Improving secretion and stress tolerance in yeast strains engineered for consolidated bioprocessing with cell- surface adhered cellulase activities.

Abstract

The consolidated bioprocessing (CBP) of lignocellulosic biomass (LCB) has been established as a leading methodology for the production of alternative fuels and value-added products, since its conceptualisation in the early 2000s. Several studies have made advancements towards the development of a CBP organism capable of complete hydrolysis of cellulose and hemi-cellulose components of LCB and subsequent fermentation of monosaccharides producing maximum product yields. This can be achieved through the endowment of the yeast Saccharomyces cerevisiae with cellulolytic capabilities through the cell free or cell-attached production of fungal cellulases. However, CBP organisms must overcome the following major challenges: low cellulase secretion titres, low hydrolysis rates and varying susceptibility to inhibitory fermentation compounds and fermentation environment conditions, which often inhibit cell viability and metabolic processes. The employment of rational strain engineering technologies using gene targets related to various activities in the secretion pathway and stress response mechanisms of yeast has proven encouraging as many studies have reported improved cellulase secretion titres and improved strain robustness. Therefore, this study aimed to improve cellulase activity and strain robustness of two CBP engineered S. cerevisiae BY4741 strains producing a cell surface attached cellulase consortium (BGL, EG CBH1 and CBH2). This was achieved through the overexpression of PSE1, YHB1, and SED5 and measuring the effect on the heterologous cellulase activity, crystalline cellulose hydrolysis and stress tolerance of S. cerevisiae strains. One of the strains used in this study underwent further engineering through the CRISPR/Cas9-mediated deletion of two cell wall protein encoding genes, resulting in an increased cellulase carrying capacity. The results demonstrated that overexpression of native yeast genes yielded varying enhancements in individual cellulase activities which collectively translated to enhanced crystalline cellulose hydrolysis. In optimal conditions the most significant increases in individual cellulase activity were demonstrated with SED5 overexpression resulting in improved β-glucosidase (BGL) activity by 75%, cellobiohydrolase (CBH) activity by 180% and endoglucanase (EG) activity by 169% when compared to the respective background strains. This led to a crystalline cellulose hydrolysis improvement of 251% when compared the BYCC background strain. Furthermore, at elevated temperatures the most significant changes in individual cellulase activities were observed in BGL activity with an increase of 275% (PSE1 overexpression), CBH activity with a significant increase of 111% (SED5 overexpression) and EG activity with an improvement of 168% (PSE1 overexpression) when compared to respective background strains. Interestingly, the most significant increase in crystalline cellulose hydrolysis in elevated temperature was observed for the BY strain overexpressing SED5. Lastly, in weak acid stress, improvements were observed in BGL activity up to 52%, CBH activity up to 225% and EG activity up to 162% as facilitated by SED5 overexpression. This translated into a significant increase in crystalline cellulose hydrolysis of 366% when compared to the respective background strains. However, metabolic burden was observed when assessing stress tolerance and growth for the various strains with superior cellulase activities, which resulted in reduced tolerance towards stress factors and reduced growth rates. Overall, this study demonstrates the potential of rational engineering using the above-mentioned genes to improve cellulolytic performance of CBP engineered strains in both optimal and harsh conditions. However, careful consideration must be given to the metabolic impact of the enhancements introduced.