Abstract Description

In an effort to sustainably meet growing global energy demands in an environmentally-friendly manner, second generation (2G) bioethanol has emerged as an alternative to finite fossil fuel-based energy, making use of non-edible lignocellulosic biomass (LCB) as its feedstock. Efficient bioconversion of LCB to ethanol by Saccharomyces cerevisiae requires its engineering to express heterologous enzymes at titres high enough to make significant impacts in industrial consolidated bioprocessing (CBP). Promoters are required for this purpose, but are reportedly influenced by various environmental factors as well as the protein specific nature of expression, warranting the need for assessment under the conditions for which they are intended. We evaluated the performances of the native SED1 and TDH3 gene promoters, and DIT1 terminator in the expression of heterologous xylan-hydrolysing enzymes in S. cerevisiae, using the ENO1 gene promoter and terminator as a benchmark. Heterologous xylosidase and xylanase encoding genes (Pyrenophora tritici-repentis xln43_SED1 and Trichoderma reesei xyn2) were individually cloned under transcriptional control of the SED1P and TDH3P promoters, and DIT1T terminator, and integrated into the genome of a S. cerevisiae strain engineered for xylose utilization (S288C-MJM121). Enzymatic assays were used to quantify the performance of the promoters when strains were cultivated on glucose- (aerobically and micro-aerobically) and xylose-containing media. Additional strains containing both xln43_SED1 and xyn2 under different promoter combinations were then used to allow direct fermentation of beechwood xylan to ethanol in a CBP. The SED1P/DIT1T and TDH3P/DIT1T significantly outperformed the benchmark ENO1P/T under all of the tested cultivation conditions, as well as during growth trials on non-native substrates (xylo-oligosaccharides/XOS and beechwood xylan) and fermentations of beechwood xylan to ethanol. Overall, TDH3P/DIT1T was the best-performing promoter/terminator combination. This study demonstrates that heterologous metabolic pathways and CBP can be significantly enhanced by employing carefully selected promoters tailored to specific conditions. Future work will involve the use of these promoter/terminator combinations in the enhancement of cellulolytic genes to enable growth on cellulose-based substrates, as well as for the direct fermentation of Avicel to ethanol in a CBP.