Yeast to directly convert cellulose and, especially, the microcrystalline cellulose into bioethanol, was engineered through display of minicellulosomes on the cell surface of Saccharomyces cerevisiae . The construction and cell surface attachment of cellulosomes were accomplished with two individual miniscaffoldins to increase the display level. All of the cellulases including a celCCA (endoglucanase), a celCCE (cellobiohydrolase), and a Ccel_2454 (β-glucosidase) were cloned from Clostridium cellulolyticum , ensuring the thermal compatibility between cellulose hydrolysis and yeast fermentation. Cellulases and one of miniscaffoldins were secreted by α-factor; thus, the assembly and attachment to anchoring miniscaffoldin were accomplished extracellularly. Immunofluorescence microscopy, flow cytometric analysis (FACS), and cellulosic ethanol fermentation confirmed the successful display of such complex on the yeast surface. Enzyme–enzyme synergy, enzyme-proximity synergy, and cellulose–enzyme–cell synergy were analyzed, and the length of anchoring miniscaffoldin was optimized. The engineered S. cerevisiae was applied in fermentation of carboxymethyl cellulose (CMC), phosphoric acid-swollen cellulose (PASC), or Avicel. It showed a significant hydrolytic activity toward microcrystalline cellulose, with an ethanol titer of 1,412 mg/L. This indicates that simultaneous saccharification and fermentation of crystalline cellulose to ethanol can be accomplished by the yeast, engineered with minicellulosome.

Bio-ethanol production from lignocellulosic raw materials could serve as a sustainable potential for improving the supply of liquid fuels in face of the food-to-fuel competition and the growing energy demand. Xylose is the second abundant sugar of lignocelluloses hydrolysates, but its commercial-scale conversion to ethanol by fermentation is challenged by incomplete and inefficient utilization of xylose. Here, we use a coupled strategy of simultaneous maltose utilization and in-situ carbon dioxide (CO₂) fixation to achieve efficient xylose fermentation by the engineered Saccharomyces cerevisiae. Our results showed that the introduction of CO₂ as electron acceptor for nicotinamide adenine dinucleotide (NADH) oxidation increased the total ethanol productivity and yield at the expense of simultaneous maltose and xylose utilization. Our achievements present an innovative strategy using CO₂ to drive and redistribute the central pathways of xylose to desirable products and demonstrate a possible breakthrough in product yield of sugars.

Consolidated bioprocessing (CBP), integrating cellulase production, cellulose saccharification, and fermentation into one step has been widely considered as the ultimate low-cost configuration for producing second-generation fuel ethanol. 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 engineering Saccharomyces 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 and ~56.37 mg cellulosic ethanol/g cell·h from phosphoric acid-swollen cellulose. 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 cellulosic biofuel production.

D-Allulose is an ultra-low calorie sweetener with broad market prospects. As an alternative to Izumoring, phosphorylation-dephosphorylation is a promising method for D-allulose synthesis due to its high conversion of substrate, which has been preliminarily attempted in enzymatic systems. However, in vitro phosphorylation-dephosphorylation requires polyphosphate as a phosphate donor and cannot completely deplete the substrate, which may limit its application in industry. Here, we designed and constructed a metabolic pathway in Escherichia coli for producing D-allulose from D-fructose via in vivo phosphorylation-dephosphorylation. PtsG-F and Mak were used to replace the fructose phosphotransferase systems (PTS) for uptake and phosphorylation of D-fructose to fructose-6-phosphate, which was then converted to D-allulose by AlsE and A6PP. The D-allulose titer reached 0.35 g/L and the yield was 0.16 g/g. Further block of the carbon flux into the Embden-Meyerhof-Parnas (EMP) pathway and introduction of an ATP regeneration system obviously improved fermentation performance, increasing the titer and yield of D-allulose to 1.23 g/L and 0.68 g/g, respectively. The E. coli cell factory cultured in M9 medium with glycerol as a carbon source achieved a D-allulose titer of ≈1.59 g/L and a yield of ≈0.72 g/g on D-fructose.