Abstract Description

Saccharomyces cerevisiae has been widely used in industrial practices as host for heterologous protein production. However, with reference to biofuel production practices, the low secretion titres often obtained by domesticated strains of S. cerevisiae hampers implementation of sustainable and economically feasible industrial bioethanol production. To mitigate this challenge, the greater phenotypic and genetic diversity offered by natural isolates of S. cerevisiae has the potential to obtain enhanced heterologous protein production titres and robustness against various process stresses. The scope of this study thus aimed to enhance the secretory capacity of strain isolates to secrete the core complex of cellulases as either free or cell-tethered entities, with the goal to hydrolyse cellulosic substrates to bioethanol in a consolidated bioprocessing (CBP) configuration. With the use of CRISPR-based technologies, natural isolates of S. cerevisiae were engineered with cellulase-encoding gene cassettes. Successful transformants were benchmarked by measuring the individual enzyme activities, evaluating the hydrolysis efficiency of the rudimentary cellulase complex, followed by fermentation of microcrystalline cellulose (Avicel). Additionally, strain robustness and growth kinetics were evaluated. Enhanced cellulolytic activity was clearly demonstrated in the cell-tethered-based systems, compared to free enzyme systems, across the same strain backgrounds. Superior enzyme activity was obtained for YI59_V2 for all individual enzymes across all process-relevant conditions tested. In addition, enhanced hydrolysis efficiency and ethanol titres were observed for this same strain, with ethanol titres exceeding 6 g/L after 96 hours, without the addition of exogenous cellulase cocktails. Interestingly, enhanced strain robustness against a multitude of process-relevant and secretion/cell wall stresses was also observed in the cell-tethered systems compared to the free enzyme systems. The choice of design strategy used for expression of cellulase-encoding genes plays a pivotal role in not just the enhancement of protein titres obtained in S. cerevisiae, but potentially also the robustness profiles of strain backgrounds. This study thus clearly highlighted the potential of these natural isolates to be used as chassis organisms in CBP bioethanol production.