Single-Step Conversion Of Cellulosic Ethanol By Saccharomyces Cerevisiae With Engineered Minicellulosome And Cellodextrin Pathway

Consolidated bioprocess (CBP) is a high integrated process, wherein all biological processes including enzyme expression, saccharification, and ethanol production are conducted in a single reactor. However, natural microorganisms are not currently available in CBP. In this work, cellulolytic yeasts were developed by engineering Saccharomyces cerevisiaewith a heterologous cellodextrin utilization pathway and bifunctional minicellulosomes. The cell-displayed minicellulosome was two-scaffoldin derived,and contained an endoglucanase and an exoglucanase, while the intracellular cellodextrin pathway consisted of a cellodextrin transporter and a P-glucosidase, which mimicked the unique cellulose-utilization system in Clostridium thermocellum and allowed S. cerevisiae to degrade and use cellulose and mixed-sugar uptake.A heterologous cellodextrin utilization pathway was expressed, and the different β-glucosidases were screened. In addition, The localization of GH1-1 and CDT-1 in yeast were characterized.The N-terminal a-factor was able to facilitate the formation of GFP complex on the displayed miniscaffoldin Ⅱ with a fluorescence increase of>30%.Two-scaffoldin-derived minicellulosomes were constructed for cellulase screening, and preferred genes were selected for degradation of carboxymethylcellulose (CMC), phosphoric acid-swollen cellulose (PASC), and Avicel. The best combinations are Eng Y and cbhB, celCCA and CA_C0911, and celCCD and cbhB.Finally, galactose concentration and miniscaffoldin Ⅱ length were optimized. Single-step co-fermentation of cellulose and galactose by the recombinant yests co-expressing the cellodextrin pathway and bifunctional cellulosome was studied. CMC-galactose co-fermentation with recombinant yeast produced 3.26g·L-1 cellulosic bioethanol, the specific ethanol productivity from CMC reached ～62.61mg cellulosic ethanol per (g cellh). While, PASC-galactose co-fermentation with recombinant yeast produced 1.09g·L-1 cellulosic bioethanol, the specific ethanol productivity from CMC reached-56.37mg cellulosic ethanol per (g cell·h). We compared the free minicellulosomes and surface-displayed minicellulosomes. The fermentation result suggested that the cellulosic ethanol of free minicellulosomes was only 2.33g·L-1, the specific ethanol productivity was 33.34mg cellulosic ethanol per (g cell·h), and a 27.41% and ～46.7% decrease compared to surface-displayed minicellulosomes.