Effects of Engineered Saccharomyces cerevisiae Fermenting Cellobiose through Low-Energy-Consuming Phosphorolytic Pathway in Simultaneous Saccharification and Fermentation

Until recently, four types of cellobiose-fermenting Saccharomyces cerevisiae strains have been developed by introduction of a cellobiose metabolic pathway based on either intracellular β-glucosidase (GH1-1) or cellobiose phosphorylase (CBP), along with either an energy-consuming active cellodextrin transporter (CDT-1) or a non-energy-consuming passive cellodextrin facilitator (CDT-2). In this study, the ethanol production performance of two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-2 (N306I) with GH1-1 or CBP were compared with two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-1 (F213L) with GH1-1 or CBP in the simultaneous saccharification and fermentation (SSF) of cellulose under various conditions. It was found that, regardless of the SSF conditions, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the best ethanol production among the four strains. In addition, during SSF contaminated by lactic acid bacteria, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the highest ethanol production and the lowest lactate formation compared with those of other strains, such as the hydrolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-1 with GH1-1, and the glucose-fermenting S. cerevisiae with extracellular β-glucosidase. These results suggest that the cellobiose-fermenting yeast strain exhibiting low energy consumption can enhance the efficiency of the SSF of cellulosic biomass.


Supplementary Materials
Comparison of cellobiose metabolic pathways in engineered S. cerevisiae strains expressing cellodextrin transporters and intracellular cellobiose degrading enzymes To develop engineered S. cerevisiae strains capable of fermenting cellobiose, the cellobiose metabolic pathway, composed of cellodextrin transporters and intracellular cellobiose degrading enzymes, has been introduced into S. cerevisiae [11][12][13].
Cellodextrin transporters identified from N. crassa are classified into two types depending on whether energy is required for cellobiose transport or not. CDT-1 is a proton symporter requiring the consumption of ATP by ATPase to export the protons that enter the cell along with cellobiose [11,14]. CDT-2 is a facilitator transporting cellobiose depending on the concentration gradient of extracellular cellobiose [11,14]. Consequently, CDT-1 spends one mole of ATP to transport one mole of cellobiose, while CDT-2 spends no ATP for cellobiose transport.
Intracellular cellobiose degrading enzymes identified from N. crassa or S. degradans are classified into two types based on the degree of energy consumption. Intracellular β-glucosidase (GH1-1) from N. crassa is involved in the hydrolysis of cellobiose (cellobiose → 2 glucose), which requires the consumption of two moles of ATP by hexokinases converting glucose to glucose-6-phosphate to initiate glycolysis from cellobiose [11]. Cellobiose phosphorylase (CBP) from S. degradans is involved in the phosphorolysis of cellobiose (cellobiose → glucose + glucose-1-phosphate), which requires only one mole of ATP consumption by hexokinases to initiate glycolysis from cellobiose due to phosphoglucomutase isomerizing glucose-1-phosphate to glucose-6-phosphate without ATP [12]. Consequently, cellobiose hydrolysis by GH1-1 spends two moles of ATP to initiate glycolysis from one mole of cellobiose, whereas cellobiose phosphorolysis by CBP spends one mole of ATP for glycolysis from one mole of cellobiose.
According to the combination of cellodextrin transporters and intracellular cellobiose degrading enzymes, four types of the cellobiose-fermenting S. cerevisiae strains have been developed [11,12,14,15]; the amounts of energy required for each strain to transport cellobiose and start glycolysis are as follow: 3 moles of ATP for D-BT1 strain (S. cerevisiae expressing CDT-1 and GH1-1); 2 moles of ATP for D-CT1 strain (S. cerevisiae expressing CDT-1 and CBP); 2 moles of ATP for D-BT2 strain (S. cerevisiae expressing CDT-2 and GH1-1); 1 mole of ATP for D-CT2 strain (S. cerevisiae expressing CDT-2 and CBP). Fig. S1 illustrates the differences in energy consumption between each cellobiose-fermenting S. cerevisiae strain according to the types of cellobiose transport and intracellular degradation.

Determination of growth kinetic parameters of the cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-2
Growth kinetic parameters, such as Monod constant (K S ) and maximum specific growth rate (μ max ), of the cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-2 (D-BT2m and D-CT2m strains) under cellobiose conditions were determined as follows. Yeast cells at the exponential growth phase in pre-cultivation were harvested, washed twice with sterilized water, and inoculated into 50 mL of minimal (SC) medium with different initial concentrations of cellobiose (0 to 2 g/L) at an initial OD600 of 0.05. The culture was carried out at 30°C and 100 rpm, and the OD values of the culture broth were checked every 2 h. The growth kinetic parameters of D-BT2m and D-CT2m strains were determined by non-linear regression of the 3 plots for the specific growth rates over various concentrations of the initial cellobiose. All cell culture experiments were performed in triplicate. cerevisiae is considered to be insignificant during SSF contaminated by lactic acid bacteria. cerevisiae with extracellular β-glucosidase; white circle, ○); D-BT1m (the hydrolytic S. cerevisiae expressing mutant CDT-1 and GH1-1; grey triangle, ▲); D-CT2m (the phosphorolytic S. cerevisiae expressing mutant CDT-2 and CBP; black square, ■). Celluclast 1.5L (10 FPU/g cellulose) was used for saccharification of cellulose. In SSF with D-56+188, Novozyme 188 (5.4 CBU/g cellulose) was added along with Celluclast 1.5L for degradation of cellobiose to glucose. Ethanol concentration was measured in three independent experiments, and the symbols in the figure indicate average values with standard deviations.