Feeding of (2S,3R)-[2,3-13C2]-2-methyl-3-hydroxypentanoyl NAC thioester (1a) to the recombinant organism Streptomyces coelicolor CH999/pCK7 harboring the complete set of eryA genes from Saccharopolyspora erythraea encoding the 6-deoxyerythronolide B synthase (DEBS) resulted in the formation of 6-deoxyerythronolide B (2a) labeled with 13C at C-12 and C-13, as evidenced by the appearance of a pair of enhanced and coupled doublets in the 13C NMR spectrum. The level of 13C enrichment was 15-20 atom% 13C, as much as 100 times higher than the usually observed efficiency of incorporation of NAC thioesters into polyketide metabolites. Similar incorporation of (2S,3R)-[3-2H,3-13C]-2-methyl-3-hydroxypentanoyl NAC thioester (1b) gave 6-deoxyerythronolide B (2b) labeled with both 13C and deuterium at C-13. The intact incorporation of both precursors confirms the normal functioning of the recombinant DEBS proteins in the heterologous host.