Engineering Yeast with Minicellulosome and Cellodextrin Pathway for Co-Utilization of Cellulose and Mixed Sugars

Consolidated bioprocessing (CBP), which integrates cellulase production, cellulose saccharification, and fermentation into one step, has been widely considered as the ultimate low-cost configuration for producing second-generation bioethanol from lignocellulose. However, the requirement of a microbial strain able to hydrolyze cellulosic biomass and convert the resulting sugars into high-titer ethanol limits CBP application. In this work, cellulolytic yeasts were developed by engineeringSaccharomyces cerevisiae with 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 β-glucosidase, which mimicked the unique cellulose-utilization system in Clostridium thermocellum and allowed S. cerevisiae to degrade and use cellulose without glucose inhibition/repression on cellulases and mixed-sugar uptake. Consequently, only a small inoculation of the non-induced yeast cells was required to efficiently co-convert both cellulose and galactose to ethanol in a single-step co-fermentation process, achieving a high specific productivity of ~62.61 mg cellulosic ethanol/g cell·h from carboxymethyl cellulose (CMC) and ~56.37 mg cellulosic ethanol/g cell·h from phosphoric acid-swollen cellulose (PASC). Our work provides a versatile engineering strategy for co-conversion of cellulose-mixed sugars to ethanol by S. cerevisiae, and the achievements in this work may further promote the application of CBP in cellulosic biofuel production.