Abstract
Natural products are widely used as pharmaceuticals and agrochemicals, or as active ingredients in food and cosmetics. Their biosynthesis typically involves a series of enzyme-controlled reactions in dedicated liquid environments. The reconstruction of these multistep transformations under in vitro conditions bears significant potential for technical utilization. However, the concurrent operation of multiple enzymes in a single reaction flask or reactor is often associated with major challenges. Herein, the difficulties in reaching high substrate conversion and product yields with in vitro enzyme cascades are summarized. Furthermore, both established and emerging concepts for improving their performance are discussed.