Abstract
BACKGROUND: 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 de novo biosynthesis of lactones, leading to the production of γ-decalactone, δ-decalactone, γ-dodecalactone and δ-dodecalactone in Escherichia coli. RESULTS: Wild-type enzymes with the appropriate substrate specificities for the pathway were not available, therefore enzyme engineering was required. Firstly, the Cuphea viscosissima FatB1 thioesterase was modified to be C10 specific, resulting in novel mutants with a 77.1% C10 specificity in E. coli. Engineered thioesterases were also found to display increased C10 specificities when expressed in Synechocystis 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 E. coli 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. CONCLUSIONS: The synthetic pathways were shown to be functional and amenable to tailoring both the chain length and ring-structure of the resultant lactones.