Disclosed herein are genetically modified microorganisms and related methods for the enhanced production and export of oligosaccharides. The microorganisms described herein express major facility superfamily proteins such as CDT-1, which allows for the export of oligosaccharides. Variants of CDT-1 exhibit higher activity regarding oligosaccharide export. The microorganisms described herein express formation enzymes for the production of oligosaccharides. Means to export oligosaccharides into the growth medium are provided herein.

An object of the present invention is to provide an enzyme preparation that efficiently catalyzes the epimerization reaction with little byproducts. The present invention provides an enzyme preparation for catalyzing an epimerization reaction of a saccharide, which contains any of the proteins (a) to (c) mentioned below, and wherein the saccharide is any selected from the group consisting of glucose, mannose, talose and galactose: (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1 or 3; (b) a protein consisting of an amino acid sequence having an amino acid sequence identity of 90% or higher to the amino acid sequence of SEQ ID NO: 1 or 3, and having an activity for catalyzing an epimerization reaction of a saccharide; and (c) a protein consisting of an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 or 3 by substitution, insertion, deletion and/or addition of one or several amino acids, and having an activity for catalyzing an epimerization reaction of a saccharide.

Compositions for producing glycoengineered proteins, e.g. antibodies, include host cells which lack the ability to produce enzymes that modulate sialic acid metabolic flux.

The invention relates to an isolated nucleic acid molecule comprising at least one promoter that is active in fungal cells of the trichoderma species, wherein a nucleic acid sequence encoding an N-acetylglucosamine-2-epimerase and/or an N-acetylneuraminic acid synthase is operatively bound to each promoter. The at least one promoter that is active in fungal cells is a constitutive promoter.

The invention discloses a genetically engineered bacterium and its application in the preparation of sialyllactose. The genetically engineered bacterium has an N-acetylneuraminic acid biosynthesis pathway, includes multiple copies of a gene neuB for encoding sialic acid synthase, and the gene neuB is initiated for expression by a strong promoter. Using the genetically engineered bacteria of the invention to produce sialyllactose has the advantages of high yield and low overall cost.

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.