Author(s): Francisco, Miguel ; Freitas, José Luís Silva ; Moura, Clara D. ; Costa, Carlos E. ; Aguiar, Tatiana Quinta ; Domingues, Lucília
Date: 2024
Persistent ID: https://hdl.handle.net/1822/93164
Origin: RepositóriUM - Universidade do Minho
Project/scholarship: info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F04469%2F2020/PT;
Subject(s): Metabolic engineering; Lactone; Precision fermentation; Biorefinery; Residue valorisation
Introduction: Lactones are volatile organic compounds derived from lipid metabolism. They have a wide range of applications, notably as fragrances and flavours, with -decalactone being particularly valuable. Lactones significantly contribute to the smell and taste of fruits like strawberries and peaches, but traditional extraction methods have many disadvantages. Microbial production has emerged as a more sustainable alternative but depends on the biotransformation of valuable limited hydroxylated fatty acids, each producing specific lactones1. Ashbya gossypii, a filamentous fungus, naturally synthetizes -lactones de novo from carbon sources2 and is a notable industrial cell factory for riboflavin (vitamin B2) production3. It can also produce phenylethyl alcohol, another valuable fragrance compound. Additionally, metabolically enhanced A. gossypii strains have been explored for lipid production, valorising biomass into biofuels4,5. Methodology: In this work, A. gossypii strains were engineered for improved de novo lactone biosynthesis, focusing on fine-tuning different steps of the lactone biosynthetic pathway, namely fatty acid desaturation. A fatty acid desaturase gene from Fragaria ananassa (strawberry) was selected to improve this step. A versatile Golden Gate-based toolbox of integrative cassettes was developed to facilitate the generation of new strains. Process optimization involved testing various carbon sources and agro-industrial residues as substrate for lactone production. Results: A strain with increased expression of a native desaturase produced significantly higher -decalactone titres, reaching 114 ± 16 mg/L using molasses as the carbon source and corn-steep liquor as supplementation. Phenylethyl alcohol production reached 18 ± 2.6 mg/L under these conditions. Bioreactor production allowed for further optimization of production conditions. Moreover, the development and optimization of a Golden Gate-based toolbox of integrative cassettes with improved transformation efficiency accelerated strain engineering. Conclusion: High titres of - decalactone were achieved with engineered strains and precision fermentation, contributing to biorefinery and the valorisation of agro-industrial residues. The developed Golden Gate-based toolbox with improved transformation efficiency is a valuable tool for A. gossypii strain engineering. Insights gained into the lactone biosynthesis pathway will be beneficial for improved production and large-scale optimization.
info:eu-repo/semantics/publishedVersion
