Protein Fusion of Biosynthetic Enzymes and a Thermo‐Responsive Polypeptide Expedites Facile Access to Biocatalysts for Nucleotide Sugars

Abstract Nucleotide sugars (NSs) are essential building blocks for the enzymatic assembly of glycans. In this study, we established a preparation and recycling avenue to the biocatalysts for the enzymatic synthesis of NSs. This approach involves fusing two enzymes into a bifunctional chimera and using elastin‐like polypeptides (ET64) as a purification tag, which allows for easy recovery of these biocatalysts without the need for chromatography. We successfully constructed and obtained five bifunctional fusion enzymes (GalK‐USP‐ET64, GlmU‐NahK‐ET64, ManC‐NahK‐ET64, FKP‐ET64, and NanA‐CSS‐ET64) for the synthesis of five common NSs (UDP‐Gal, UDP‐GlcNAc, GDP‐Man, GDP‐Fuc, and CMP‐Neu5Ac). These enzymes were obtained using the Inverse Transition Cycling (ITC) process in yields ranging from 60 to 124 mg per liter of fermentation. The enzymatic synthesis of NSs was carried out on a scale from hundreds of milligrams to multiple grams using these biocatalysts. Furthermore, we investigated the reusability of these biocatalysts by recycling them from the reaction solution using the ITC process. The recycling of GalK‐USP‐ET64, GlmU‐NahK‐ET64, FKP‐ET64, and NanA‐CSS‐ET64 was effectively achieved for 15, 13, 3, and 4 times, respectively. These biocatalysts could be used not only for the enzymatic synthesis of NSs but also for the chemoenzymatic synthesis of glycan biomolecules when coupled with glycosyltransferases.