Engineered Pentafunctional Minicellulosome for Simultaneous Saccharification and Ethanol Fermentation in Saccharomyces cerevisiae

Several yeast strains have been engineered to express different cellulases to achieve simultaneous saccharification and fermentation of lignocellulosic materials. However, successes in these endeavors were modest, as demonstrated by the relatively low ethanol titers and the limited ability of the engineered yeast strains to grow using cellulosic materials as the sole carbon source. Recently, substantial enhancements to the breakdown of cellulosic substrates have been observed when lytic polysaccharide monooxygenases (LPMOs) were added to traditional cellulase cocktails. LPMOs are reported to cleave cellulose oxidatively in the presence of enzymatic electron donors such as cellobiose dehydrogenases. In this study, we coexpressed LPMOs and cellobiose dehydrogenases with cellobiohydrolases, endoglucanases, and β-glucosidases in Saccharomyces cerevisiae. These enzymes were secreted and docked onto surface-displayed miniscaffoldins through cohesin-dockerin interaction to generate pentafunctional minicellulosomes. The enzymes on the miniscaffoldins acted synergistically to boost the degradation of phosphoric acid swollen cellulose and increased the ethanol titers from our previously achieved levels of 1.8 to 2.7 g/liter. In addition, the newly developed recombinant yeast strain was also able to grow using phosphoric acid swollen cellulose as the sole carbon source. The results demonstrate the promise of the pentafunctional minicellulosomes for consolidated bioprocessing by yeast.