Amino acids as a route to biofuel production in Saccharomyces cerevisiae

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Abstract

Biofuels are considered to be part of the solution to dwindling global fossil fuel reserves. Bioethanol offers commercial viability, but has substantial disadvantages in terms of its fuel properties. Higher alcohols, such as n-butanol, isobutanol, isoamyl alcohol (3-methyl-1-butanol) and active amyl alcohol (2-methyl-1-butanol) possess better fuel characteristics than bioethanol, but their production lags behind in terms of commercial viability. While much research has concentrated on using Escherichia coli and Clostridium species, here we use yeast as a vehicle for the production of these higher alcohols. In Saccharomyces cerevisiae, higher alcohol production occurs via amino acid catabolism through the Ehrlich pathway, but the yield is relatively low. Upregulation of amino acid biosynthesis should therefore increase higher alcohol production. The first objective of the project was to identify which groups of amino acid when added in excess could be directed to production of higher alcohols. We conducted a preliminary assessment of higher alcohol production from different excess (150 mM) amino acid groups in a wild-type yeast strain under aerobic condition. The aliphatic amino acid group was identified as the most metabolised group to isobutanol and amyl alcohols. This result was also established at different temperatures (25°C and 37°C) and under semi-anaerobic condition. Excess alanine and leucine proved to be most metabolised aliphatic amino acids to isobutanol and amyl alcohols respectively. In addition, we show that resistance to higher alcohol resulted in 3-fold increase in amyl alcohol production when metabolism of excess leucine was compared between a butanol resistant strain (GCD1-P180) and a butanol sensitive strain (GCD1-S180). The main goal of the project was to increase higher alcohol production by overexpressing GCN4 in a prototrophic strain of S. cerevisiae. Yeast strains bearing wild-type (GCN4), uORF-less form (4uÃŽ”-GCN4) and deleted (gcn4ÃŽ”) alleles of GCN4 were made. The genes were integrated from plasmids into a high expression site of a prototrophic strain of S. cerevisiae bearing a deleted chromosomal copy of GCN4. This led to a moderate increase in overall isobutanol production (~30 mg/L) in the uORF-less strain compared to the other strains tested. To increase production further, three genes ALD6, BAT1 and PDC1 representing competing pathways in yeast were deleted singly, in pairs and together. Results obtained showed an increase in isobutanol production (40 mg/L) in the triple deletion strain overexpressing the transcription factor almost twice the values obtained in the wild-type (21 mg/L) and delete strains (17 mg/L). On the whole, this project has shown that production of isobutanol can be significantly increased in a prototrophic yeast strain by overexpressing GCN4 and deleting genes involved in competing pathways. The levels obtained however suggest that more strategic genetic manipulations will be required in order to attain industrially significant levels.

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