DE102018116200A1 - METHOD FOR PRODUCING A NUCLEOTIDE SUGAR - Google Patents

Abstract

In a method for producing a monosaccharide-1-nucleoside diphosphate, a monosaccharide, a monosaccharide-1-phosphate kinase, a nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase, a pyrophosphatase and at least one nucleoside triphosphate or a deoxynucleoside and a deoxynucleoside tripod are used in a reaction vessel contacted in an aqueous solution. The specificity of the monosaccharide-1-phosphate kinase allows phosphorylation of the monosaccharide and the specificity of the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase allows the transfer of a nucleoside monophosphate unit to a monosaccharide-1-phosphate of the monosaccharide. When the monosaccharide is converted to the monosaccharide 1-nucleoside diphosphate in a temperature range from 0 ° C to 95 ° C, the protein concentration is at least 20 µg / ml. The monosaccharide-1-nucleoside diphosphate is removed by means of an ultra and / or nanofiltration membrane in such a way that the total volume is maintained or restored, the enzymes remain and the at least one nucleoside triphosphate or the deoxynucleoside triphosphate and the nucleoside triphosphate and the monosaccharide are added again.

Description

area

The present disclosure relates to a method for producing a monosaccharide-1 nucleoside diphosphate.

thanksgiving

The invention on which this patent application is based was created in a project funded by the BMBF under grant number 031A557A.

background

Glycosylation of natural products is becoming increasingly important in the discovery and development of active pharmaceutical ingredients and the development of food ingredients. The glycosylation is often decisive for the effectiveness of a natural product in a desired application. Glycosylation is usually a post-translational modification in the protein biosynthesis of a glycoprotein, as N-glycosylation in the endoplasmic reticulum and Golgi device or as O-glycosylation in the Golgi device. Glycosyltransferases catalyze the transfer of monosaccharide units from an activated donor substrate to an acceptor molecule, usually a hydroxy group (alcohol, monosaccharide), and thus produce glycosidic compounds.

Glycosyltransferases that use nucleotide sugar as the donor molecule are called Leloir enzymes, after Luis F. Leloir, the scientist who discovered the first sugar nucleotide and received the Nobel Prize in Chemistry in 1970 for his work on carbohydrate metabolism. Due to their specificity, regioselectivity and quantitative substrate conversion, Leloir glycosyltransferases are the enzymes of choice for glycan and glycoside synthesis ( Schmaltz, RM, et al., Chemical Reviews (2011), 111, 4259-4307 ). Nucleotide sugars were also used for the synthesis of oligosaccharides, which were formed from monosaccharides ( US 2014/235575 A1 ).

Nucleotide sugars are still considered to be cost-intensive substrates and thus a bottleneck for the large-scale application of Leloir glycosyltransferases as standard procedures in synthetic carbohydrate chemistry. However, both multi-enzyme cascades and in-situ regeneration systems have been developed that cover a range of nucleotide-activated sugars ( Schmaltz et al., 2011, supra; Engels, L., et al., Adv. Synth. Catal. (2015) 357, 1751-1762 ; Rupprath, C., et al., Adv. Synth. Catal. (2007), 349, 1489-1496 ; Zervosen, A., et al., J. Mol. Catal. B: enzyme. (1998) 5, 25-28 ; Zervosen, A., et al., Tetrahedron (1996) 52, 2395-2404; Zhao, G., et al., Nat. Protoc. (2010) 5, 636-646 ; Rupprath, C., et al., Curr. Med. Chem. (2005) 12, 1637-1675 ). The development of enzyme cascades with a combination of monosaccharide-1-phosphate kinases and UDP-sugar pyrophosphorylases was a breakthrough in the field of enzymatic nucleotide sugar synthesis and its analogues (Zhao et al., 2010, supra). The synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc) was made possible by the discovery of an N-acetylhexosamine-1 kinase from Bifidobacterium longum (NahK, EC 2.7.1.162) (Zhao et al., 2010, supra). Their combination with a human UDP-N-acetylhexosamine pyrophosphorylase (AGX1, EC 2.7.7.83) gave UDP-GlcNAc in a relatively high product yield (75%) within two hours reaction time (Guan, W., et al., Chem. - Eur. J. (2010) 16, 13343-13345.). This enzyme cascade was also used for the synthesis of UDP-N-acetylgalactosamine (UDP-GalNAc) GalNAc with 79% yield in two hours reaction time ( Guan, W., et al., Chem. Commun. (2009) 6976-6978 ).

However, the production of suitable amounts of enzymes for large-scale synthesis is a cost factor and complex. The maximization of enzyme productivity in relation to the mass-based turnover number (TTN _mass , g enzyme per g product) and the specific product performance (space-time yield, STY) is therefore a challenge to exploit the full potential of the enzyme cascade.

Summary

In a first aspect, a method for producing a monosaccharide-1-nucleoside diphosphate is disclosed here. In this method, a monosaccharide, a monosaccharide-1-phosphate kinase, a nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase, a pyrophosphatase and at least one nucleoside triphosphate or a deoxynucleoside triphosphate and a Nucleoside triphosphate contacted in an aqueous solution. The nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase can also be referred to as nucleoside diphosphate sugar pyrophosphorylase. An aqueous reaction mixture is formed by contacting in aqueous solution. The monosaccharide-1-phosphate kinase is selected so that the specificity of the monosaccharide-1-phosphate kinase allows phosphorylation of the monosaccharide by means of the deoxynucleoside triphosphate or one of the at least one nucleoside triphosphate. Furthermore, the nucleoside triphosphate, or one of the at least one nucleoside triphosphate, is selected such that the specificity of the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase allows the transfer of a nucleoside monophosphate unit to a monosaccharide 1 phosphate of the monosaccharide. The method also includes allowing the monosaccharide to be converted into a monosaccharide-1-nucleoside diphosphate according to the following scheme in a temperature range from 0 ° C to 95 ° C.

In this scheme (1) indicates the monosaccharide. NTP and NuTP denote a nucleoside triphosphate and dNTP a deoxynucleoside triphosphate. (2) indicates the nucleoside triphosphate which is provided for the reaction with the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase, which is denoted by (6). NTP and NuTP can be identical or different. Nu denotes the nucleosidyl group of the nucleoside triphosphate which is suitable for reacting with the monosaccharide 1-phosphate by means of which the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase.

In the previous scheme (3) indicates the monosaccharide 1-phosphate. (4) indicates the monosaccharide-1 nucleoside diphosphate and (5) indicates the monosaccharide-1-phosphate kinase. The radical Z stands for H, a -CH ₂ OH-, a -CH ₃ or a -COOH group. Y is an OH group, an NH ₂ group or an N-acetyl group. The protein concentration in the reaction mixture during the reaction is 5 µg / ml or more. The method also includes removing the monosaccharide-1 nucleoside diphosphate from the reaction vessel in such a way that the monosaccharide-1-phosphate kinase, the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase and the pyrophosphatase remain in the reaction vessel. The monosaccharide 1 nucleoside diphosphate is removed from the reaction vessel by means of an ultra and / or nanofiltration membrane. To remove the monosaccharide-1 nucleoside diphosphate while retaining the monosaccharide-1-phosphate kinase, the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase and the pyrophosphatase, it is important to maintain or restore the total volume of the reaction mixture in the reaction vessel. Finally, the method includes feeding the at least one nucleoside triphosphate or the deoxynucleoside triphosphate and the nucleoside triphosphate and the monosaccharide to the reaction mixture in the reaction vessel.

The monosaccharide-1-phosphate kinase and the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase are chosen independently of one another. In some embodiments, the monosaccharide-1-phosphate kinase and the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase are the same enzyme. For example, functionalities of the same enzyme can be involved. It can also be a single reaction in which a direct conversion of the the monosaccharide into that Monosaccharide-1 nucleoside diphosphate takes place. In some embodiments, the monosaccharide-1-phosphate kinase and the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase are different enzymes.

Removing the monosaccharide 1 nucleoside diphosphate means that it is part of the process to let the monosaccharide 1 nucleoside diphosphate pass through the ultra and / or nanofiltration membrane.

In some embodiments, the monosaccharide contains a plurality of monosaccharides. In some such embodiments, the monosaccharide-1-phosphate kinase contains a plurality of monosaccharide-1-phosphate kinases. In some such embodiments, the nucleoside triphosphate sugar 1 phosphate nucleosidyl transferase contains a plurality of nucleoside triphosphate sugar 1 phosphate nucleosidyl transferases.

In some embodiments, the reaction vessel contains the ultra and / or nanofiltration membrane. In some embodiments, the reaction vessel is in fluid communication with the ultra and / or nanofiltration membrane. In some embodiments, the reaction vessel is in fluid communication with the ultra and / or nanofiltration membrane. In some such embodiments, removal of the monosaccharide-1 nucleoside diphosphate may include a pressure differential of 100 between the side of the ultra and / or nanofiltration membrane that is in fluid communication with the reaction vessel and the opposite side of the ultra and / or nanofiltration membrane to generate kPa or more, including 250 kPa or more. For example, an excess pressure of 100 kPa or more, including 250 kPa or more, can be provided in the reaction vessel compared to the ambient pressure. In all such embodiments, the removal of the monosaccharide-1-nucleoside diphosphate can also include allowing a force of at least 2.5 x g to act on the reaction mixture located above the ultra and / or nanofiltration membrane.

In some embodiments, the reaction vessel is an ultrafiltration concentrator for centrifugation.

In some embodiments, the at least one nucleoside triphosphate is removed from the reaction mixture along with the monosaccharide 1 nucleoside diphosphate.

In some embodiments, the method includes contacting a monosaccharide, a monosaccharide-1-phosphate kinase, a nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase, a pyrophosphatase, and at least one nucleoside triphosphate. At least one nucleoside triphosphate is ATP in some embodiments.

For example, one of the methods disclosed here may include allowing the monosaccharide to be converted to a monosaccharide-1 nucleoside diphosphate according to the following scheme:

The designations in this scheme are the same as those given above. In such embodiments, the nucleoside triphosphate or one of the at least one nucleoside triphosphate is selected such that the specificity of the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase allows the transfer of a nucleoside monophosphate unit to a monosaccharide 1 phosphate of the monosaccharide.

The enzymes used are typically used in a non-immobilized form. The enzymes used are typically used in a non-crosslinked form. As a rule, the enzymes used are in aqueous solution as a soluble, free protein.

In some embodiments, one or more additional cofactors are used that are necessary for the function of one or more enzymes used. In particular, this may be a magnesium salt which forms a complex with a nucleoside triphosphate and / or a deoxynucleoside triphosphate. A manganese salt can also be used as a cofactor for a number of the enzymes which can be used in a process and a use disclosed here. In some embodiments, a monosaccharide, a monosaccharide-1-phosphate kinase, a nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase, a pyrophosphatase, at least one nucleoside triphosphate and a magnesium salt or a manganese salt in aqueous solution are contacted in the reaction vessel. In some embodiments, essentially only a monosaccharide, a monosaccharide-1-phosphate kinase, a nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase, a pyrophosphatase, at least one nucleoside triphosphate, and a magnesium salt are contacted in the reaction vessel. Typically, the aqueous solution contains one or more buffer compounds such as Trisaminomethane (Tris), 2- (4- (2-Hydroxyethyl) -1-piperazinyl) ethanesulfonic acid (HEPES), 4- (2-Hydroxyethyl) piperazine-1-propanesulfonic acid (HEPPS), an acetate, or a phosphate. In some embodiments, in an aqueous phase containing such a buffer compound, only a monosaccharide, a monosaccharide 1-phosphate kinase, a nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase, a pyrophosphatase, at least one nucleoside triphosphate and a magnesium salt are contacted brought.

In some embodiments, the process described here is carried out without the presence of another protein in the reaction mixture. In some embodiments, the method described here is carried out without adding another protein to the reaction mixture. In some embodiments, the process described here is carried out without the presence of another saccharide in the reaction mixture. In some embodiments, the process described here is carried out without adding another saccharide to the reaction mixture. In some embodiments, the process described here is carried out without the presence of another monosaccharide in the reaction mixture. In some embodiments, the process described here is carried out without adding another monosaccharide to the reaction mixture. In some embodiments, the process described here is carried out without the presence of a disaccharide in the reaction mixture. In some embodiments, the process described here is carried out without adding another disaccharide to the reaction mixture.

In some embodiments, in which at least one nucleoside triphosphate is used, the at least one nucleoside triphosphate contains, in addition to ATP, one or more of the nucleoside triphosphates UTP, GTP and CTP. In some embodiments, the at least one nucleoside triphosphate consists of ATP and UTP. In some embodiments, the at least one nucleoside triphosphate consists of ATP and GTP. In some embodiments, the at least one nucleoside triphosphate consists of ATP and CTP.

In some embodiments using a deoxynucleoside triphosphate and a nucleoside triphosphate, the nucleoside triphosphate is or contains one or more of the nucleoside triphosphates ATP, UTP, GTP and CTP. In some embodiments using a deoxynucleoside triphosphate and a nucleoside triphosphate, the nucleoside triphosphate contains one or more of the nucleoside triphosphates UTP, GTP and CTP. In some embodiments, in which a deoxynucleoside triphosphate and a nucleoside triphosphate are used, the nucleoside triphosphate consists of UTP. In some embodiments in which a deoxynucleoside triphosphate and a nucleoside triphosphate are used, the nucleoside triphosphate consists of GTP. In some embodiments, the nucleoside triphosphate consists of CTP.

In some embodiments, the protein concentration in the reaction mixture during the reaction is 10 µg / ml or more. In some embodiments, the protein concentration in the reaction mixture during the reaction is 20 µg / ml or more.

The process is typically carried out as a repetitive batch process. In this case there is usually no need to add reagents. Apart from taking samples as part of the analysis, there is usually no need to take anything from the process. In some embodiments, the process is carried out as a continuous process. In this case, care must typically be taken to ensure that the removed nucleotide, monosaccharide and magnesium salt are replaced or returned to the reaction mixture.

In some embodiments, the monosaccharide is galactose or glucose. In some embodiments, the monosaccharide is N-acetylgalactosamine. In some embodiments, the monosaccharide is mannose or xylose. In some embodiments, the monosaccharide is glucuronic acid. In some embodiments, the monosaccharide is galacturonic acid. In some embodiments, the monosaccharide is fucose.

In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a UDP sugar pyrophosphorylase from Hordeum vulgare (barley). In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is the human UDP-GaINAc pyrophosphorylase. In some embodiments, the monosaccharide-1-phosphate kinase is an E. coli galactokinase. In some embodiments, the monosaccharide-1-phosphate kinase is an N-acetylhexosamine-1-kinase from Bifidobacterium longum.

In some embodiments, the original volume of the aqueous reaction mixture is restored when the deoxynucleoside triphosphate and the nucleoside triphosphate, or the at least one nucleoside triphosphate, and the monosaccharide are added again. In some embodiments, the original volume is exactly restored. In some embodiments, the original volume is restored with a tolerance of ± 2% or with a tolerance of ± 5%. In some embodiments, the original volume is restored with an error tolerance of ± 10%.

In some embodiments, the original volume of the aqueous reaction mixture is restored after the deoxynucleoside triphosphate and the nucleoside triphosphate, or the at least one nucleoside triphosphate, have been added again. In some embodiments, the original volume is restored exactly or as stated above with an error tolerance of ± 2%. In some embodiments, the original volume is restored with an error tolerance of ± 5% or ± 10%.

In some embodiments, the method of the first aspect involves repeating a cycle at least 10 times or at least 20 times, which includes, as previously described, allowing the conversion of the monosaccharide to a monosaccharide-1 nucleoside diphosphate, the monosaccharide Remove -1-nucleoside diphosphate from the reaction vessel and feed the deoxynucleoside triphosphate and the nucleoside triphosphate, or the at least one nucleoside triphosphate, to the reaction mixture again. In some embodiments, the method according to the first aspect involves repeating a cycle as described above 40 times.

In a second aspect, the use of the combination of a reaction vessel, an enzyme mixture and an ultra and / or nanofiltration membrane for producing a monosaccharide-1-nucleoside diphosphate on a gram scale is disclosed here. The enzyme mixture contains a monosaccharide 1-phosphate kinase, a nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase and a pyrophosphatase. In use, the enzyme mixture is provided in the form of an aqueous solution in the reaction vessel. Furthermore, a monosaccharide and at least one nucleoside triphosphate or a monosaccharide, a deoxynucleoside triphosphate and a nucleoside triphosphate are added to this aqueous solution. In use, a conversion of the monosaccharide to a monosaccharide-1-nucleoside diphosphate according to the above scheme is also permitted in a temperature range from 0 ° C to 95 ° C

The names in this scheme are the same as those given in the explanation of the method of the first aspect. During the reaction, the protein concentration in this reaction mixture is 5 µg / ml or more. The ultra and / or nanofiltration membrane serves to remove the monosaccharide-1 nucleoside diphosphate from the reaction vessel during or after the reaction. Through this membrane, the monosaccharide-1-nucleoside diphosphate is removed from the reaction vessel in such a way that the total volume of the aqueous solution in the reaction vessel is retained or restored. Furthermore, the membrane removes the monosaccharide 1-nucleoside diphosphate from the reaction vessel in such a way that the enzyme mixture remains in the reaction vessel. The at least one nucleoside triphosphate, or the deoxynucleoside triphosphate and the nucleoside triphosphate, and the monosaccharide are added to the reaction mixture again.

The monosaccharide 1 nucleoside diphosphate is removed by letting it pass through the ultra and / or nanofiltration membrane.

In some embodiments, the enzyme mixture consists of the monosaccharide 1-phosphate kinase, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase, and the pyrophosphatase. The monosaccharide-1-phosphate kinase can be a single enzyme or consist of several monosaccharide-1-phosphate kinases, for example of several monosaccharide-1-phosphate kinases, each with different sequences. The nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase can also be a single enzyme or consist of several nucleoside triphosphate sugar 1 phosphate nucleosidyl transferase. In some embodiments, the pyrophosphatase may be a single enzyme, e.g., a particular enzyme with a particular sequence. In some embodiments, the pyrophosphatase can consist of multiple pyrophosphatase.

In the use according to the second aspect, the monosaccharide 1-phosphate kinase and the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase are selected independently of one another. In some embodiments, the monosaccharide-1-phosphate kinase and the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase are the same enzyme. For example, functionalities of the same enzyme can be involved. In some embodiments, the monosaccharide-1-phosphate kinase and the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase are different enzymes.

In some embodiments, use according to the second aspect includes adding a monosaccharide and at least one nucleoside triphosphate to the aqueous solution containing the enzyme mixture. At least one nucleoside triphosphate is ATP in some embodiments.

In some embodiments of use according to the second aspect, the monosaccharide contains a plurality of monosaccharides. In some such embodiments, the monosaccharide-1-phosphate kinase contains a plurality of monosaccharide-1-phosphate kinases. In some such embodiments, the nucleoside triphosphate sugar 1 phosphate nucleosidyl transferase contains a plurality of nucleoside triphosphate sugar 1 phosphate nucleosidyl transferases.

In some embodiments, the protein concentration in the reaction mixture during the reaction is 10 µg / ml or more. In some embodiments, the protein concentration in the reaction mixture during the reaction is 20 µg / ml or more.

In some embodiments, the ultra and / or nanofiltration membrane is contained in the reaction vessel. In some embodiments, the ultra and / or nanofiltration membrane is in fluid communication with the reaction vessel, including in fluid communication. In some such embodiments, the removal of the monosaccharide-1 nucleoside diphosphate may include a pressure difference of at least between the side of the ultra and / or nanofiltration membrane that is in fluid communication with the reaction vessel and the opposite side of the ultra and / or nanofiltration membrane To generate 100 kPa. In some embodiments, a vacuum of at least 100 kPa can be generated on the side of the membrane facing away from the reaction mixture. In some embodiments, removal of the monosaccharide-1 nucleoside diphosphate may include creating an excess pressure of at least 100 kPa in the reaction vessel compared to the ambient pressure. In some embodiments, removal of the monosaccharide-1 nucleoside diphosphate may include exposing the reaction mixture located over the ultra and / or nanofiltration membrane to a force of 2.5 x g or more.

As previously indicated, in some embodiments, the reaction vessel is an ultrafiltration concentrator for centrifugation. In some embodiments, the at least one nucleoside triphosphate can be removed from the reaction mixture together with the monosaccharide-1 nucleoside diphosphate. As also previously stated, in some embodiments, one or more additional cofactors can be used which are / are necessary for the function of one or more enzymes used (see above). In some embodiments, when used in accordance with the second aspect, no further protein is present in addition to the enzyme mixture. In some embodiments, one or more buffer connections are still used (see above).

Typically, the enzymes of the enzyme mixture are in non-immobilized form. Typically, the enzymes of the enzyme mixture are in non-cross-linked form.

In some embodiments, in which at least one nucleoside triphosphate is added to the aqueous solution, the at least one nucleoside triphosphate contains ATP and UTP. In some embodiments in which at least one nucleoside triphosphate is added to the aqueous solution, the at least one nucleoside triphosphate contains ATP and GTP. In some embodiments, in which at least one nucleoside triphosphate is added to the aqueous solution, the at least one nucleoside triphosphate contains ATP and CTP. In some embodiments, in which at least one nucleoside triphosphate is added to the aqueous solution, the at least one nucleoside triphosphate contains ATP, GTP and UTP. In some embodiments, in which at least one nucleoside triphosphate is added to the aqueous solution, the at least one nucleoside triphosphate contains ATP, CTP and UTP. In some embodiments, the at least one nucleoside triphosphate contains ATP, CTP, GTP and UTP.

In some embodiments, in which at least one nucleoside triphosphate is added to the aqueous solution, the at least one nucleoside triphosphate consists of ATP and UTP. In some embodiments, in which at least one nucleoside triphosphate is added to the aqueous solution, the at least one nucleoside triphosphate consists of ATP and GTP. In some embodiments, in which at least one nucleoside triphosphate is added to the aqueous solution, the at least one nucleoside triphosphate consists of ATP and CTP. In some embodiments, in which at least one nucleoside triphosphate is added to the aqueous solution, the at least one nucleoside triphosphate consists of ATP, GTP and UTP. In some embodiments, in which at least one nucleoside triphosphate is added to the aqueous solution, the at least one nucleoside triphosphate consists of ATP, CTP and UTP. In some embodiments, the at least one nucleoside triphosphate consists of ATP, CTP, GTP and UTP.

In some embodiments in which a deoxynucleoside triphosphate and a nucleoside triphosphate are added to the aqueous solution, the nucleoside triphosphate is or contains one or more of the nucleoside triphosphates ATP, UTP, GTP and CTP. In some embodiments in which a deoxynucleoside triphosphate and a nucleoside triphosphate are added to the aqueous solution, the nucleoside triphosphate contains one or more of the nucleoside triphosphates UTP, GTP and CTP. In some embodiments in which a deoxynucleoside triphosphate and a nucleoside triphosphate are added to the aqueous solution, the nucleoside triphosphate consists of UTP. In some embodiments in which a deoxynucleoside triphosphate and a nucleoside triphosphate are added to the aqueous solution, the nucleoside triphosphate consists of GTP. In some embodiments, the nucleoside triphosphate consists of CTP.

A repetitive batch process is typically used in the use according to the second aspect. Regarding the restoration of the original volume and the re-supply of the at least one nucleoside triphosphate, cf. the foregoing. In some embodiments, a continuous process is used.

In some embodiments, the monosaccharide is galactose or glucose. In some embodiments, the monosaccharide is N-acetylgalactosamine. In some embodiments, the monosaccharide is mannose or xylose. In some embodiments, the monosaccharide is glucuronic acid. In some embodiments, the monosaccharide is galacturonic acid.

In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is the UDP sugar pyrophosphorylase from Hordeum vulgare (barley). In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is the human UDP-GaINAc pyrophosphorylase. In some embodiments, the monosaccharide-1-phosphate kinase is the galactokinase from E. coli. In some embodiments, the monosaccharide-1-phosphate kinase is the N-aacetylhexosamine-1-kinase from Bifidobacterium longum.

In some embodiments, in use according to the second aspect, a cycle is repeated at least 10 times or at least 20 times, which includes, as described above, allowing the conversion of the monosaccharide to a monosaccharide-1 nucleoside diphosphate, the monosaccharide-1 -Nucleoside diphosphate to be removed from the reaction vessel and the at least one nucleoside triphosphate to be added to the reaction mixture again. In some embodiments, such a cycle is repeated 40 times in use.

The summary described above is not restrictive, and further features and advantages of the method and the use described here should be apparent from the following detailed description, the figures and the claims.

list of figures

• 1 shows an overview of exemplary multi-enzyme cascades with the respective substrates. The respective configurations of pyranoses chosen as monosaccharide are shown in stereochemical representation. (1) = monosaccharide, (2) = nucleoside triphosphate, (3) = monosaccharide 1-phosphate, (4) = monosaccharide 1-nucleoside diphosphate, (5) = monosaccharide 1-phosphate kinase, (6) = sugar- 1-phosphate nucleosidyl transferase, (7) = pyrophosphatase.
• 2 shows multi-enzyme cascades used in the exemplary embodiments for the synthesis of UDP-Gal (A), UDP-GlcNAc (B) and UDP-GalNAc (C), which can be used in a method disclosed here. EcGalK: galactokinase from E. coli; HvUSP: UDP-sugar pyrophosphorylase from Hordeum vulgare (barley); NahK: N-acetylhexosamine-1-kinase from Bifidobacterium longum; AGX1: human UDP-GalNAc pyrophosphorylase; PP _i ase: pyrophosphatase from Saccharomyces cerevisiae.
• 3 shows examples of other suitable multi-enzyme cascades for the synthesis of a monosaccharide-1-nucleoside diphosphate. 3A : Implementation of fucose using the bifunctional enzyme L-fucokinase / GDP-fucose-pyrophosphorylase (FKP). 3B : Implementation of L-galactose using FKP. 3C : Implementation of L-mannose using the enzymes N-acetylhexosamine-1-kinase from Bifidobacterium longum (NahK) and mannose-1-phosphate-guanylyl transferase (rfbM). 3D : Implementation of L-mannose using the enzymes N-acetylhexosamine-1-kinase from Bifidobacterium longum (NahK) and UDP-sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP). 3E : Conversion of D-glucuronic acid using Arabidopsis thaliana glucuronokinase (AtGlcAK) and Arabidopsis thaliana UDP-sugar pyrophosphorylase (AtUSP). 3F : Reaction of N-acetylneuraminic acid with CMP-sialic acid synthetase from Neisseria meningitidis (NmCSS).
• 4 shows data on product enrichment in a repetitive batch synthesis of UDP-Gal with batches on a 20 mL scale (Vivaspin® 20 enzyme reactor) (n = 3). The product was determined using MP-CE at the beginning and at the end of the respective batch cycles (days 1-5).
• 5 shows data on product enrichment in a repetitive batch synthesis of UDP-GlcNAc with batches on a 20 mL scale (Vivaspin® 20 enzyme reactor) (n = 3). The product analysis was carried out using MP-CE at the beginning and at the end of each batch cycle (days 1-5).
• 6 shows data on the kinetics of the enzyme AGX1 with GaINAc-1-P, measured by MP-CE. The synthesis was carried out in 50 mM Tris-HCl (pH 8.0) with 2 mM MgCl ₂ and UTP (constant).
• 7 shows data on the kinetics of AGX1 with UTP, measured by MP-CE. The synthesis was carried out in 50 mM Tris-HCl (pH 8.0) with 2 mM GaINAc-1-P (constant). The concentration of MgCl ₂ was chosen differently in order to maintain a ratio of 1: 1.
• 8th summarizes kinetic data of AGX1 with GaINAc-1-P and UTP, measured using MP-CE.
• 9 shows the relative activity of the UDP-HexNAc producing enzyme cascade as a function of the ratio NTPs / Mg ²⁺ . Reaction conditions: 50 mM Tris pH 8.0 (HCl), 5 mM ATP, UTP, GalNAc, 2 U / mL PP _i ase, 0.2 U / mL NahK and AGX1, at 37 ° C, measured with MP-CE.
• 10 shows the product yield of the UDP-HexNAc-producing enzyme cascade after a reaction time of 30 minutes as a function of the AGX1 / PP _i ase ratio. Reaction conditions: 50 mM Tris pH 8.0 (HCl), 5 mM ATP, UTP, GalNAc, 10 mM MgCl ₂ , 0.3 U / mL NahK and AGX1, at 37 ° C, measured with MP-CE.
• 11 shows the product yield of the UDP-GalNAc producing enzyme cascade, scaled up to 100 mL batches, under the conditions improved for 300 µL. Reaction conditions: 50 mM Tris buffer pH 8.0 (HCl), 10 mM ATP, UTP, GalNAc, 20 mM MgCl ₂ , 2 U / mL NahK, AGX1 and PP _i ase, at 37 ° C, measured with MP-CE , The STY values for the 300 µL batches were 24.3 [g / L * h] with complete conversion after 15 minutes, 6.4 [g / L * h] for the 100 mL batches after 60 min and 4 , 2 [g / L * h] for the upscaled 1 L batches after 90 min.
• 12 shows the MP-CE analysis of the state for nucleotides and monosaccharide-1-nucleoside diphosphate during repetitive batch cycles at the respective starting points S 1-4 and the reaction end points E1-4.
• 13 shows data on product enrichment in a repetitive batch synthesis of UDP-GlcNAc with approaches on a 200 mL scale (Vivacell® 250) (n = 1). The product was determined using MP-CE at the beginning and at the end of the respective batch cycles (days 1-5).
• 14 shows average yield, total quantity, product concentration, specific product performance and mass-related turnover number were calculated for triplicate samples for the 20 mL scale (n = 3) and with single sample (n = 1) for the 200 mL scale. The mass-based sales figures take into account all the enzymes involved.
• 15 shows data on product enrichment in a repetitive batch synthesis of UDP-GalNAc with approaches on a 20 mL scale (Vivaspin® 20 enzyme reactor) (shown: n = 2). The product was determined by means of MP-CE at the beginning and at the end of the respective batch cycles (shown day 1-4).
• 16 shows a mass-based calculation of the total turnover ([g _{product /} g _enzyme ]) for the individual enzymes of the UDP-Gal, UDP-GIcNAc and UDP-GalNAc-producing enzyme cascades in repetitive batch mode. TTN _mass was calculated based on the specific, direct product of the respective enzyme reaction.
• 17 summarizes the mass spectroscopic analysis of the monosaccharide-1-nucleoside diphosphates produced.
• 18A shows the ESI mass spectrum of UDP-GalNAc, [MH] ^- , m / z 606.2.
• 18B shows the ESI mass spectrum of UDP-GlcNAc, [MH] ^- , m / z 606.2.
• 18C shows the ESI mass spectrum of UDP-Gal, [MH] ^- , m / z 564.9.

Detailed description

Unless otherwise stated, the following terms and expressions, when used in this document, including the description and claims, have the meanings given below.

The term "consisting of" as used in this document means inclusive and limited to what follows the term "consisting of". The term "consisting of" thus indicates that the elements listed are necessary or necessary and that no further elements may be present. The term "consisting essentially of" is understood to mean that it includes any element defined by the expression and that other elements, for example in a sample or composition, may be present that reflect the activity or effect that are specified for the relevant elements in this document, do not alter them and do not affect them and do not contribute to them. In other words, the expression "consisting essentially of" indicates that the defined elements are necessary or required, but that further elements are optional and may or may not be present, depending on whether or not they are important for the effect or effectiveness of the defined elements.

The word “about” when used herein refers to a value that is within an acceptable range of error for a particular value, as determined by one of ordinary skill in the art. This will depend in part on how the respective value was determined or measured, i.e. of the limitations of the measurement system. For example, "About" can mean within a standard deviation of 1 or more, depending on the use in the area. The term “about” is also used to indicate that the amount or value can be the designated value or another value that is approximately the same. The term is intended to express that similar values favor equivalent results or effects as disclosed in this document. In this context, “about” can refer to a range of up to 10% above and / or below a certain value. In some embodiments, "about" refers to a range up to 5% above and / or below a certain value, such as 2% above and / or below a certain value. In some embodiments, "about" refers to a range up to 1% above and / or below a certain value. In some embodiments, "about" refers to a range up to 0.5% above and / or below a certain value. In one embodiment, "about" refers to a range up to 0.1% above and / or below a certain value.

The term “substantially identical” when used here in connection with two nucleic acids or polypeptides refers to a sequence that has at least 95% sequence identity with a reference sequence. The percentage identity can be any integer from 95% to 100%. In some embodiments, the identity is at least 96% or at least 97% compared to a reference sequence using BLAST with standard parameters. In some embodiments, the identity is at least 98% or at least 98.5% using BLAST with standard parameters. In some embodiments, the identity is at least 99% or at least 99.5% compared to a reference sequence using BLAST with standard parameters.

Nucleic acid or protein sequences that are essentially identical to a reference sequence usually contain one or more "conservatively modified variants". In the case of nucleic acid sequences, a conservatively modified variant relates to those nucleic acids which encode identical or essentially identical amino acid sequences or, if the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneration of the genetic code, there is a significant number of functionally identical nucleic acids that code for a specific protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at any point where an alanine is specified by a codon, the codon can be changed to each of the corresponding other codons without the coded polypeptide changing. Such nucleic acid variants are "silent variations", which are a form of conservatively modified variations. Every possible silent variation of the nucleic acid can also be determined on the basis of each nucleic acid sequence described here which encodes a polypeptide. Accordingly, any silent variation of a nucleic acid encoding a polypeptide is implicitly disclosed in any sequence described.

The term "nucleotide sugar" as used in this document refers to a monosaccharide that contains a nucleoside diphosphate group. The monosaccharide in question can be a hexose or pentose, including a monodeoxy hexose or a monodeoxy pentose. Hexoses include, for example, glucose (Glc) and glucosamine (2-amino-2-deoxy-glucose, GlcNH ₂ ). Hexoses also include N-acetylglucosamine (2-acetamido-2-deoxy-glucose, GlcNAc). Hexoses also include galactose (Gal) and galactosamine (2-amino-2-deoxy-galactose, GaINH ₂ ). N-acetylgalactosamine (2-acetamido-2-deoxy-galactose, GalNAc) is also an example of a hexose. Two other examples of hexose are mannose (Man) and mannosamine (2-amino-2-deoxy-mannose, ManNH ₂ ). Hexoses also include N-acetylmannosamine (2-acetamido-2-deoxy-mannose; ManNAc). Another example of a hexose is glucuronic acid (GlcA). Hexoses also include iduronic acid (IdoA) and galacturonic acid (GalA). Another example of a hexose is 6-deoxy-galactose called fucose.

Pentoses include, for example, ribose and xylose. Arabinose (Arb) is also an example of a pentose. The monosaccharide in question can be a D-monosaccharide or an L-monosaccharide. The monosaccharide can be unsubstituted or substituted with one or more groups. Possible substituents include, but are not limited to, an amino group or an amido group. An acylamido group is also an example of a suitable substituent. Further examples of possible substituents are an O-sulfate group or an N-sulfate group (sulfamate).

A nucleotide sugar can, for example, be a UDP sugar, that is to say a nucleotide sugar whose nucleoside diphosphate group is UDP. Examples of a UDP sugar include, but are not limited to, UDP-Glc, UDP-GlcNAc, UDP-GIcNH ₂ , UDP-GlcA, UDP-IdoA, UDP-GalA, UDP-Gal, UDP-GalNAc, UDP-GalNH ₂ , UDP-Man, UDP-ManNAc and UDP-ManNH ₂ . The UDP sugar can be unsubstituted or contain substituents as described above.

The term “salvage pathway” (German salvage route) is a term known to the person skilled in the art. It describes a metabolic pathway that accomplishes the synthesis of a biomolecule from its degradation products and can therefore be viewed as a form of recycling. In particular, the salvage pathway means that for nucleotide sugars, which usually contain purine and pyrimidine nucleotides.

The conjunctional expression "and / or" between multiple elements, when used here, is understood to be broadly understood as both individual and combined options. If, for example, two elements are linked by "and / or", a first option concerns the use of the first element without the second. A second option involves using the second element without the first. A third option involves using the first and second elements together. It is understood that any of these options fall within the meaning of the term and thus meets the terms of the term "and / or" as used in this document.

Singular forms such as "a", "an", "the", "the" or "that" include the plural form when used in this document. For example, a reference to “a cell” refers to both an individual cell and a plurality of cells. In some cases, the expression "one or more" is used explicitly to indicate in each case that the singular form also includes the plural form. Such explicit references do not limit the general meaning of the singular form. Unless otherwise stated, the terms "at least", "at least" and "at least" when preceded by a sequence of elements are understood to refer to each of these elements. For example, the terms “at least one”, “at least one”, “at least one” or “at least one of” include one, two, three, four or more elements.

Disclosed are a method and a use with which a monosaccharide-1 nucleoside diphosphate can be produced on a gram scale. Surprisingly, it was observed that when a protein concentration of about 1 µg / ml or higher was maintained, the enzymes used were so stable that a repeated sequence of reaction, separation of the product and replacement of the nucleoside triphosphate or the nucleoside triphosphates could be carried out for days. Amazingly, it was not necessary to add a saccharide, e.g. to add a disaccharide such as sucrose or a protein such as serum albumin to ensure reaction conditions under which the enzymes used were sufficiently stable.

The protein concentration in the reaction mixture during the reaction is at least 10 µg / ml, including 20 µg / ml or higher. In some embodiments, the protein concentration in the reaction mixture during the reaction is 40 µg / ml or higher, including 75 µg / ml or higher. In some embodiments, the protein concentration in the reaction mixture during the reaction is 100 µg / ml or higher. For example, in some embodiments, the protein concentration in the reaction mixture during the reaction may range from about 2 ug / ml to about 250 ug / ml, including from about 5 ug / ml to about 200 ug / ml. In some embodiments, the protein concentration ranges from about 15 µg / ml to about 150 µg / ml. In some embodiments, the protein concentration in the reaction mixture during the reaction is 110 or 130 µg.

Depending on the enzyme used, the reaction can be carried out within the entire range in which Waser is in a liquid state, possibly after the addition of salts. For example, when using thermostable enzymes, you can work in areas near the boiling point. In some embodiments, the method or use can be performed in a temperature range from about 30 ° C to about 95 ° C, including, for example, in a temperature range from about 35 ° C to about 95 ° C. In some embodiments, the method or use can be performed in a temperature range from about 18 ° C to about 70 ° C, including, for example, in a temperature range from about 22 ° C to about 45 ° C. In some embodiments, the reaction using the enzymes used can take place in a temperature range from about 20 ° C. to about 38 ° C., for example a range from 27 ° C. to 35 ° C. can be selected. In some embodiments, a temperature range of 28 ° C to 32 ° C can be selected, including a temperature of 30 ° C ± 1 ° C or ± 0.5 ° C. It was also observed that, with a possibly lower reaction rate, reaction conditions near the freezing point of water - possibly after the addition of salts, so - enable a sufficiently effective implementation in the protein amounts that can be used here. In some embodiments, the method or use can be performed in a temperature range from about 2 ° C to about 28 ° C, including, for example, in a temperature range from about 4 ° C to about 18 ° C.

In some embodiments, the conversion of the monosaccharide to a monosaccharide-1-nucleoside diphosphate proceeds according to the following scheme in the method and the use disclosed here:

As previously indicated, (1) indicates the monosaccharide, (2) the nucleoside triphosphate, (3) the monosaccharide 1-phosphate, (4) the monosaccharide 1 nucleoside diphosphate, (5) the monosaccharide 1-phosphate kinase and (6) the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase. Nu also denotes the nucleosidyl group of the nucleoside triphosphate. The rest X stands for H or an OH group. The radical Y represents an OH group, an NH ₂ group or an N-acetyl group.

The method or use disclosed here can be applied to numerous monosaccharides by selecting suitable enzymes for the reaction. As a rule, one or two enzymes of a “salvage pathway” can be found among the enzymes used. This is typically a metabolic pathway for recycling nucleotide degradation products that result from the degradation of RNA and DNA. In a salvage pathway for the recycling of purines, the bases released during the degradation are mainly used, in the salvage pathway for the recycling of pyrimidines, however, the nucleosides are mainly recycled.

This is typically a metabolic pathway for recycling polysaccharide degradation products that result from the degradation of glycans. In a salvage pathway for the recycling of monosaccharides, the monosaccharides released during the breakdown are mainly used for the synthesis of nucleotide sugars.

As already indicated above, in some embodiments the monosaccharide can contain several different monosaccharides or consist of several different monosaccharides. For example, the monosaccharide can contain N-acetyl-galactosamine and N-acetyl-glucosamine. In some embodiments, the monosaccharide can contain fucose, arabinose, and galactose. In some embodiments, the monosaccharide can contain glucuronic acid and galacturonic acid. In some such embodiments, the monosaccharide-1-phosphate kinase contains a plurality of monosaccharide-1-phosphate kinases. In some such embodiments, the nucleoside triphosphate sugar 1 phosphate nucleosidyl transferase contains a plurality of nucleoside triphosphate sugar 1 phosphate nucleosidyl transferases.

In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a UDP sugar pyrophosphorylase from Hordeum vulgare (barley). In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is the enzyme derived from the RNA sequence with GenBank accession number AK366137.1 from Hordeum vulgare subsp. vulgare is encoded, version 1 of the Sequence from March 18, 2011, version 2 of the database entry from May 23, 2011 (specified: PLN from May 20, 2011). This is the protein from Hordeum vulgare with the Swissprot / Uniprot accession number F2DQG9, version 1 of the sequence from May 31, 2011, version 19 of the database entry from May 23, 2018. In some embodiments, the nucleoside triphosphate sugar is 1- Phosphate nucleosidyltransferase is a protein whose sequence is essentially identical to the sequence of the previous Swissprot / Uniprot accession number F2DQG9.

In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a D-galactose 1-phosphotransferase (galactokinase) from Bifidobacterium longum. This enzyme showed broad substrate acceptance in specificity studies. For example, Li et al. (Li, Y., et al., Molecules 2011, 16, 6396-6407) two suitable N-acetylhexosamine-1-kinases from Bifidobacterium longum subsp. infantis, strain ATCC 15697 and strain ATCC 55813 cloned and characterized.

In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is the enzyme from Bifidobacterium longum subsp. infantis (strain ATCC 15697) with the Swissprot / Uniprot accession number B7GUI0, version 1 of the sequence of February 10, 2009, version 79 of the database entry of May 23, 2018. In some embodiments, the nucleoside triphosphate sugar 1-phosphate is -Nucleosidyltransferase for a protein, the sequence of which is essentially identical to the sequence of the Swissprot / Uniprot accession number B7GUI0. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a galactokinase with Swissprot / Uniprot accession number C5E862 from Bifidobacterium longum subsp. infantis CCUG 52486, version 1 of the sequence of July 28, 2009, version 50 of the database entry of October 25, 2017. As Li et al. (Li, L. et al., Carbohydrate Research (2012) 355, 35-39, who disclose the sequence of a corresponding galactokinase from Bifidobacterium longum subsp. Infantis, report that this enzyme shows a broad substrate specificity with regard to the monosacchaid and that Nucleoside triphosphate or deoxynucleoside triphosphate.

In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a UDP sugar pyrophosphorylase from sugar cane, namely Saccharum hybrid cultivar R570, with the Swissprot / Uniprot accession number A0A059PZS7, version 1 of the sequence of September 3, 2014, version 7 of the database entry dated February 15, 2017. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a protein whose sequence is substantially identical to the sequence of Swissprot / Uniprot accession number A0A059PZS7. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a UDP-sugar corn pyrophosphorylase with Swissprot / Uniprot accession number C0P727, version 1 of the sequence of May 5, 2009, version 57 of the database entry of May 23, 2018. In In some embodiments, the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase is a protein the sequence of which is substantially identical to the sequence of Swissprot / Uniprot accession number C0P727. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a UDP-sugar corn pyrophosphorylase with Swissprot / Uniprot accession number A0A1D6LNL3, version 1 of the sequence of November 30, 2016, version 13 of the database entry of 23. May 2018. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a UDP-sugar corn pyrophosphorylase with Swissprot / Uniprot accession number A0A1D6GV15, version 1 of the sequence of November 30, 2016, version 9 of the database entry of May 23 2018. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a protein whose sequence is substantially identical to the sequence of the previous Swissprot / Uniprot accession number A0A1D6LNL3 or the sequence of the previous Swissprot / Uniprot accession number A0A1D6GV15. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a Gras UDP-sugar pyrophosphorylase from Oryza brachyantha with Swissprot / Uniprot accession number J3MHA9, version 1 of the sequence of October 3, 2012, version 28 of the database entry dated March 28, 2018. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a protein whose sequence is substantially identical to the sequence of Swissprot / Uniprot accession number J3MHA9.

In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is the enzyme UTP glucose 1-phosphate uridylyl transferase from Hordeum vulgare with Swissprot / Uniprot accession number Q43772, version 1 of the sequence of July 15, 1999, version 74 of the database entry of May 23, 2018. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a protein whose sequence is associated with the sequence of the previous Swissprot / Uniprot accession number Q43772 is essentially identical. In some embodiments, the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase is the enzyme UTP-glucose-1-phosphate uridylyl transferase from Hordeum vulgare with Swissprot / Uniprot accession number Q43772, version 1 of the sequence of July 15, 1999, version 74 of Database entry of May 23, 2018. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a protein whose sequence is substantially identical to the sequence of the previous Swissprot / Uniprot accession number Q43772.

In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a human UDP-GaINAc pyrophosphorylase. In some embodiments, the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase is the human enzyme UDP-N-acetylhexosamine pyrophosphorylase with Swissprot / Uniprot accession number Q16222, version 3 of the sequence of July 5, 2005, version 174 of the database entry of July 22. November 2017. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a protein whose sequence is substantially identical to the sequence of the previous Swissprot / Uniprot accession number Q16222. In some embodiments, the nucleoside triphosphate sugar 1 phosphate nucleosidyl transferase is the human enzyme UTP glucose 1 phosphate uridylyl transferase with Swissprot / Uniprot accession number Q168512, version 5 of the sequence of January 23, 2007, version 160 of the database entry dated May 23, 2018. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a protein the sequence of which is substantially identical to the sequence of Swissprot / Uniprot accession number Q168512. In some embodiments, the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase is the human protein with Swissprot / Uniprot accession number Q3KQV9, version 2 of the sequence of March 18, 2008, version 97 of the database entry of November 22, 2017. In some embodiments the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a protein whose sequence is essentially identical to the sequence of the Swissprot / Uniprot accession number Q3KQV9.

In some embodiments, the nucleoside triphosphate sugar-1-phosphate nucleosidyltransferase is a human enzyme called UDP-N-acetylhexosamine pyrophosphorylase with Swissprot / Uniprot accession number Q16222, version 3 of the sequence of July 5, 2005, version 175 of the database entry of July 20 June 2018. In some embodiments, it is an isoform AGX1 (identifier Q16222-2) of the database entry with the Swissprot / Uniprot access number Q16222. In some embodiments, it is an isoform AGX2 (identifier Q16222-1) of the database entry with the Swissprot / Uniprot access number Q16222. In some embodiments, it is an isoform AGX3 (identifier Q16222-3) of the database entry with the Swissprot / Uniprot access number Q16222. One day expression of AGX1 isoform was reported by Bourgeaux et al. (Bourgeaux, V., et al., Bioorganic & Medicinal Chemistry Letters (2005) 15, 5459-5462).

In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is the murine enzyme UDP-N-acetylhexosamine pyrophosphorylase with Swissprot / Uniprot accession number Q91YN5, version 1 of the sequence of December 1, 2001, version 125 of the database entry of June 30, 2001. August 2017. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a protein whose sequence is substantially identical to the sequence of the previous Swissprot / Uniprot accession number Q91YN5.

In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a UDP-sugar pyrophosphorylase from Arabidopsis thaliana (thale cress, pod cress), which was described by Liu et al. (Liu, J., et al., Bioorganic & Medicinal Chemistry Letters (2013) 23, 3764-3768) was cloned and characterized. For example, the protein with the Swissprot / Uniprot accession number Q9C5I1, version 1 of the sequence from June 1, 2001, version 102 of the database entry from April 25, 2018 or the protein with the Swissprot / Uniprot accession number O64765, version 1 of the sequence from August 1, 1998, version 127 of the database entry dated April 25, 2018. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a protein whose sequence is substantially identical to the sequence of the previous Swissprot / Uniprot accession number O64765. One as described by Liu et al. (2013, supra) described enzyme combination can also be used in a method disclosed here.

In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a protein encoded by the melon RNA (Cucumis melo) with GenBank accession number NM_001297525, version 1 of the July 2, 2014 sequence. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a protein whose sequence is substantially identical to the sequence of the protein encoded by the nucleic acid sequence with GenBank accession number NM_001297525.

In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is the enzyme E. coli mannose 1-phosphate guanylyl transferase with Swissprot / Uniprot accession number P37741, version 1 of the sequence of October 1, 1994, version 91 of the database entry dated May 23, 2018. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a protein whose sequence is substantially identical to the sequence of the previous Swissprot / Uniprot accession number P37741. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is the enzyme mannose-1-phosphate-guanylyl transferase from Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) with the Swissprot / Uniprot accession number P26404, version 1 of the sequence of 1 August 1992, version 123 of the database entry dated March 28, 2018.

In some embodiments, the monosaccharide-1-phosphate kinase is an E. coli galactokinase. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a galactokinase encoded by the nucleic acid from Escherichia coli strain TB182A with GenBank accession number EU903918.1, version 1 of the sequence of April 30, 2009. In some embodiments, the monosaccharide-1-phosphate kinase is a protein whose sequence is substantially identical to the sequence of the protein encoded by the nucleic acid sequence with GenBank accession number EU903918.1.

In some embodiments, the monosaccharide-1-phosphate kinase is an N-acetylhexosamine-1-kinase from Bifidobacterium longum. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyltransferase is the N-acetylhexosamine-1-kinase from Bifidobacterium longum subsp. Longum with Swissprot / Uniprot accession number E8MF12, version 1 of the sequence of April 15, 2011, version 31 of the database entry of February 28, 2018. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is a protein , whose sequence is essentially identical to the sequence of the Swissprot / Uniprot accession number E8MF12.

In some embodiments, the monosaccharide-1-phosphate kinase is the enzyme galactokinase from Lactococcus lactis, for example one of the enzymes described by Grossiord et al. ( Grossiord, BP, et al., Journal of Bacteriology (2003) 185, 3, 870-878 ) have been described. The galactokinase can be the enzyme from Lactococcus lactis Subsp. cremoris (strain MG1363), with the Swissprot / Uniprot accession number Q9S6S2, version 1 of the sequence of May 1, 2000, version 108 of the database entry of October 25, 2017, or for the enzyme from Lactococcus lactis Subsp. lactis (Stam IL1403), with the Swissprot / Uniprot access number Q9R7D7, version 1 of the sequence from May 1, 2000, version 123 of the database entry from June 20, 2018. In some embodiments, the monosaccharide-1-phosphate kinase is a protein, the sequence of which is substantially identical to the sequence of one of the preceding Swissprot / Uniprot accession numbers Q9S6S2 or Q9R7D7.

In typical embodiments, the monosaccharide 1-phosphate kinase and the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase are two different enzymes. Like for example in 3A and 3B shown, in some embodiments, both catalytic functions can be performed by the same enzyme. Thus, in some embodiments, the monosaccharide-1-phosphate kinase and the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase are the same enzyme.

Numerous pyrophosphatases are known which can be used in a process and a use described here. An example of a pyrophosphatase is the enzyme from S. cerevisiae with the Swissprot / Uniprot accession number P00817, version 4 of the sequence of July 24, 2007, version 193 of the database entry of May 23, 2018. Another example of a pyrophosphatase is the enzyme from Pasteurella multocida by Muthana ( Muthana, MM, et al., Chem. Commun. (2012) 48, 2728-2730 ) was used. The enzyme from Pasteurella multocida can be, for example, the enzyme with the Swissprot / Uniprot accession number Q9CKF5, version 1 of the sequence from June 1, 2001, version 89 of the database entry from June 20, 2018. In some embodiments, the pyrophosphatase is a protein whose sequence is substantially identical to one of the sequences of the previous Swissprot / Uniprot accession numbers P00817 or Q9CKF5. Another example of a pyrophosphatase is that of Li et al. ( Li, L., et al. Org. Lett. (2013) 15, 21, 5528-5530 ) used enzyme from E. coli. For example, the pyrophosphatase can be the enzyme with the Swissprot / Uniprot accession number P0A7A9, version 2 of the sequence from January 23, 2007, version 113 of the database entry from March 28, 2018. In some embodiments, the pyrophosphatase can be a protein, the sequence of which is essentially identical to the sequence of the preceding Swissprot / Uniprot accession number P0A7A9.

In some embodiments, the monosaccharide used is N-acetylgalactosamine. As suggested by Bourgeaux et al. ( Bourgeaux, V., et al., Bioorganic & Medicinal Chemistry Letters (2005) 15, 5459-5462 ) can be used for this monosaccharide as monosaccharide-1-phosphate kinase, for example an N-acetylgalactosamine kinase from porcine. Bourgeaux et al. describe the cloning and expression of the enzyme. For example, it can be an enzyme from pork called galactokinase with the Swissprot / Uniprot accession number A0A287BB25, version 1 of the sequence from November 22, 2017, version 5 of the database entry from March 28, 2018. In some embodiments, the galactokinase is a protein whose sequence is substantially identical to the sequence of the previous Swissprot / Uniprot accession number A0A287BB25. For example, human UDP-N-acetylhexosamine pyrophosphorylase with the Swissprot / Uniprot accession number Q16222 (supra) can be used as nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase (Bourgeaux et al., 2005, supra).

In some embodiments, the monosaccharide used is galacturonic acid. In such embodiments, for example, the nucleoside triphosphate sugar 1-phosphate nucleosidyltransferase described by Muthana et al. ( Muthana, MM, et al., Chem. Commun. (2012) 48, 2728-2730 ) cloned and characterized UDP-sugar pyrophosphorylase from Bifidobacterium longum strain ATCC55813 can be used. As described by Muthana et al. investigated, can as monosaccharide-1-phosphate kinase, for example, the enzyme aminoglycoside phosphotransferase, also called N-acetylhexosamine-1-kinase, from Bifidobacterium longum subsp. Use infantis strain ATCC 15697, e.g. with the Swissprot / Uniprot access number B7GN78, version 1 of the sequence from February 10, 2009, version 61 of the database entry from May 23, 2018. Two suitable enzymes have been developed by Li et al. described and cloned ( Li, Y., et al., Molecules (2011) 16, 6396-6407 ). The enzyme galactokinase from Streptococcus pneumoniae can also be used as monosaccharide-1-phosphate kinase (ibid.). This can be, for example, the enzyme with the Swissprot / Uniprot access number Q97NZ6, version 1 of the sequence from October 1, 2001, version 108 of the database entry from February 28, 2018, or the enzyme with the Swissprot / Uniprot access number Q8DNK7 , Version 1 of the sequence of March 1, 2003, version 97 of the database entry of June 20, 2018. All of these enzyme combinations can be used in a method and a use disclosed here.

In some embodiments, the monosaccharide used is fucose. One of Engels et al. ( Engels, L., and Elling, L., Glycobiology (2014) 24, 170-178 ) described enzyme combination with a fucokinase is a suitable combination. This can be, for example, the fucokinase from Bacteroides fragilis with the Swissprot / Uniprot accession number Q58T34, version 1 of the sequence from April 26, 2005, version 59 of the database entry from March 28, 2018.

As described by Ohashi et al. described ( Ohashi, H., et al., Adv. Synth. Catal. (2017) 359, 4227-4234 ) Due to its substrate specificity, this fucokinase can also be used for galactose as a monosaccharide. In some embodiments, the fucokinase may be the protein from Arabidopsis thaliana (thale cress, pod cress) with Swissprot / Uniprot accession number Q9LNJ9, version 2 of the sequence of May 3, 2011, version 102 of the database entry of March 25, 2018. In some embodiments, the galactokinase is a protein whose sequence is substantially identical to the sequence of the previous Swissprot / Uniprot accession number Q9LNJ9. In some embodiments, the fucokinase can be the protein from Bacteroides fragilis (strain ATCC 25285 / DSM 2151 / JCM 11019 / NCTC 9343) with Swissprot / Uniprot accession number Q5LC59, version 1 of the sequence of January 21, 2005, version 77 of the database entry of 28 February 2018. In some embodiments, the galactokinase is a protein whose sequence is substantially identical to the sequence of the previous Swissprot / Uniprot accession number Q5LC59.

In some embodiments, the monosaccharide used is mannose. In this case, the enzyme N-acetylhexosamine-1-kinase from Bifidobacterium longum is a suitable monosaccharide-1-phosphate kinase due to its substrate specificity (Nishimoto, M., and Kitaoka, M., Appl. Environ. Microbiol. (2007) 73, 20, 6444-6449; Cai, L., et al., Bioorg. Med. Chem. Lett. (2009) 19, 5433-5435).

In some embodiments, the monosaccharide used is N-acetylneuraminic acid. The monosaccharide-1-nucleoside diphosphate formed can be CMP-N-acetyl-neuraminic acid. For example, the enzyme described by Karwaski et al. (Karwaski, M.-F., et al., Protein Expression and Purification 25 (2002) 237-240) expressed and isolated CMP sialic acid synthetase from Neisseria meningitidis as described in 3G illustrates a direct conversion to a monosaccharide-1 nucleoside monophosphate catalyzed. In some embodiments, the enzyme may be the Neisseria meningitidis serogroup B protein (strain MC58) with Swissprot / Uniprot accession number P0A0Z7, version 1 of the sequence of February 15, 2005, Version 69 of the database entry dated February 28, 2018. In some embodiments, the enzyme may be the Neisseria meningitidis protein with Swissprot / Uniprot accession number P0A0Z8, version 1 of the sequence of February 15, 2005, version 51 of the database entry of March 28, 2018.

In some embodiments, the monosaccharide used is glucuronic acid. A UDP-sugar pyrophosphorylase from Leishmania major is suitable for this monosaccharide as nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase because of its substrate specificity. For example, it can be the enzyme with the Swissprot / Uniprot accession number D3G6S4, version 1 of the sequence from March 23, 2010, version 29 of the database entry from August 30, 2017. For example, a monosaccharide-1-phosphate kinase is a glucuronokinase, for example the enzyme from Arabidopsis thaliana with the Swissprot / Uniprot accession number Q93ZC9, version 1 of the sequence from December 1, 2001, version 106 of the database entry from April 26, 2018.

In some embodiments, an enzyme mixture consisting of a monosaccharide-1-phosphate kinase, one and a pyrophosphatase is used as the only proteins in the method and in an application described here. The enzyme mixture can be provided as an aqueous solution. The enzyme mixture can also be provided as a lyophilisate, which is dissolved in an aqueous solution. The enzyme mixture can also be provided from the lyophilizates of the individual enzymes, which are taken up in succession or simultaneously in an aqueous solution. A corresponding aqueous solution is typically buffered using a suitable buffer substance, see above.

In some embodiments, the enzymes used are used in a largely purified form. In some embodiments, the enzymes used have been purified by at least one chromatography process. In some embodiments, each of the enzymes used is used in a form in which less than 10%, including less than 5%, of foreign protein is present. In some embodiments, each of the enzymes used is used in a form in which less than 2%, including less than 1%, of foreign protein is present. In some embodiments, an enzyme mixture in the form of a crude extract or a mixture of crude extracts is provided in the method and application described here. Such a crude extract can be obtained, for example, by expressing the enzymes in a eukaryotic or prokaryotic host cell. Such a crude extract can also be obtained from inclusion bodies by expression of the enzymes in a prokaryotic host cell.

The monosaccharide-1-nucleoside diphosphate formed is removed from the reaction vessel by means of an ultra and / or nanofiltration membrane. This can be done discontinuously at one or more specific times, for example at one or more predetermined times. As an example, the process step described above can be converted into a monosaccharide-1-nucleoside diphosphate by means of a monosaccharide-1-phosphate kinase, a nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase, a pyrophosphatase, a monosaccharide and the at least one nucleoside triphosphate in the reaction vessel can be carried out for a certain period, for example a predetermined period. During this period, apart from taking samples for analysis, there is essentially no need to take the monosaccharide -1-nucleoside diphosphate. The period during which a monosaccharide and a The ability to convert nucleoside triphosphate to monosaccharide-1 nucleoside diphosphate depends on the conditions chosen. In particular, the volume of the reaction mixture, the dimensions of the reaction vessel and the selected temperature influence the period of time required to achieve the most complete possible reaction. The reaction can be achieved by analyzing the reaction mixture, in particular by a separation process. The detection of individual components can be done spectroscopically. The implementation can be checked and also monitored by a combination of a rapid chromatographic or electrophoretic separation with a spectrometric method. For example, capillary electrophoretic or HPLC isolation can be combined with mass spectrometric analysis.

In some embodiments, the time period for which a monosaccharide and a nucleoside triphosphate can be converted to a monosaccharide-1 nucleoside diphosphate ranges from about 1 minute to about 25 minutes, including up to about 10 minutes. In some embodiments, the time period for which the reaction can be allowed to range is from about 5 minutes to about 30 minutes. In some embodiments, the time period for which a monosaccharide and a nucleoside triphosphate can be converted to a monosaccharide-1 nucleoside diphosphate ranges from about 10 minutes to about 60 minutes, including up to about 30 minutes. In some embodiments, this time range is from about 20 minutes to about 3 hours, including from about 25 minutes to about 5 hours. In some embodiments, the time period for which a monosaccharide and a nucleoside triphosphate can be converted to a monosaccharide-1 nucleoside diphosphate ranges from about 45 minutes to about 8 hours, for example, from about 50 minutes to about 7 hours. In some embodiments, this period is greater than 8 hours.

The monosaccharide-1-nucleoside diphosphate formed is removed from the reaction vessel by means of an ultra and / or nanofiltration membrane. The monosaccharide-1 nucleoside diphosphate is allowed to pass through the membrane. The pore size of the membrane is chosen so that the monosaccharide 1 nucleoside diphosphate can pass through the membrane, while the monosaccharide 1 phosphate kinase, the nucleoside triphosphate sugar 1 phosphate nucleosidyl transferase and the pyrophosphatase cannot pass through the membrane , The monosaccharide and the at least one nucleoside triphosphate can usually also pass through the membrane due to their mass.

In order to allow the monosaccharide-1-nucleoside diphosphate to pass through the membrane, it is generally necessary to exert a certain force on the reaction mixture located in front of the membrane in question. This can take place, for example, by means of a relative excess pressure which, in comparison to the side of the membrane on which the monosaccharide 1 nucleoside diphosphate is to be collected, is applied to the side of the membrane on which the enzymes monosaccharide 1 phosphate kinase, nucleoside triphosphate, are located -Sugar-1-phosphate nucleosidyltransferase and pyrophosphatase are located. The pressure difference between the side of the membrane on which the reaction mixture is located and the side to which the monosaccharide-1 nucleoside diphosphate passes can be 100 kPa or more in some embodiments, e.g. 250 kPa or more. In some embodiments, the pressure difference between the two membrane sides can be 500 kPa or more, including 1000 kPa or more.

A certain force that acts on the reaction mixture in front of the membrane in question can also be a centrifugal force. A sufficient force can be achieved in this way by centrifuging the reaction mixture in a centrifugal concentrator in a suitable centrifuge after the time interval provided for the reaction. For example, it may be part of the method or use to allow a force of at least 250 xg, including a force of at least 1000 xg, to act on the side of the membrane from which the monosaccharide-1-nucleoside diphosphate is to pass through the membrane , In some embodiments, a force of at least 2500 x g, for example at least 4500 x g, is allowed to act on the corresponding side of the membrane. In some embodiments, the method or use includes applying a force of approximately 7500 x g to the corresponding side of the membrane.

In some embodiments, a centrifugal concentrator is already used as the reaction vessel. In such embodiments, the centrifugal concentrator can be subjected to centrifugation in a centrifuge without a transfer of the reaction mixture after a certain time interval for removing the monosaccharide-1 nucleoside diphosphate.

Any conventional ultrafiltration membrane can be used in a method and use disclosed herein. Suitable ultrafiltration membranes are commercially available, for example from Millipore Corp. (Bedford, Mass., USA), Sartorius (Göttingen, DE), Hyflux (SG), MEMOS Membranes Modules Systems GmbH (Pfullingen, DE) or Atech Innovations (Gladbeck, DE).

If the monosaccharide-1-nucleoside diphosphate is allowed to pass through the membrane, it is possible in some embodiments to at least substantially pass the nucleoside triphosphate (s) also passing through the membrane and the monosaccharide also passing through the membrane to let another membrane pass through, while the monosaccharide-1-nucleoside diphosphate cannot pass through this additional membrane. A suitable nanofiltration membrane with an appropriate pore size can be used for this. A suitable charged membrane, which can be an ultrafiltration membrane, can also be used. In some embodiments, it is possible with the aid of a further membrane to allow the nucleoside triphosphate (s) and / or cofactors, which also pass through the membrane, to pass through a further membrane, while the monosaccharide 1-nucleoside diphosphate does not pass this further Membrane can pass. In such embodiments, the monosaccharide cannot pass through the membrane. Such a further membrane typically has a smaller pore diameter than the first membrane, which serves to remove the monosaccharide-1-nucleoside diphosphate from the reaction vessel. Such a further membrane can be, for example, a nanofiltration membrane.

A nanofiltration membrane used in a method and a use disclosed here can be any conventional nanofiltration membrane. Suitable nanofiltration membranes are available from Millipore Corp. (Bedford, Mass., USA), Sartorius (Göttingen, DE), Atech Innovations (Gladbeck, DE), Ionopor (Veilsdorf, DE) or Osmonics Inc. (Minnetonka, Minn., USA).

The monosaccharide-1 nucleoside diphosphate can then be processed using common techniques such as crystallization and chromatography, including e.g. Gel filtration and / or ion exchange chromatography, enriched and purified.

A method and use, which are illustratively described herein, can be suitably practiced and implemented without a single element or elements, limitation or limitations, which are not explicitly disclosed here. The terms and expressions used herein are also used as descriptive terms and not as limitations, and there is no intent to exclude any equivalents of the features and parts or parts thereof shown and described when using such terms and expressions. It is recognized that various modifications are possible within the scope of the claimed invention. Thus, it should be understood that those skilled in the art can resort to modifications and variations of the disclosed embodiments, although the method and uses disclosed herein have been described and illustrated in sufficient detail to those skilled in the art to employ them, and such modifications and variations are considered to be within the scope of the invention.

It is to be understood that the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the subsequent claims for protection. Other embodiments are within the scope of the following claims.

The invention has been expanded and generally described here. Each of the narrower species and subgenus groupings that fall under the general disclosure also form part of the process. This includes the general description of the method with a condition or negative limitation that excludes an item from the genus, regardless of whether the excluded item is explicitly reproduced here.

Further embodiments are given in the following patent claims. If features or aspects of the invention are given in the form of Markush groups, the person skilled in the art will recognize that the corresponding subject is also described in this way with regard to each individual member or each individual subgroup of members of Markush groups.

EXAMPLES

The following examples illustrate the implementation of a repetitive batch process for the synthesis of a monosaccharide-1-nucleoside diphosphate. The synthesis of UDP-Gal, UDP-GIcNAc and UDP-GalNAc are shown as examples.

Unless otherwise stated, established methods have been used in microbial culture and for recombinant genetic material.

A screening for parameters for the fast reaction and the enzyme stability of the UDP-HexNAc enzyme cascade was carried out by multiplex capillary electrophoresis (MP-CE). As an example, an upscaling from an analytical 300 µL batch to a synthesis on a preparative scale of 20 mL or 200 mL is shown. Centrifugal concentrators are present in every biochemical laboratory and were therefore chosen as reactors and as devices for product extraction. They show how it is possible to set up a repetitive batch system without great technical effort. Under the partially optimized synthesis conditions, a high stability of enzyme cascades can be obtained, which leads to a high specific product performance or space-time yield (STY) and high mass-based turnover numbers (TTN _mass ) in the repetitive batch process. As can be seen, amounts of monosaccharide-1-nucleoside diphosphate in the gram or multi-gram range can be achieved with an ordinary technical laboratory equipment on an eight-hour working day. The monosaccharide-1-nucleoside diphosphates obtained can be used directly in a glycosyltransferase reaction for the synthesis of glycoconjugates.

Materials and processes

Production of recombinant enzymes

Recombinant galactokinase from E. coli (EcGalK, EC 2.7.1.6) and recombinant UDP-sugar pyrophosphorylase (EC 2.7.7.64) from Hordeum vulgare (barley, HvUSP) were prepared as previously described ( Wahl, C., et al., Biotechnol. J. (2016) 11, 1298-1308 ; Wahl, C., et al., Journal of Biotechnology (2017) 258, 20, 51-55 ). Fusion proteins of the N-acetylhexosamine-1 kinase from Bifidobacterium longum (NahK, EC 2.7.1.162) and human pyrophosphorylase (AGX1, EC 2.7.7.7.23), which contained an N-terminal His ₆ tag, were found in E. coli BL21 (DE3) expressed. The recombinant enzymes were produced in fermentation batches of 1 L. Overnight cultures (20 ml, LB + antibiotics, 37 ° C., 120 rpm) were used for inoculation. Protein expression was induced by adding 0.1 mM IPTG to the main culture. Batch fermentation at 25 ° C for 18-20h at 80 rpm gave 20-25 g cells (wet weight). The cells were harvested by centrifugation for 30 minutes at 7000 rpm and 4 ° C. A 40% (w / w) cell suspension was sonicated (3 x 30 s) and after a centrifugation step (30 min, 15,000 rpm) the supernatant was filtered (0.8 μm) and chromatography on an immobilized metal ion - Affinity matrix (IMAC) subjected. After washing with 50mM Tris / HCl (pH 8.0) containing 300mM NaCl and 10mM imidazole, the desired protein was made with 50mM Tris / HCl (pH 8.0) containing 300mM NaCl and 300mM imidazole , eluted. The purified enzymes were concentrated in a 30 kDa centrifugal concentrator (Vivaspin® 20, Sartorius, Göttingen) for the buffer exchange with 50 mM Tris / HCl pH 7.5 and diafiltered. EcGalK and AGX1 were stored at -20 ° C, NahK, HvUSP at 4 ° C.

MP-CE analysis

Multiplex capillary electrophoresis (MP-CE) was used as a high-throughput analysis tool for nucleotides, nucleosides and nucleotide sugar as previously published (Wahl, C., et al., Biotechnol. J. (2016) 11, 1298-13082; Wahl, C., et al., 2017, supra). A cePRO 9600 ™ system (Advanced Analytical Technologies, Ames, IA, USA) with an array of 96 quartz glass capillaries (effective separation length 55 cm, total length 80 cm, 50 µm inner diameter) was used. Air cooled to 15 ° C by a Peltier element was blown over the inlet side of the capillary array.

The separation buffer (50 mM NH ₄ Ac, pH 9.2) contained 1 mM EDTA. The samples were adjusted to a final concentration of 7 mM SDS by adding sodium dodecyl sulfate (SDS) solution for immediate denaturation of the enzymes. The solution also contained 1 mM 4-aminobenzoic acid (PABA) and 1 mM 4-aminophthalic acid (PAPA) as internal standards. After a centrifugation step, an aliquot of each sample was injected by vacuum for 10 s at - 0.7 psi (approx. -0.05 bar).

The separation took place at 12 kV. The analytes were detected by UV-VIS at 254 nm. The peak areas were standardized to the internal standard PABA. The concentrations were determined by calibration curves as previously described (Wahl, C., et al., 2016, supra; Wahl, C., et al., 2017, supra; Fischöder, T., et al., Molecules (Basel, Switzerland ) 2017, 22, 1320). In between, the array was rinsed with 0.1 M NaOH or distilled water for 5 min at 4.8 bar (70 psi), followed by an equilibration step with the electrophoresis buffer (50 mM ammonium acetate pH 9.2 with 1 mM EDTA). The samples were injected hydrodynamically with a vacuum of -0.05 bar (-0.7 psi) for 5 or 10 s.

Enzyme Activity Assay

Enzyme activity was performed in a 96-well plate with a total volume of 300 µL as previously described (Wahl, C., et al., 2017). The composition of the reaction mixtures (300 µL) was selected from the optimized published data or synthesis conditions (Wahl, C., et al., 2016, supra; Wahl, C., et al., 2017; Guan, W., et al. , Chem. - Eur. J. (2010) 16, 13343-13345; Cai, L., et al., Bioorg. Med. Chem. Lett. (2009) 19, 5433-5435; Li, Y., et al., Molecules (Basel, Switzerland) (2011) 16, 6396-6407 , All enzyme reactions were carried out at 37 ° C. The NahK reaction mixture contained 50 mM Tris / HCl (pH 8.0), 2 mM MgCl ₂ , 2 mM GlcNAc, 2 mM ATP and 1 U pyrophosphatase from Saccharomyces cerevisiae (Sigma-Aldrich (Roche), Mannheim, Germany). For AGX1, the reaction mixture consisted of Tris / HCl (pH 8.0) with 2 mM MgCl ₂ , 2 mM GaINAc-1-P, 2 mM UTP and pyrophosphatase. The EcGalK mixture contained 50 mM Tris / HCl (pH 7.5) and was supplemented with 2 mM MgCl ₂ , 2mM Gal, 2 mM ATP and pyrophosphatase. The activity test for HvUSP was carried out in 50 mM Tris / HCl (pH 7.5), containing 2 mM MgCl ₂ , 2 mM UTP, 2 mM Gal-1-P and pyrophosphatase. The reaction was carried out at various times by denaturing the enzymes in a 7 mM SDS solution with 1 mM PABA and 1 mM PAPA as an internal standard stopped for MP-CE analysis. The samples were analyzed with MP-CE (see above). The activity of the NahK and EcGalK kinases was determined by the decrease in ATP or increase in ADP over time. The activity of the pyrophosphorylases HvUSP and AGX1 was determined by the rise in UDP sugar over time. One enzyme unit was defined as a conversion of µmol substrate per minute under standard assay conditions.

Screening of the enzyme cascade parameters

The UDP-Gal producing enzyme cascade consists of an EcGalK (E. coli), an HvUSP (H. vulgare) and a PP _i ase. This module was already thoroughly characterized and STY-optimized in our previous study (Wahl, C., et al., 2017, supra). The UDP-HexNAc-producing enzyme module includes NahK (B. longum), AGX1 (human) and a PP _i ase. The enzymes involved are already well characterized. Optimal conditions for pH, temperature and ion preference are known and are reported elsewhere [ Bourgeaux, V., et al., Bioorg. Med. Chem. Lett. (2005) 15, 5459-5462 ; Li, Y., et al., Molecules (Basel, Switzerland) 2011, 16, 6396-6407 ; Nishimoto, M., et al., Appl. Environ. Microbiol. 2007, 73, 6444-6449 ; Wang-Gillam, A., et al., J. Biol. Chem. (1998) 273, 27055-27057 ). No further studies on the entire enzyme cascade with the aim of STY optimization have been carried out to date. Therefore, we performed a quick parameter screening for STY-optimized synthesis conditions using MP-CE as a high-throughput analysis method, as described previously (Wahl, C., et al., 2017, supra; Wahl, C., et al., 2016, supra). The kinetic data obtained ( 6 ) for AGX1 are comparable to literature data ( Bourgeaux, V., et al., Bioorg. Med. Chem. Lett. (2005) 15, 5459-5462; Wang-Gillam, A., et al., J. Biol. Chem. (1998) 273, 27055-27057 ). Our results show that the adjustment of the NTP ion ratio leads to significantly better product yields. How 7 shows, a slight increase or a decrease in the magnesium concentration can reduce the product yield. Furthermore, the AGX1 / PP _i ase ratio proved to be an important parameter for achieving high product yields ( 8th ). In summary, a ratio of 1: 1 NTP / metal ion [mM] and 1: 1: 1 enzyme [U / mL] offer the best prerequisites for STY-optimized synthesis. With these adjusted parameters and increased amounts of enzyme, we were able to achieve a complete conversion on a 300 µL scale after 15 minutes ( 9 ). Furthermore, we were able to demonstrate the successful upscaling up to a batch of 100 mL, which achieved a complete conversion after 60 minutes with an STY of 6.4 [g / L * h].

Substrate and product monitoring during repetitive batch synthesis

As a high-throughput method, MP-CE (see above) enables the parallel analysis of 96 samples and the monitoring of the nucleotide concentrations and the concentrations of monosaccharide-1-nucleoside diphosphate during the synthesis cycles. Samples were taken at each starting point (S 1-40) and at the end of the batch reactions (E1-40) as technical triple samples for each of the three parallel tests (n = 3). Using the example of UDP-GlcNAc synthesis ( 10A-D ) we were able to monitor the expected product and residual substrate enrichment by the remaining reaction volume in the repetitive batch mode shown. With regard to the overall synthesis, this effect is balanced.

Repetitive batch synthesis of UDP-Gal on a 20 mL scale

The enzymatic UDP-Gal synthesis was carried out in a reaction volume of 20 mL in centrifugal concentrators (Vivaspin® 20, Sartorius, Göttingen, 30kDa). The reaction mixture contained 10 mM D-galactose (Gal), 10 mM ATP, 10 mM UTP and 20 mM MgCl ₂ , 4 U / mL EcGalK or HvUSP and 4 U / mL pyrophosphatase from Saccharomyces cerevisiae (PP _i ase, Sigma-Aldrich (Roche), Mannheim) in 50 mM Tris / HCl (pH 7.5). After incubation at 37 ° C. for 30 min, the reaction mixture was concentrated to an exact residual volume of 5 ml (25% of the reaction volume) by centrifugation at 4 ° C. and 5000 rpm for 30-45 min. The remaining 15 mL permeate (75% of the reaction volume) were collected and stored at -20 ° C. If the centrifugation time was over 45 minutes, the filtration module was replaced by a new one. For the next synthesis run, the reaction volume was made up to 20 mL with a defined reaction mixture, which contained the initial concentrations of substrates, cofactors and buffer. The reaction mixture, including the retained enzyme, was mixed gently and the reaction mixture was incubated again at 37 ° C. for 30 min. Eight repetitive cycles per day were carried out with three parallel trials for 5 days. For overnight storage, the reaction mixture from the last incubation was kept at 4 ° C each day. A total of 120 batches (15 mL each) were obtained.

Repetitive batch synthesis of UDP-Gal on a 20 mL scale

Nucleotide sugar synthesis for UDP-GlcNAc and UDP-GalNAc was carried out as previously described for UDP-Gal synthesis. The reaction mixture contained 10 mM GlcNAc / GalNAc, 10 mM ATP, 10 mM UTP and 20 mM MgCl ₂ in a total volume of 20 ml of a 50 mM Tris / HCl buffer, pH 8.0. The enzyme concentrations of NahK, AGX1 and PP _i ase were each 2 U / mL. Each synthesis batch was carried out at 37 ° C for 30 minutes. Product recovery and ongoing repetitive steps were performed as described above. Eight repetitive batch cycles with three parallel attempts (ie 24 batches) were carried out for five days. A total of 120 batches (15 mL each) were obtained.

mass spectroscopy

The product samples were treated overnight at room temperature with 14 U / mL alkaline phosphatase. After removal of the enzyme by ultrafiltration, 0.5 µL ammonium hydroxide was added. The product was verified using HPLC / ESI-MS, i.e. HPLC coupled to electrospray ionization mass spectroscopy. A Multospher 120 RP 18 HP-3µ HPLC column (60 x 2 mm, CS-Chromatographie Service GmbH, Langerwehe) with isocratic 50% (v / v) acetonitrile: water as a mobile phase at a flow rate of 0.2 mL / min used. The proof of the product is based on the mass-to-charge ratio (m / z), which was determined with a Finnigan Surveyor MSQ Plus (Thermo Scientific) in negative mode with -4 kV needle voltage at 400 ° C and 100 V cone voltage.

Determination of the average yield

The average yields are given as total mol-based yields. The product amounts for all batches / batches of a synthesis were therefore determined by means of MP-CE, added up and related to the theoretical yield of the corresponding monosaccharide-1-nucleoside diphosphate on the basis of the amount of monosaccharide used.

Results and discussion

Cascades of recombinant enzymes optimized for specific product performance

The term “specific product performance” of a reaction vessel indicates the amount of product formed in the vessel per space and time. Since the time that is required to process a volume of liquid in the reaction vessel under the chosen conditions flows in, the term space-time yield is also used.

The enzyme cascades of the salvage pathway provide an uncomplicated biocatalytic synthesis route. Starting from readily available monosaccharides, the kinase-mediated phosphorylation reactions with adenosine triphosphate (ATP) as the phosphate donor give the respective sugar 1-phosphates ( 1A-C ).

The further implementation by a nucleotide sugar pyrophosphorylase using uridine triphosphate (UTP) and the removal of the inhibitory by-product pyrophosphate (PP _i ) from the equilibrium by means of a PP _i ase leads to the synthesis of the corresponding monosaccharide-1-uridine diphosphate. A fundamental optimization of the enzyme cascade for the synthesis of UDP-Gal ( 1 A) has already been carried out in a previous study by the same laboratory (Wahl, C., et al., 2017, supra). A specific product performance of 17 g / L * h was achieved for the complete conversion of 10 mM substrate within 20 min at 1.2 U of each enzyme.

The flexible enzyme cascade containing NahK, AGX1 and PP _i ase was developed with MP-CE for the synthesis of UDP-GlcNAc and UDP-GalNAc ( 1B and 1C ) optimized for high throughput analysis. A rapid screening of the reaction parameters (inhibitors, kinetics, cofactors and amounts of enzyme) based on microtiter plates led to improved reaction conditions with a complete conversion of 10 mM substrate within 30 min. With the improved synthesis conditions, a high STY of 6.4 [g / L * h] could be achieved on a 100 mL scale for the production of UDP-GalNAc ( 9 ).

Repetitive batch workflow for the production of monosaccharide-1-nucleoside diphosphates

For the production of UDP-Gal, UDP-GlcNAc and UDP-GalNAc in repetitive batch mode, the improved reaction conditions for high STY in 96-well plate batches were used ( 1A-C ). The aim was to maximize the enzyme productivity of the synthesis cascades through a high number of repetitive batches. The use of conventional laboratory equipment and the adaptation of the production workflow to an average 8-hour working day and a 5-day week should facilitate multi-gram synthesis.

For this purpose, centrifugal concentrators were chosen as the enzyme reactors. After maximum substrate conversion in 37 min at 37 ° C per batch reaction, the products are separated by ultrafiltration. Each 20 mL batch was carried out in parallel batches (n = 3) and showed high reproducibility. The addition of new substrate solution to the retained enzymes started the next batch reaction. Eight consecutive batches / batches were run in this way per day and continued for five days, resulting in an effective, repetitive batch cycle number of 120 (3 × 8 × 5). At the end of each day, the supernatants from the reaction batches were stored at 4 ° C. overnight. It was shown that this had no harmful effects on the biocatalysts. Although the remaining enzyme activities would have allowed further repetitive batches, the syntheses were terminated after five days of operation. At each start and end point, the product concentration was determined using triple samples using MP-CE. The initial sample was only taken after a short mixing, which was the easiest to handle in the workflow. For this reason, however, the initial sample cannot be strictly equated with zero reaction time.

The MP-CE analysis method made it possible to monitor the product enrichment and the effective substrate concentration of each batch based on the remaining reaction volume after separation of 75% of the product-containing reaction solution ( 10A-D ).

Practical repetitive batch synthesis of nucleotide activated monosaccharides

The repetitive batch system for UDP-Gal synthesis showed a high average yield of 94% over 40 cycles (eight cycles per day over five days, 2 ). It can be concluded from this that surprisingly all enzymes have a high degree of stability. Obviously, the enzyme system also compensates for physical stress during the centrifugation step for product separation very well. The nightly storage between synthesis and centrifugation also had no influence on the synthetic performance. Without wishing to be bound by theory, the high enzyme stability may be due to the high protein concentrations in the improved protocol. This explanation would fit previous studies where stabilization was shown by the addition of bovine serum albumin (BSA) ( Zervosen, A., et al., J. Mol. Catal. B: enzyme. (1998) 5, 25-28 ). However, good reproducibility (n = 3) was achieved over the entire duration of the synthesis. The improved enzyme approach (300 µL) was successfully scaled up and transferred to the repetitive batch mode. A total of 12.8 g of UDP-Gal was obtained. The product concentration and STY of the combined product fractions of all batches were calculated as 7 g / L and 10.7 [g / L * h] ( 12 ). The mass-based turnover number (TTN _mass ) was 494 [g product / g enzyme] ( 12 ). This underscores the efficiency of scaling up from an improved enzyme module (300 µL) to a repetitive batch mode. The TTN _mass is in a range that is typical for solid-phase bioprocesses, where a TTN _mass of 1000 is the limit. for the enzyme module system with the three biocatalysts involved, almost half of it has already been achieved without further process or enzyme optimization.

The TTN _mass values for the individual enzymes in the cascade ( 14 ) point to HvUSP as a potential target for optimization. In summary, we present a practical system for the simple and fast in-house laboratory production of UDP-Gal, with the potential for further development into an industrially interesting process. Compared to an earlier repetitive batch synthesis of UDP-Gal by Bülter et al. (J. Mol. Catal. B: Enzym. (2000) 8, 281-284) we achieve a 30-fold higher STY and a 6-fold higher product quantity due to the increased number of repetitive batches / batches without loss of enzyme activity.

The transfer of the UDP-GlcNAc producing enzyme module ( 1B) in a repetitive batch mode was also successful with 40 cycles (n = 3) and a total reaction time of 20 hours. The product yields of repeated batches remained constant at an average level of 81% ( 12 ), resulting in a total of 11.9 g UDP-GlcNAc. The N-acetylhexosamine-1 kinase used (NahK) from Bifidobacterium longum converts N-acetylglucosamine (GlcNAc) and N-acetlygalactosamine (GalNAc) with almost the same activity. In contrast, UDP-GalNAc pyrophosphorylase (AGX1) has slightly higher activity for GalNAc-1 phosphate (Wang-Gillam, A., et al., J. Biol. Chem. (1998) 273, 27055-27057). Nevertheless, we achieved a total STY of 9.9 [g / L * h] and a TTN _mass of 522 [g product / g enzyme] for the synthesis of UDP-GlcNAc. In summary, this is the first example of a synthesis of UDP-GlcNAc based on a repetitive batch on such a scale.

Regarding the optimized conditions for the UDP-GalNAc producing enzyme module ( 1C ) with seven of eight batches in triplicate, we achieved a yield of over 90% for the first day ( 13 ). In comparison to UDP-GlcNAc synthesis, the higher average yield can be attributed to the higher activity of AGX1 with the substrate GaINAc-1-phosphate (Wang-Gillam et al., 1998, supra). Two replicates showed a very stable conversion of over 90% for at least 16 cycles in two days. In summary, we achieved a total of 9 g UDP-GalNAc with a STY of 7.5 [g / L * h] and a TTN _mass of 398 [g product / g enzyme] ( 12 ).

The repetitive batch synthesis for UDP-GalNAc was scaled up to a 200 mL batch reaction volume in a 250 mL pressure concentrator (Vivacell® 250, Sartorius, Göttingen). Due to the increased time required for pressure-controlled product separation, the number of batches per day was reduced to four cycles per day over a period of five days. A total of 20 repetitive batch cycles were carried out within a response time of ten hours with this approach ( 11 ). An average yield of 95.5% was achieved, which corresponds to 23.3 g of UDP-GalNAc. Accordingly, the STY of 19.4 [g / L * h] is in a high range. However, the reduced number of cycles leads to a lower TTN _mass (103 [g product / g enzyme]). Due to the higher substrate costs of GalNAc, a high yield will make more economic sense instead of high enzyme productivity.

conclusion

To the best of the inventors' knowledge, this study is the first example of a successful UDP-GalNAc and UDP-GlcNAc synthesis via enzymatic cascades in repetitive batch mode on a gram scale. The repetitive batch approach presented proved to be a simple and reproducible procedure for nucleotide sugar production on a laboratory scale using the example of UDP-Gal, UDP-GlcNAc and UDP-GalNAc. A synthesis of nucleotide-activated monosaccharides down to the multi-gram scale can be achieved with the aid of, for example, centrifugal and / or pressure concentrators without requiring special technical equipment.

The transfer of enzyme modules, which are adapted to a high specific product performance (STY) in microtiter plates, proved to be a suitable strategy for setting up a production process in a repetitive batch mode. The high enzyme stability leads to a significant increase in enzyme productivity with TTN _mass values in a number of industrially interesting processes. In these examples, production was stopped after five days regardless of the remaining enzyme activity. In particular for the enzyme cascades producing UDP-Gal and UDP-GlcNAc, even higher enzyme productivity is expected, since the product yield did not decrease in 40 cycles over five days after a reaction time of 20 h.

For product isolation, however, a cascade with the help of enzymes from the salvage pathway for nucleotide sugar synthesis offers the possibility of controlling the efficiency of the substrate usage and the composition of the product solution. This opens up a direct coupling with glycosyltransferase modules without time-consuming and costly cleaning steps for nucleotide sugar.

QUOTES INCLUDE IN THE DESCRIPTION

This list of documents listed by the applicant has been generated automatically and is only included for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

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Claims (10)

A method for producing a monosaccharide-1 nucleoside diphosphate (4), comprising: - in a reaction vessel, a monosaccharide (1), a monosaccharide-1-phosphate kinase (5), a nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase (6) , a pyrophosphatase and at least one nucleoside triphosphate, or a deoxynucleoside triphosphate and a nucleoside triphosphate, in contact in an aqueous solution, so that an aqueous reaction mixture is formed, the monosaccharide-1-phosphate kinase (5) being chosen such that the specificity of the Monosaccharide-1-phosphate kinase allows phosphorylation of the monosaccharide (1) by means of the deoxynucleoside triphosphate or one of the at least one nucleoside triphosphate, and the nucleoside triphosphate or one of the at least one nucleoside triphosphate (2) is selected such that the specificity of the nucleoside triphosphate sugar -Phosphate nucleosidyl transferase (6) the transfer of a nucleoside monophosphate unit to a mon Osaccharide-1-phosphate (3) of the monosaccharide (1) allows, and wherein the monosaccharide-1-phosphate kinase (5) and the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase (6) are selected independently of one another and from the same enzyme or can be represented by different enzymes, and - allow conversion of the monosaccharide (1) to a monosaccharide-1-nucleoside diphosphate (4) according to the following scheme in a temperature range from 0 ° C to 95 ° C

where NTP and NuTP denote a nucleoside triphosphate and dNTP a deoxynucleoside triphosphate, Nu denotes the nucleosidyl group of the nucleoside triphosphate (2), where NTP and NuTP are selected independently of one another, Z stands for -CH ₂ OH-, -CH ₃ , H or -COOH and Y stands for OH, NH ₂ or N-acetyl, the protein concentration in the reaction mixture being 20 μg / ml or more during the reaction, - removing the monosaccharide-1-nucleoside diphosphate (4) from the reaction vessel by means of an ultra and / or nanofiltration membrane that the total volume of the reaction mixture is retained or restored, the monosaccharide-1-phosphate kinase (5), the nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase (6), and the pyrophosphatase remain in the reaction mixture and that the at least one nucleoside triphosphate or the deoxynucleoside triphosphate and the nucleoside triphosphate and the monosaccharide are fed back into the reaction mixture. Procedure according to Claim 1 comprising a monosaccharide (1), a monosaccharide-1-phosphate kinase (5), a nucleoside triphosphate sugar-1-phosphate nucleosidyl transferase (6), a pyrophosphatase and at least one nucleoside triphosphate in contact, at least one nucleoside triphosphate being ATP , Procedure according to Claim 2 , wherein the at least one nucleoside triphosphate in addition to ATP has one or more of the nucleoside triphosphates UTP, GTP and CTP. Method according to one of the preceding claims, wherein the reaction vessel has the ultra and / or nanofiltration membrane or is in fluid communication with the ultra and / or nanofiltration membrane and wherein the monosaccharide-1 nucleoside diphosphate (4) has: a) generate a pressure difference of at least 100 kPa between the side of the ultra and / or nanofiltration membrane that is in fluid communication with the reaction vessel and the opposite side of the ultra and / or nanofiltration membrane, or b) to the pressure difference above the ultra and / or reaction mixture or nanofiltration membrane can exert a force of at least 250 xg. Procedure according to Claim 4 , wherein the reaction vessel is an ultrafiltration concentrator for centrifugation. Method according to one of the preceding claims, wherein the monosaccharide is galactose, glucose, glucuronic acid, galacturonic acid, N-acetylgalactosamine, N-acetylglucosamine, mannose, fucose, or xylose. Procedure according to one of the Claims 2 to 6 , wherein the at least one nucleoside triphosphate consists of ATP and UTP. Procedure according to Claim 7 , wherein the nucleoside triphosphate sugar 1-phosphate nucleosidyl transferase is UDP-sugar pyrophosphorylase from Hordeum vulgare (barley) or the human UDP-GaINAc pyrophosphorylase and / or wherein the monosaccharide-1-phosphate kinase is from E. coli or the N-acetylhexosamine-1-kinase from Bifidobacterium longum. Method according to one of the preceding claims, wherein with or after the renewed addition of the at least one nucleoside triphosphate, the original volume of the aqueous reaction mixture is restored with an error tolerance of 10%. Procedure according to Claim 9 , the method comprising a cycle of a) allowing the conversion of the monosaccharide to a monosaccharide-1 nucleoside diphosphate, b) removing the monosaccharide-1 nucleoside diphosphate from the reaction vessel and c) the deoxynucleoside triphosphate and the nucleoside triphosphate, or the at least one Repeat the nucleoside triphosphate and the monosaccharide in the reaction mixture, repeat 20 times, preferably repeat 40 times.

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