A synthetic metabolic pathway for the de novo biosynthesis of medium chain length γ- and δ-lactones
File(s)
Published version
Author(s)
Muller, Jonathan
Xia, Angel
Jones, Patrik
Type
Journal Article
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.
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.
Date Acceptance
2025-10-18
Citation
Journal of Biological Engineering, 19
ISSN
1754-1611
Publisher
BMC
Journal / Book Title
Journal of Biological Engineering
Volume
19
Copyright Statement
© The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
License URL
Publication Status
Published
Article Number
104
Date Publish Online
2025-11-25