A one-pot enzymatic total synthesis of angucycline antibiotic rabelomycin was accomplished, starting from acetyl-CoA and malonyl-CoA, using a mixture of polyketide synthase (PKS) enzymes of the gilvocarcin, ravidomycin, and jadomycin biosynthetic pathways. The in vitro results were compared to in vivo catalysis using analogous sets of enzymes.