Fucosylated
chondroitin sulfate (FuCS), a unique glycosaminoglycan
derived from the body walls of sea cucumbers, plays a vital role in
various physiological processes. Although the physiological importance
of FuCS is well-documented, the biosynthesis pathway, especially α1–3
fucosylation, is still unclear, thus hindering the development of
an enzymatic synthesis approach to access both it and its derivatives.
Herein, we report a programmable enzymatic toolbox for the synthesis
of fucosylated chondroitin derivatives. Two distinct microbial α1–3-fucosyltransferases
were identified for the synthesis of fucosylated chondroitin. Through
precise control over chondroitin backbone extension and fucosylation,
13 fucosylated chondroitin oligosaccharides were assembled. Additionally,
the synthetic toolbox has been further adapted for the fucosylation
of chondroitin sulfate A, setting the stage for the formulation of
a FuCS glycan library. This work demonstrates a possible biosynthetic
pathway of FuCS and paves the way for the synthesis of this intriguing
biomolecule.