The present invention relates to genetically engineered organisms, especially microorganisms such as bacteria and yeasts, for the production of added value bio-products such as specialty saccharide, activated saccharide, nucleoside, glycoside, glycolipid or glycoprotein. More specifically, the present invention relates to host cells that are metabolically engineered so that they can produce said valuable specialty products in large quantities and at a high rate by bypassing classical technical problems that occur in biocatalytical or fermentative production processes.

The present invention provides a one-pot multi-enzyme method for preparing UDP-sugars from simple sugar starting materials. The invention also provides a one-pot multi-enzyme method for preparing oligosaccharides from simple sugar starting materials.

The present invention relates to a process for attaching an N-acetylgalactosamine-(hetero)arylmoiety to an N-acetylglucosaminemoiety, the process comprising the step of contacting the N-acetylgalactosamine-(hetero)arylmoiety with the N-acetylglucosaminemoiety in the presence of a mutant galactosyltransferase, wherein the N-acetylglucosaminemoiety is according to Formula (1) the N-acetylgalactosamine-(hetero)arylmoiety is according to Formula (2): In a particularly preferred embodiment of the process according to the invention, the N-acetylgalactosamine-(hetero)arylmoiety comprises a 1,3-dipole functional group, and the N-acetylglucosaminemoiety is a terminal GlcNAc moiety of a glycoprotein glycan. The invention further relates to a product obtainable by the process according to the invention, in particular to glycoproteins. Also, the invention relates to several compounds comprising an N-acetylgalactosamine-(hetero)arylmoiety.

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A process for making a nucleotide analog includes combining a first substrate that includes a linker and a base with a second substrate to form a substrate composition. An enzyme contacts the substrate composition and catalyzes formation of the nucleotide analog from the first substrate and the second substrate. Additionally, a composition includes the first substrate, second substrate, the enzyme, the nucleotide analog, and optional additives.

The invention provides the production of sugar-nucleotides and the isolation of sugar-nucleotides. In some embodiments the production or isolation are accomplished under acidic conditions. The production is a cell-free synthesis using enzymes, including immobilized enzymes. They may be accomplished using a one-pot reaction protocol. The synthesis may be used as a highly customizable and highly efficient cell-free manufacturing process. In some embodiments, the sugar-nucleotides are used to prepare UDP-Gal, lactose derivatives, and human milk oligosaccharides (HMOs).

A recombinant engineered strain for de novo synthesis of CDP-choline using glucose as a substrate and its preparation method and application are provided. Using BS168N as the starting strain, firstly, the phosphatidylethanolamine N-methyltransferase gene PEM1 and phosphatidylethanolamine/phosphatidyl-N-methylethanolamine N-methyltransferase gene PEM2 from S. cerevisiae are integrated into the genome of the BS168N for induced expression, thereby opening up the synthesis pathway from phosphatidylethanolamine to phosphatidylcholine; subsequently, the CKI and CCT genes of S. cerevisiae are further integrated into the BS168N genome expressing PEM1-PEM2, opening up the synthesis pathway of choline to CDPC, thereby obtaining the recombinant engineered strain. Further, the recombinant engineered strain is subjected to shake flask fermentation to achieve de novo synthesis of CDP-choline using glucose as a substrate. The method of the present disclosure provides a fundamental research and theoretical basis for the construction of efficient cell factories for de novo synthesis of CDP-choline through synthetic biology.