It relates to a LfliZ gene and its application. The sequence of gene LfliZ is shown in SEQ ID NO.1. This invention also relates to the recombinant protein LfliZ encoded by LfliZ gene and its application in preparation of 2, 3-cNMPs. The recombinant LfliZ protein encoded by gene LfliZ can bind four kinds of 2, 3-cNMPs during its expression in Escherichia coli. Four kinds of 2, 3-cNMPs can be prepared simultaneously from the recombinant E. coli by extracting the recombinant protein LfliZ. The yield of 2, 3-cNMPs reaches 2.5 mg/L fermentation broth by the method in the present invention, indicating that this method has a good application potential.

Disclosed herein are oligosaccharides and intermediates useful for the production thereof. The compounds are useful as analytical standards and as intermediates for the preparation of more complex oligosaccharide and N-glycan products. The compounds may be prepared in high purity using the selective stop/go synthetic methods disclosed herein.

The present invention relates to the cell-based production of bacterial nonulosonates and their biosynthetic precursors. Specifically, the present invention provides recombinant cells for the production of pseudaminic acid, legionaminic acid, UDP-2,4-diacetamido-2,4,6-trideoxy--L-altropyranose, and UDP-2,4-diacetamido-2,4,6-trideoxy--D-glucopyranose. Methods for producing the sugars are also provided.

The present invention includes novel systems, methods, and compositions for the enzymatic/chemical production of pseudouridine (?) and its variants, such as N1-methyl-pseudouridine-5-triphosphate (m1?TP).

Provided herein is a method of labeling the epigenetic modification 5-hydroxymethyl-cytosine along a DNA molecule that involves attaching a 5-hydroxymethyl-cytosine specific labeling agent to the DNA molecule, which is effected by enzymatically synthesizing UDP-6-N3-Glucose by subjecting 6-azidoglucose to an enzymatic catalysis by N-acetylhexoseamine 1-kinase (NahK) in the presence of ATP to thereby obtain a phosphorylated 6-azidoglucose, followed by subjecting the phosphorylated 6-azidoglucose to enzymatic catalysis by uridyltransferase (GlmU) in the presence of UTP to thereby obtain UDP-6-N3-Glucose, followed by incubating the DNA molecule with UDP-6-N3-Glucose and ?-glucosyltransferase, to thereby obtain a 5-hydroxymethyl-cytosin e glycosylated by a 6-azidoglucose residue in the DNA molecule, and reacting the modified DNA molecule with a labeling agent which is chemically compatible with the azide group of the 6-azidoglucose residue.

It relates to a method for preparation of four kinds of 2, 3-cNMPs (2, 3-cAMP, 2, 3-cGMP, 2, 3-cCMP and 2, 3-cUMP), comprising steps of: (1) extract genomic DNA and amplify gene If3; (2) ligate If3 gene to expression plasmid to construct a recombinant vector, and transfer the recombinant vector to E. coli to obtain a recombinant strain. Cultivate the recombinant strain and collect the fermentation broth; (3) collect the cells form the fermentation broth and disrupt the cells, and then purify the recombinant protein IF3 from the cell extract by Ni.sup.2+-nitrilotriacetic acid resin. Incubate the recombinant protein IF3 solution at 0 C. for 3 days to release 2, 3-cNMPs from IF3, and centrifuge the solution; (4) Ultrafiltrate the supernatant to remove proteins, and prepare four kinds of 2, 3-cNMPs by high-performance liquid chromatographic (HPLC) on a C.sub.18 reversed-phase column.

It relates to a LfliZ gene and its application. The sequence of gene LfliZ is shown in SEQ ID NO.1. This invention also relates to the recombinant protein LfliZ encoded by LfliZ gene and its application in preparation of 2,3-cNMPs. The recombinant LfliZ protein encoded by gene LfliZ can bind four kinds of 2,3-cNMPs during its expression in Escherichia coli. Four kinds of 2,3-cNMPs can be prepared simultaneously from the recombinant E. coli by extracting the recombinant protein LfliZ. The yield of 2,3-cNMPs reaches 2.5 mg/L fermentation broth by the method in the present invention, indicating that this method has a good application potential.

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.

A process for the synthesis of Sofosbuvir ##STR00001##
is provided comprising the steps of selectively mono-deacetylating a compound of formula (V) ##STR00002##
enzymatically using a resin supported lipase B derived from Candida Antarctica to obtain a compound formula (IV), ##STR00003##
then converting the compound of formula (IV) to a compound of formula (II) ##STR00004##
by reacting the compound of formula (IV) with a compound of formula (III), and ##STR00005##
then converting the compound of formula (II) to Sofosbuvir of formula (I) by deacetylation reaction.

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.