The economical and efficient commercial production of second-generation bioethanol requires fermentation microorganisms capable of entirely and rapidly utilizing all sugars in lignocellulosic hydrolysates. In this study, we developed a recombinant Saccharomyces cerevisiae strain, BLH510, through protoplast fusion and metabolic engineering to enhance its ability to co-ferment glucose, xylose, cellobiose, and xylooligosaccharides while tolerating various inhibitors commonly found in lignocellulosic hydrolysates. The parental strains, LF1 and BLN26, were selected for their superior glucose/xylose co-fermentation capabilities and inhibitor tolerance, respectively. The fusion strain BLH510 demonstrated efficient utilization of mixed sugars and high ethanol yield under oxygen-limited conditions. Under low inoculum conditions, strain BLH510 could completely consume all four kinds of sugars in the medium within 84 h. The fermentation produced 33.96 g/L ethanol, achieving 84.3% of the theoretical ethanol yield. Despite the challenging presence of mixed inhibitors, BLH510 successfully metabolized all four sugars above after 120 h of fermentation, producing approximately 30 g/L ethanol and reaching 83% of the theoretical yield. Also, strain BLH510 exhibited increased intracellular trehalose content, particularly under conditions with mixed inhibitors, where the intracellular trehalose reached 239.3 mg/g yeast biomass. This elevated trehalose content contributes to the enhanced stress tolerance of BLH510. The study also optimized conditions for protoplast preparation and fusion, balancing high preparation efficiency and satisfactory regeneration efficiency. The results indicate that BLH510 is a promising candidate for industrial second-generation bioethanol production from lignocellulosic biomass, offering improved performance under challenging fermentation conditions. Our work demonstrates the potential of combining protoplast fusion and metabolic engineering to develop superior S. cerevisiae strains for lignocellulosic bioethanol production. This approach can also be extended to develop robust microbial platforms for producing a wide array of lignocellulosic biomass-based biochemicals.

As a potential alternative to fossil-based fuels, cellulosic ethanol has attracted much attention due to its great benefit to energy sustainability and environmental friendliness. However, at present, the industrial competitiveness of cellulosic ethanol production is still insufficient compared with fossil-based fuels because of the higher costs. Expanding the range of lignocellulosic biomass may be a promising measure to promote the economical production of cellulosic ethanol. Corn fiber, a byproduct from the corn deep-processing, is an attractive feedstock for cellulosic ethanol production because of its rich carbohydrate content (generally exceeding 65% of dry weight), almost no transportation cost, and low lignin content allow it to be easily handled. This study first optimized the hydrolysis conditions, including the pretreatment and enzymolysis process based on dilute sulfuric acid, to achieve a high sugar yield. Then, the corn fiber hydrolysates obtained under different hydrolysis conditions were suitably fermented by different C5/C6 co-fermentation Saccharomyces cerevisiae, indicating that the hydrolysate at high solid loading (20%) needs to detoxification to a certain extent but not low solid loading (10%) to achieve high ethanol yield. Finally, the fermentation of the 20% solid loading hydrolysates with resin detoxification was performed in a 50 L bioreactor, achieving the sugar (glucose and xylose) metabolic rate of 2.24 g L ⁻¹ h ⁻¹ and ethanol yield of 92% of the theoretical value, which are the highest reported levels to date. This study provided a potential process route for cellulosic ethanol production from corn fiber from the perspective of the suitability between the upstream hydrolysis process and the downstream fermentation strain.