Abstract: The present invention provides a novel biosynthetic production process which converts L-galactose into 2?-fucosyllactose via four enzymatically catalyzed reaction steps. The present process is designed such that co-factors required by the process are regenerated within the four reaction steps, hence making the process cost-effective and efficient. The process can be performed in vitro in a cell-free system. The present invention also provides mutant enzymes that can be used to increase production levels of 2?-fucosyllactose, whether using the novel pathway described herein or the mannose-dependent pathway known in the art.