Prototyping and development of synthetic metabolic pathways for the biosynthesis of flavour & fragrance compounds
File(s)
Author(s)
Muller, Jonathan
Type
Thesis
Abstract
There is an urgent need to develop sustainable methods for producing renewable chemicals. Photosynthetic cyanobacteria could be used to produce sustainable chemicals from CO2 and sunlight. However, bioproduction using cyanobacteria has yet to be commercialised due to the high cultivation costs, and low product titres and values. To develop this technology, biosynthesis of higher value compounds should be targeted first. Flavour and fragrance compounds are a promising target, due to their high-value and large global market. In this work, synthetic flavour and fragrance biosynthesis pathways were prototyped in Escherichia coli for a faster Design-Build-Test-Learn Cycle, with the intention to thereafter transfer them to cyanobacteria. A novel pathway was proposed for the biosynthesis of decalactones, which are of commercial interest. The first step in the pathway was the biosynthesis of decanoic acid by a C10 specific acyl-ACP thioesterase. Such thioesterases had not yet been reported, so one with a C10 specificity of 77.1% was engineered. The second step in the pathway was hydroxylation of the decanoic acid by BM3 to generate 4-hydroxy and 5-hydroxydecanoic acid, which spontaneously condensed to form γ-decalactone and δ-decalactone, respectively. Native BM3 did not hydroxylate decanoic acid at the required positions, so mutants of BM3 were prepared that showed altered regioselectivity. The complete biosynthetic pathway was then expressed in E. coli and biosynthesis of γ-decalactone (3.53 mg/L) and δ-decalactone (0.53 mg/L) was achieved. The biosynthesis of other flavour and fragrance compounds using synthetic pathways that required less enzyme engineering was also investigated, to reduce the amount of work required in E. coli prior to transfer to cyanobacteria. The biosynthesis of butyl acetate (153 mg/L), medium chain length methyl ketones (3.73mg/L) and 2-dodecenol (22 mg/L) was achieved by adapting previously developed synthetic pathways, while biosynthesis of ethyl butyrate (224 mg/L) was achieved using a novel pathway.
Version
Open Access
Date Issued
2022-11-04
Date Awarded
01/09/2023
License URL
Advisor
Jones, Patrik
Hellgardt, Klaus
Sponsor
Biotechnology and Biological Sciences Research Council (Great Britain)
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)