Abstract <p>Engineering yeast strains for use as chassis organisms in second-generation (2G) bioethanol is a promising strategy to improve process economics. Natural isolates of <i>Saccharomyces cerevisiae</i> offer strain backgrounds with greater genetic diversity and enhanced robustness, with the potential for improved heterologous protein production capabilities. In this study, heterologous cellulase production using different expression strategies was evaluated in various process-relevant conditions. Enhanced cellulolytic activity was clearly demonstrated in a cell-tethered enzyme system, compared to a free enzyme system, across identical strain backgrounds. Superior secretory capacity was obtained for YI59_V2 for all individual enzymes across all process-relevant conditions tested. In addition, this strain exhibited improved hydrolysis efficiency and ethanol production from crystalline cellulose, achieving ~10&#xa0;g/L after 96&#xa0;h (~88% of the maximum theoretical yield) without the need for exogenous cellulase supplementation. Interestingly, enhanced strain robustness against process-relevant, secretion, and cell wall stresses was also observed in transformants with cell-tethered cellulase systems compared to those with free enzyme systems. This study highlights that the expression design strategy for cellulase-encoding genes in this natural isolate was pivotal for increasing protein titres and for influencing strain robustness. Strains exhibiting elevated cellulase activity and increased robustness represent a key step toward the industrial deployment of consolidated bioprocessing (CBP).</p> Key points <p>•&#xa0;<i>Cell-tethered expression greatly boosted cellulase activity and cellulose breakdown.</i></p> <p>•&#xa0;<i>YI59_V2 yielded ~ 10 g/L ethanol from crystalline cellulose without added enzymes.</i></p> <p>•&#xa0;<i>Tethered enzymes reshaped cell walls and altered stress tolerance.</i> </p>