Background <p>Medium chain length γ- and δ-lactones, in particular γ-decalactone, are potent aroma compounds used by the flavour and fragrance industry. The native pathways for their biosynthesis are unknown so to facilitate future biomanufacturing, we designed and implemented a novel metabolic pathway, using engineered enzymes, for the <i>de novo</i> biosynthesis of lactones, leading to the production of γ-decalactone, δ-decalactone, γ-dodecalactone and δ-dodecalactone in <i>Escherichia coli</i>.</p> Results <p>Wild-type enzymes with the appropriate substrate specificities for the pathway were not available, therefore enzyme engineering was required. Firstly, the <i>Cuphea viscosissima</i> FatB1 thioesterase was modified to be C10 specific, resulting in novel mutants with a 77.1% C10 specificity in <i>E. coli</i>. Engineered thioesterases were also found to display increased C10 specificities when expressed in <i>Synechocystis</i> Sp. PCC 6803. The cytochrome P450 BM3 was then used to hydroxylate decanoic acid to 4- or 5-hydroxydecanoic acid which spontaneously condensed to γ- and δ-decalactone, respectively. Feeding methyl decanoate to <i>E. coli</i> cells expressing BM3 mutants led to improved production of both γ- and δ-decalactone. Expressing the complete pathway using the engineered enzymes enabled the accumulation of γ- and δ-decalactone with maximum titres of 3.53 mg/L and 0.51 mg/L, respectively. The pathway was also modified to biosynthesise dodecalactones by using a C12-specific thioesterase and different BM3 mutants leading to γ- and δ-dodecalactone titres of 1.21 mg/L and 3.29 mg/L, respectively.</p> Conclusions <p>The synthetic pathways were shown to be functional and amenable to tailoring both the chain length and ring-structure of the resultant lactones.</p>