Harnessing native nitrogen in lignocellulosic biomass for cellulosic ethanol production by ancestral xylose isomerase-engineered Saccharomyces cerevisiae.

Efficient xylose-utilizing Saccharomyces cerevisiae, straightforward pretreatment, elimination of detoxification steps, reduced cellulase dosage, and cost-effective nutrients are critical for the commercialization of lignocellulosic ethanol production. In this study, three highly efficient xylose-utilizing S. cerevisiae, which were capable of consuming 40 g/L xylose within 14 h and consuming a mixture of 80 g/L glucose and 40 g/L xylose within 18 h, were developed by integrating artificial ancestral xylose isomerases into diploid S. cerevisiae genome, followed by laboratory evolution and colony screening. Thereafter, a practical lignocellulosic ethanol process was established, which incorporated DLC(sa) pretreatment (densifying lignocellulosic biomass using sulfuric acid as the reagent), a low cellulase dosage of 14.81 FPU per gram of cellulose, and the elimination of washing or detoxification steps, as well as the need for additional nitrogen sources. Using this approach, 54.8 g/L ethanol was produced from 30 wt% hydrolysate prepared from unwashed corn stover. Further analysis revealed that S. cerevisiae utilized the native nitrogen sources present in the hydrolysate for cell growth and metabolism. In summary, this study offers a practical framework and valuable insights for advancing the commercial production of lignocellulosic ethanol.