The present disclosure relates to engineered RNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered RNA polymerase polypeptides. The present disclosure also provides methods of using the engineered RNA polymerase polypeptides or compositions thereof for producing RNA.

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.

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).

The present disclosure provides a ribose-modified cap analog and a use thereof, and belongs to the technical field of chemical and biological engineering. The ribose-modified cap analog has a structure of formula (I). The ribose-modified cap analog described herein can improve the stability of mRNA and/or the translation efficiency of mRNA.

The present disclosure provides a ribose-modified cap analog and a use thereof, and belongs to the technical field of chemical and biological engineering. The ribose-modified cap analog has a structure of formula (I). The ribose-modified cap analog described herein can improve the stability of mRNA and/or the translation efficiency of mRNA.