Enhancement of Galactose Uptake from Kappaphycus alvarezii Hydrolysate Using Saccharomyces cerevisiae Through Overexpression of Leloir Pathway Genes.

A total 42.68 g/L monosaccharide with 0.10 g/L HMF was obtained from 10% (w/v) Kappaphycus alvarezii with thermal acid hydrolysis using 350 mM HNO3 at 121 °C for 60 min and enzymatic saccharification with a 1:1 mixture of Viscozyme L and Celluclast 1.5 L for 72 h. To enhance the galactose utilization rate, fermentation was performed with overexpression of GAL1 (galactokinase), GAL7 (galactose-1-phosphate uridyltransferase), GAL10 (UDP-glucose-4-epimerase), and PGM2 (phosphoglucomutase 2) in Saccharomyces cerevisiae CEN.PK2 using CCW12 as a strong promoter. Among the strains, the overexpression of PGM2 showed twofold high galactose utilization rate (URgal) and produced ethanol 1.4-fold more than that of the control. Transcriptional analysis revealed the increase of PGM2 transcription level leading to enhance glucose-6-phosphate and fructose-6-phosphate and plays a key role in ensuring a higher glycolytic flux in the PGM2 strain. This finding shows particular importance in biofuel production from seaweed because galactose is one of the major monosaccharides in seaweeds such as K. alvarezii.

Enhancement of Galactose Uptake from Kappaphycus alvarezii Using Saccharomyces cerevisiae through Deletion of Negative Regulators of GAL Genes.

This study was aimed at enhancing galactose consumption from the red seaweed Kappaphycus alvarezii. The optimal pretreatment condition of thermal acid hydrolysis was treated with 350 mM HNO3 for 60 min at 121 °C. The enzymatic saccharification with a 1:1 mixture of Celluclast 1.5 L and Viscozyme L showed the maximum yield of glucose; 42-g/L monosaccharide concentration was obtained with the highest yield of pretreatment and enzymatic saccharification (EPS) and the lowest inhibitory compound concentration. The deletion of the GAL80, MIG1, CYC8, or TUP1 gene was performed to improve the galactose consumption rate. The strains with the deletion of the MIG1 gene (mig1Δ) showed higher galactose consumption rate and ethanol yield than other strains. High transcription levels of regulatory genes revealed that the mig1Δ relieved glucose repression. These results show that the mig1Δ enhances galactose consumption rate from K. alvarezii.

Enhancement of bioethanol production from Gracilaria verrucosa by Saccharomyces cerevisiae through the overexpression of SNR84 and PGM2.

A total monosaccharide concentration of 47.0 g/L from 12% (w/v) Gracilaria verrucosa was obtained by hyper thermal acid hydrolysis with 0.2 M HCl at 140°C for 15 min and enzymatic saccharification with CTec2. To improve galactose utilization, we overexpressed two genes, SNR84 and PGM2, in a Saccharomyces cerevisiae CEN-PK2 using CRISPR/Cas-9. The overexpression of both SNR84 and PGM2 improved galactose utilization and ethanol production compared to the overexpression of each gene alone. The overexpression of both SNR84 and PGM2 and of PGM2 and SNR84 singly in S. cerevisiae CEN-PK2 Cas9 produced 20.0, 18.5, and 16.5 g/L ethanol with ethanol yield (YEtOH) values of 0.43, 0.39, and 0.35, respectively. However, S. cerevisiae CEN-PK2 adapted to high concentration of galactose consumed galactose completely and produced 22.0 g/L ethanol at a YEtOH value of 0.47. The overexpression of both SNR84 and PGM2 increased the transcriptional levels of GAL and regulatory genes; however, the transcriptional levels of these genes were lower than those in S. cerevisiae adapted to high galactose concentrations.

Enhancement of galactose consumption rate in Saccharomyces cerevisiae CEN.PK2-1 by CRISPR Cas9 and adaptive evolution for fermentation of Kappaphycus alvarezii hydrolysate.

Ethanol ferrmentation of Kappaphycus alvarezii hydrolysates was performed using wild-type (WT) Saccharomyces cerevisiae CEN.PK2-1, hexokinase 2 deleted (Δhxk2) and adapted strain on high galactose concentrations. The WT and Δhxk2 strains produced 8.9 and 14.67 g/L of ethanol with yield coefficient (YEtOH) of 0.20 and 0.33 (g/g), respectively. However, neither the WT nor Δhxk2strain could utilize all of the galactose, leaving 16.4 and 6.2 g/L of galactose in the fermentation broth, respectively. Therefore, fermentation with S. cerevisiae CEN.PK2-1 adapted to galactose was carried out to increase the ethanol yield coefficient (YEtOH), producing a maximum ethanol concentration of 20.0 g/L with a YEtOH of 0.44 (g/g). Ethanol concentration of adapted strain was 1.36-2.25 times higher than WT and Δhxk2 strains. The adapted yeast exhibited the highest transcript levels of GAL genes. The yeast strain via adaptive yeast strain produced ethanol with a higher titer and yield due to a modular activation of GAL genes than WT or the hxk2 deleted strains.