| dc.description.abstract | Microbial contamination is a major challenge in fuel ethanol facilities, especially during the fermentation stage. Bacterial contaminants compete with yeast for fermentable sugars and nutrients, which obstructs starch to ethanol conversion. This leads to a reduction in ethanol yield. At large production scales, prior sterilization of the feedstocks is economically unfeasible. Therefore, antibiotic supplementation is common practice to limit the growth of contaminating bacteria. In addition to the high cost of the antibiotics, concerns and evidence are mounting that antibiotic use in non-clinical settings is driving the emergence of drug resistant microbes. This study focused on heterologous expression of antimicrobial peptides (AMP) as an alternative strategy for combating microbial contamination. This work aimed to engineer an industrial Saccharomyces cerevisiae strain that produces AMPs active against lactic acid bacteria and Enterobacteriaceae which are some of the main bacterial contaminants found in industrial biofuel fermenters. Seven candidate AMPs were selected from curated online databases, and their amino acid sequence was analyzed using bioinformatic tools. AlphaFold, Clustal Omega, and SNAP2 were used to predict AMP tertiary structures, construct a phylogenetic tree, and analyse mutation effects, respectively. The amino acid sequences of the selected AMPs were used to produce codon optimized genes for expression in S. cerevisiae on expression plasmids. These plasmids were successfully cloned into S. cerevisiae Y294. The soft agar-overlay and well diffusion method was used to determine antimicrobial activity, however no antimicrobial activity was found for any of the genes. The inducible GAL1P-CYC1T was replaced with the constitutive ENO1P/T and the newly constructed plasmids were cloned into S. cerevisiae Y294. These transformants were subjected to antimicrobial activity testing against a range of microorganisms. Transformants expressing the AMPs Garvieacin Q, Carnobacteriocin BM1 and Piscicolin 126 respectively, showed antimicrobial activity against Listeria spp. and Enterococcus spp. An attempt was made to create antimicrobial industrial S. cerevisiae strains. The Carnobacteriocin BM1 gene was integrated into the genome of the industrial strain S. cerevisiae Ethanol Red, targeting either δ-sequences or the intergenic regions of chromosome 11. | en_US |
