CRISPR/Cas9-engineered thermotolerant Saccharomyces cerevisiae JAYET for enhanced 3G-ethanol production from Chlorella vulgaris biomass

Abstract

The production of third-generation (3G) ethanol from microalgae-derived carbohydrates requires a cost-effective hydrolysis strategy and a robust fermenting microorganism. In this study, the amyA gene encoding α-amylase from Aspergillus tubingensis was integrated into the genome of the thermotolerant Saccharomyces cerevisiae JAYET strain using CRISPR/Cas9. JAYET was previously derived from the industrial PE-2 strain and engineered for cellulase production. The CRISPR/Cas9 system enabled efficient genome editing and construction of a synthetic yeast strain, named JAYCA. The performance of the engineered strain was evaluated under different fermentation strategies (SHF, SSF, pSSF), temperatures (30 °C and 40 °C), and compared with parental strains. JAYCA showed strong growth on starch agar, confirming successful expression of extracellular α-amylase. During fermentation of carbohydrate-rich Chlorella vulgaris biomass, JAYCA achieved 92.1 % of the theoretical ethanol yield using Separate Hydrolysis and Fermentation (SHF) at 30 °C. The highest ethanol concentration (16.41 g·L⁻¹) was obtained under Simultaneous Saccharification and Fermentation (SSF), combining α-amylase and amyloglucosidase activity. These results demonstrate that the engineered yeast effectively converted microalgal carbohydrates into ethanol, achieving high 3G ethanol titers through an eco-friendly Consolidated Bioprocessing (CBP) strategy based on enzymatic hydrolysis and renewable biomass. This is the first report of applying the engineered JAYCA strain in a microalgae-based CBP strategy for 3G ethanol production.