Identifying promoters to enhance heterologous gene expression in recombinant Saccharomyces cerevisiae strains cultivated on non-native substrates

Efficient bioconversion of lignocellulosic biomass (LCB) to ethanol by Saccharomyces cerevisiae requires its engineering to express heterologous enzymes at titres high enough to make significant impacts on 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. Heterologous xylosidase- and xylanase-encoding genes (xln43_SED1 and xyn2) were individually cloned under transcriptional control of the SED1_P and TDH3_P promoters, and DIT1_T terminator, and integrated into the genome of an a S. cerevisiae strain engineered for xylose utilization. Enzymatic assays were used to quantify the performance of the promoters when strains were cultivated on glucose (aerobically and micro-aerobically) and xylose. 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 SED1_P/DIT1_T and TDH3_P/DIT1_T combinations significantly outperformed the benchmark ENO1_P/T under all of the tested cultivation conditions, as well as with regard to growth trials on non-native substrates (xylo-oligosaccharides/XOS and beechwood xylan) and fermentations of beechwood xylan to ethanol. Overall, TDH3_P was the best-performing promoter. This study demonstrates that heterologous metabolic pathways and CBP can be significantly enhanced by employing carefully selected promoters tailored to specific conditions. KEY POINTS: • Promoters are unpredictable and must be tested under their intended conditions. • TDH3_P, SED1_P, and DIT1_T were effective in enhancing heterologous xylanase activity. • Optimized xylanolytic enzyme expression improved CBP of xylan to ethanol.