Screening, cloning, enzymatic properties of a novel thermostable cellulase enzyme, and its potential application on water hyacinth utilization.

Cellulose is the cheapest, natural, renewable organic substance that is used as a carbon source in various fields. Water hyacinth, an aquatic plant rich in cellulose, is often used as a raw material in fuel production. However, natural cellulase can be hardly used in industrial production on account of its low thermal stability and activity. In this study, a metagenomic library was constructed. Then, a new cellulase gene, cel1029, was screened by Congo red staining and expressed in the prokaryotic system. Enzymatic properties of Cel1029 were explored, including optimum temperature and pH, thermal and pH stability, and tolerance against organic solvents, metal ions, and salt solutions. Finally, its ability of degrading water hyacinth was identified and evaluated. Cel1029 displayed high homology with endoglucanase in the glycoside hydrolase family 5 (GH5) and had high stability across a broad temperature range. More than 86% of its enzymatic activities were retained between 4 and 60 °C after 24 h of incubation. Single-factor analysis and orthogonal design were further conducted to determine the optimal conditions for the highest reducing sugar yield of water hyacinth. Interestingly, Cel1029 efficiently transformed water hyacinth with a reducing sugar yield of 430.39 mg/g in 22 h. These findings may open the door for significant industrial applications of a novel GH5 cellulase (NCBI Reference Sequence: MK051001, Cel1029) and help identify more efficient methods to degrade cellulose-rich plants.

Engineering the robustness of Saccharomyces cerevisiae by introducing bifunctional glutathione synthase gene.

Robust, high-yielding Saccharomyces cerevisiae is highly desirable for cost-effective cellulosic ethanol production. In this study, the bifunctional glutathione (GSH) synthetase genes GCSGS at high copy number was integrated into ribosomal DNA of S. cerevisiae by Cre-LoxP system. Threefold higher GSH contents (54.9 µmol/g dry weight) accumulated in the engineered strain BY-G compared to the reference strain. Tolerance of BY-G to H2O2 (3 mM), temperature (40 °C), furfural (10 mM), hydroxymethylfurfural (HMF, 10 mM) and 0.5 mM Cd(2+) increased compared to reference strain. Twofold higher ethanol concentration was obtained by BY-G in simultaneous saccharification and fermentation of corn stover compared to the reference strain. The results showed that intracellular GSH content of S. cerevisiae has an influence on robustness. The strategy is used to engineer S. cerevisiae strains adaptive to a combination of tolerance to inhibitors and raised temperature that may occur in high solid simultaneous saccharification and fermentation of lignocellulosic feedstocks.

Characterization of cattle fecal Streptomyces strains converting cellulose and hemicelluloses into reducing sugars.

To characterize Streptomyces isolated from cattle feces for converting lignocellulose into reducing sugars, five Streptomyces strains were screened. All the strains could convert lignocellulose into reducing sugars. The strain A16 accumulate 3.3-folds more reducing sugars on cottonseed shells treated with ethanol than without the treatment (P < 0.05). The five strains did not accumulate more reducing sugars on rice straws and wheat brans than those on cottonseed shells. Compared with A10 alone, the microbial combination of F1 + A10 accumulated 19, 61, and 25 % less reducing sugars on cottonseed shell, rice straw, and wheat bran than those by A10 solely, respectively (P < 0.05). Further studies indicated that the activities of avicelase and xylanase were not correlated with the reducing sugar amount accumulated by the test strains. Strain A7 could produce more cellular lipids with xylose and glucose as the sole carbon sources. This study shows the potential for Streptomyces strains from herbivore feces to convert lignocelluloses into lipids and reducing sugars for fuel production.