Use of Saccharomyces cerevisiae for the production of industrially relevant alcohols

Abstract

Saccharomyces cerevisiae is an established biotechnological workhorse for the production of various compounds including food products, biopharmaceuticals and biofuels. Previously, the Ashe lab has demonstrated that the biofuel n-butanol can be produced by overexpressing a clostridial butanol synthetic pathway in the yeast cytoplasm. However, the titre of n-butanol produced is below what would be viewed as industrially significant. Hence, the first objective of this project was to compartmentalise the butanol synthetic pathway into yeast peroxisomes in order to concentrate the enzymes to investigate whether this improved butanol titres. A yeast strain was constructed bearing a yeast endogenous gene ERG10 and four other Clostridium beijerinckii genes carrying a peroxisome targeting signal (PTS1). Although peroxisome morphology was visualised, there was little evidence that the butanol synthetic enzymes were localised to peroxisomes and little evidence for improved butanol production. However, the localisation studies suggested that the butanol synthetic enzymes may be present in protein aggregates. In order to test this, the solubility of each butanol synthetic enzyme was biochemically quantified to reveal that all of the enzymes involved in the butanol synthesis pathway were present in insoluble aggregates, regardless of the presence of PTS1. As a result, for one of the butanol production enzymes, AdhE2, strategies such as chaperone overexpression, altered growth temperature and analysis of different protein domains were undertaken with the aim of reducing protein aggregation. A small reduction in the level of insoluble protein was observed for AdhE2 at lower growth temperatures, yet these lower temperatures did not improve butanol levels from the butanol production strains. Interestingly, in some of these butanol production strains, 2,3-butanediol (2,3-BD) accumulates to high levels. 2,3-BD is an important high value bulk chemical with numerous industrial applications. Indeed, the presence of the clostridial bifunctional aldehyde-alcohol dehydrogenases, AdhE2, leads to significantly higher levels of 2,3-BD. In particular, the alcohol dehydrogenase domain plays a key role in 2,3-BD accumulation. On the whole, the project has applied several metabolic engineering strategies to improve bioalcohols production in yeast. However, further optimisation would be required to explore and understand the dynamic system of cell metabolism in yeast especially in the context of highly expressed exogenous proteins and their effect on cell homeostasis.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Christopher Grant (Supervisor) & Mark Ashe (Supervisor)