE 14 Enzymes and vectors for the production of the cascade in one single strain. Uniprot Restric- Gene Enzyme Source acc. No. Plasmid tion site UMK3 UMPK Arabidopsis O04905 pACYCDuet Ncol, thaliana Notl SPO1727 PPK3 Ruegeria Q5LSN8 pACYCDuet Ndel, pomeroyi Kpnl neuA CSS Neisseria P0A0Z8 pET100/D- meningitidis TOPO
[0799] The biomass from a 200 mL culture was lysed by high pressure homogenizer in 40 mL lysis buffer containing 25 mM Tris-HCl (pH 7.1), 400 NaCl and 5% glycerol. After centrifugation, the supernatant containing the overexpressed enzymes was used to initiate a synthesis reaction. The 100 mL scale reaction was carried out in a spinner flask. The reaction matrix contained 150 mM Tris-HCl (pH 8.5), 75 mM MgCl.sub.2, 50 mM CMP, 51 mM Neu5Ac, 5 mM ATP, 16 mM PolyP.sub.n. After 6.6 h of incubation at 37 C. and 50 rpm, CMP-Neu5Ac was produced with a final concentration of 45.3 mM (27.8 g/L) and a yield of around 90%. The productivity was 4.2 g/(L*h). The chromatogram of the reaction mixture at the end of the reaction is shown in
Example 3: Coupling of the Cascade
[0800] The cascade can be coupled to sialyltransferase to transfer CMP-Neu5Ac to acceptor molecules. Acceptor molecules can be for example monoclonal antibodies. For the coupling soluble sialyltransferase can be added, a sialyltransferase can be co-immobilized on the same support and/or the sialyltransferase can be immobilized on an additional support and then be added to reaction.
Example 4: Production of Neu5Acylated Biomolecules
[0801] The synthesis of Neu5Acylated biomolecules is facilitated by producing CMP-Neu5Ac in a one pot multi-enzyme cascade reaction first and then mixing it with the biomolecule substrate as well as a sialyltransferase to transfer Neu5Ac from CMP-Neu5Ac to the substrate. In the examples below the biomolecules are human milk oligosaccharides (HMOs).
Methods
[0802] All experiments were performed in 1.5 mL Eppendorf safe lock tubes and at 37 C. under shaking (550 rpm). For the identification of compounds, high performance anion chromatography (HPAEC) with pulsed amperometric detection (PAD) was used. The HPAEC system was equipped with Dionex CarboPac PA200 guard and analytical columns (in series, Thermo Scientific, USA). Aqueous solution with various concentrations of sodium hydroxide and sodium acetate were used as eluents.
Sample Preparation for Mass Spectrometry:
[0803] Before performing mass spectrometry (MS) on the samples, cotton hydrophilic interaction liquid chromatography (HILIC) was carried out to remove salts from the solution. In short, 15 L of the samples were mixed with 85 L of 100% acetonitrile (ACN). Approximately, one fifth of a 200 L pipette tip was filled with cotton. Afterwards, the cotton was washed with water to remove any contamination. After equilibration with 85% ACN, samples were loaded by pipetting up and down, followed by washing steps (five times) with 85% ACN and 1% trifluoroacetic acid (TFA). Oligosaccharides were eluted from the cotton matrix with 50 L of water in three steps (final volume:150 L).
Mass Spectrometry:
[0804] The UltraFlextreme matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-MS (Bruker Daltonics, Germany) was used for the analyses of HMOs and sugar nucleotides. For the analyses of HMOs, super-DHB (Merck, Germany) mixed with 10 mg/mL TA30 (2 mM NaCl in a solution consisting of 70% H.sub.2O, supplemented with 0.1% TFA, and 30% ACN) was used the matrix. Briefly, 1 L of matrix was spotted on a AnchorChip 384 BC MALDI target plate (Bruker Daltonics) and left to dry by air. Afterwards, a sample aliquot of 1 L was added to the spots. After drying, 0.2 L of ethanol per spot was added to allow rapid and homogenous recrystallization. The HMOs and sugar nucleotides analyses were measured in positive-ion, and negative-ion reflector mode, respectively. The calibration for positive-ion mode was carried out with a Dextran ladder.
Enzyme Preparation:
[0805] The list of genes, their origin and the vectors used are described in Table 15. The LOBSTR E. coli (Kerafast, USA) strain was used as the expression host. The cells harboring the plasmids were cultivated in terrific broth media supplemented with 1.5 mM MgSO.sub.4 and selection markers at 37 C. At an OD of 0.8-1 the gene expression was induced by the addition of 0.4 mM IPTG, followed by 20-24 hours of incubation at 16 C. (15 C. for the -2,6-sialyltransferase).
TABLE-US-00017 TABLE 15 List of genes, their origin, and plasmid used for heterologous expression Enzyme Gene Full name origin Plasmid LGTA LgtA -1,3-N-acetylglucosamine Neisseria pMAL-c4X transferase meningitidis LGTB LgtB -1,4- Neisseria pET-15b galactosyltransferase meningitidis -2,6-ST P145-ST -2,6-sialyltransferase Photobacterium pCold II leiognathi
[0806] At the end of cultivation, the cells were precipitated by centrifugation (7000g, 30 minutes) and lysed by high-pressure homogenization (3 to 5 passages at 800-1000 psg). Afterwards, cell debris was removed by centrifugation (7000g, 45 minutes) and filtration of supernatant through a 0.45 m cellulose acetate filter. For the purification of enzymes, common His-tag purification method were used. The binding (lysis) buffer was: 50 mM MOPS (pH 7.5). 10 mM MgCl.sub.2, 300 mM NaCl, 5% glycerol, and 10 mM imidazole. The elution buffer was 50 mM MOPS (pH 7.5). 10 mM MgCl.sub.2, 300 mM NaCl, 5% glycerol, and 250 mM imidazole.
[0807] After elution, fractions containing the enzymes of interest were pooled together. A buffer exchange and enzyme concentration was carried out using 3 kDa Amicon filter units. Enzymes were mixed 1:1 with glycerol and stored at 20 C.
Example 4-1: Production of sialyllacto-N-neotetraose c (LSTc) and disialyllacto-N-neotetraose (DSLNnT)
[0808] CMP-Neu5Ac was produced in one-pot multi-enzyme reaction using the cascade described earlier (for the reaction condition see Table 16). Afterwards, an aliquot (70 L) of the latter was mixed with a buffered (Tris-HCl) solution (198 L, pH 8.5) containing LNnT, alkaline phosphatase (30 units) and -2,6-sialyltransferase (-2,6-ST). The production of LSTc and DSLNnT was confirmed by MALDI-TOF-MS (see
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TABLE-US-00018 TABLE 16 Reaction condition for the production of CMP-Neu5Ac. Concentration Enzyme (g/mL) Reactants Concentration (mM) UDK 56 cytidine 33 URA6/PPK3 71 GlcNAc 75 CSS 1094 pyruvate 80 AGE 33 ATP 2.6 NANA 1002 PolyP.sub.25 24 PPA 27 Buffer Concentration (mM) Tris (8.5) 150 Co-factor Concentration (mM) MgCl.sub.2 75 Total volume (L) 233.5
Example 4-2: Production of 6-Sialyllactose (6-SL)
[0809] For the synthesis of 6-SL from lactose and CMP-Neu5Ac, 70 L of previously detailed one-pot reaction mix containing CMP-Neu5Ac (see Table 16) as the product was mixed with lactose (20 mM), MnCl.sub.2 (20 mM), Tris-HCl (150 mMpH 8), and 0.3 g/L -2,6-ST to a final volume of 200 L. The successful production of 6-SL is confirmed by the HPAEC chromatogram and MS/MS spectra of the reaction mix at the reaction end point (see
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