PhD Research Project: Structure and function of the alcohol acyltransferases from yeast and fruit

Volatile esters are produced in yeast and fungi during fermentation, in plants during fruit ripening, and in higher organisms such as humans in response to ethanol stress. These compounds play an important role in the flavour of fruits and the taste of fermented beverages such as beer and wine. For example, isoamyl acetate tastes of banana; ethyl hexanoate tastes of apples. These esters are produced by enzymes that act as acyl-CoA:ethanol O-acyltransferases (AATases), and understanding the molecular biochemistry of yeast and plant AATases may lead to designer yeast strains with tailored fermentation products and improvements to fruit crops, fruit flavour and shelf life. There is also increasing interest in using AATases for metabolic engineering to produce renewable biofuels and fine chemicals.

However, these enzymes have only been partially characterized to date because of a lack of methods for isolating recombinant protein and for studying enzyme function. We have recently established methods that can be used to purify and study these proteins (Knight et al, (2014) Yeast 31:464; Nancolas et al, (2015) in preparation). The student will use and extend these methods to pursue two linked research strands:

(i) Use molecular biochemistry and biophysics to characterize the structure and function of two yeast AATases from different protein families. This will use X-ray crystallography and classical enzyme kinetics, combined with computational ligand docking and site-directed mutagenesis. The outcome of this strand will be to define the location and features of the catalytic active site of these proteins. This will provide the first detailed insights into enzyme mechanism, including how these enzymes discriminate between different substrates.

(ii) Use our established ‘toolkit’ of methods to begin studying other AATases associated with the ripening of commercial fruits such as strawberry, banana, apple, papaya and kiwifruit. This will include cloning these genes prior to protein expression in a recombinant host. The recombinant protein will be purified and analysed by a suite of biochemical and biophysical methods. The outcome of this project strand will be the first detailed study of this enzyme family.

Skills training for the student will include: Protein Biochemistry (immobilized metal affinity chromatography, gel filtration, SDS-PAGE and native-PAGE, circular dichroism, site-directed mutagenesis, enzyme kinetics); Structural Biology (including high-throughput crystallization screening with state-of-the art liquid handling robotics and structure determination using synchrotron radiation); and Computational Biochemistry (ligand docking). The student will be introduced to each of these methods during laboratory rotation projects.

Funding Notes

This project is part of the BBSRC-funded South West Biosciences Doctoral Training Partnership (SWBio DTP).

Link to SWBio DTP website: View Website