Expression of a periplasmic β-glucosidase from Yarrowia lipolytica allows efficient cellobiose-xylose co-fermentation by industrial xylose-fermenting Saccharomyces cerevisiae strains

Abstract

This study aimed to compare the effects of cellobiose hydrolysis, whether occurring inside or outside the cell, on the ability of Saccharomyces cerevisiae strains to ferment this sugar and then apply the most effective strategy to industrial S. cerevisiae strains. Firstly, two recombinant laboratory S. cerevisiae strains were engineered: CEN.PK-X-Bgl1YL, expressing the periplasmic β-glucosidase BGL1 from Yarrowia lipolytica; and CEN.PK-X-B7-T2, co-expressing the intracellular β-glucosidase SpBGL7 from Spathaspora passalidarum and the cellobiose transporter MgCBT2 from Meyerozyma guilliermondii. Both engineered strains were able to grown in media with cellobiose and to ferment this disaccharide. However, CEN.PK-X-Bgl1YL, which hydrolyzes cellobiose extracellularly, exhibited faster growth and superior batch fermentation performance. Furthermore, enzymatic and transport activities revealed that sugar uptake was possibly the limiting factor in cellobiose fermentation by CEN.PK-X-B7-T2. Since extracellular hydrolysis with the periplasmic β-glucosidase was more efficient for cellobiose fermentation, we integrated the BGL1 gene into two industrial xylose-fermenting S. cerevisiae strains. The resulting strains (MP-C5H1-Bgl1YL and MP-P5-Bgl1YL) efficiently co-consumed ∼ 22 g L^{- 1} of cellobiose and ∼ 22 g L^{- 1} of xylose in 24 h, achieving high ethanol production levels (∼ 17 g L^{- 1} titer, ∼ 0.50 g L^{- 1} h^{- 1} volumetric productivity, and 0.40 g g^{- 1} ethanol yield). Our findings suggest that the expression of periplasmic β-glucosidases in S. cerevisiae could be an effective strategy to overcome the disaccharide transport problem, thus enabling efficient cellobiose fermentation or even cellobiose-xylose co-fermentation.

Keywords: 2G biorefineries; Bioethanol; Cellobiose; Co-fermentation; Xylose; Yeasts.