The invention provides modular cell-free de-novo synthesis of glycans with immobilized bionanocatalysts. The invention provides materials, and in particular, magnetic materials, for producing glycans of defined length and sequences using one or more enzymes that are immobilized within bionanocatalysts (BNCs) which in turn are embedded within scaffolds to control the synthesis in batch or continuous processes manufacturing. In some embodiments, the scaffolds are high magnetism and high porosity composite blends of thermoplastics or thermosets comprising magnetic particles that form powders. In some embodiments, Selective Laser Sintering (SLS) is used to design and produce objects via 3D printing by sintering composite magnetic powders. The modular flow cells may be mixed and matched for a highly customizable and highly efficient cell-free manufacturing process. In some embodiments the elementary and system modules provided by the invention are employed. In preferred embodiments, human milk oligosaccharides (HMOs) are produced.

Provided herein, in some embodiments, are methods and composition for the production of nucleoside triphosphates and ribonucleic acids.

The present invention relates to a method for producing cytidine 5-monophospho-N-acetyl-neuraminic acid (CMP-Neu5Ac, 1) from low-cost substrates N-acetyl-D-glucosamine (GlcNAc), pyruvate, cytidine and polyphosphate in a single reaction mixture with a set of optionally immobilized or optionally co-immobilized enzymes comprising N-acylglucoamine 2-epimerase (AGE), an N-acetylneuraminate lyase (NAL), an N-acylneuraminate cytidylyltransferase (CSS), a uridine kinase (UDK), a uridine monophosphate kinase and a polyphosphate kinase 3 (PPK3). Further, said process may be adapted to produce Neu5Acylated i.e. sialylated biomolecules and biomolecules including a saccharide, a peptide, a protein, a glycopeptide, a glycoprotein, a glycolipid, a glycan, an antibody, and a glycoconjugate, in particular, an antibody drug conjugate, and a carbohydrate conjugate vaccine, or a flavonoid.

The present invention relates to a method for producing cytidine 5-monophospho-N-acetyl-neuraminic acid (CMP-Neu5Ac, 1) from low-cost substrates N-acetyl-D-glucosamine (GlcNAc), pyruvate, cytidine and polyphosphate in a single reaction mixture with a set of optionally immobilized or optionally co-immobilized enzymes comprising N-acylglucosamine 2-epimerase (AGE), an N-acetylneuraminate lyase (NAL), an N-acylneuraminate cytidylyltransferase (CSS), a uridine kinase (UDK), a uridine monophosphate kinase and a polyphosphate kinase 3 (PPK3). Further, said process may be adapted to produce Neu5Acylated i.e. sialylated biomolecules and biomolecules including a saccharide, a peptide, a protein, a glycopeptide, a glycoprotein, a glycolipid, a glycan, an antibody, and a glycoconjugate, in particular, an antibody drug conjugate, and a carbohydrate conjugate vaccine, or a flavonoid.