Abstract
The development of lignocellulosic bioethanol plays an important role in the substitution of petrochemical energy and high-value utilization of agricultural wastes. The safe and stable expression of cellulase gene sestc was achieved by applying the clustered regularly interspaced short palindromic repeats-Cas9 approach to the integration of sestc expression cassette containing Agaricus biporus glyceraldehyde-3-phosphate-dehydrogenase gene (gpd) promoter in the Saccharomyces cerevisiae chromosome. The target insertion site was found to be located in the S. cerevisiae hexokinase 2 by designing a gRNA expression vector. The recombinant SESTC protein exhibited a size of approximately 44 kDa in the engineered S. cerevisiae. By using orange peel as the fermentation substrate, the filter paper, endo-1,4-β-glucanase, exo-1,4-β-glucanase activities of the transformants were 1.06, 337.42, and 1.36 U/mL, which were 35.3-fold, 23.03-fold, and 17-fold higher than those from wild-type S. cerevisiae, respectively. After 6 h treatment, approximately 20 g/L glucose was obtained. Under anaerobic conditions the highest ethanol concentration reached 7.53 g/L after 48 h fermentation and was 37.7-fold higher than that of wild-type S. cerevisiae (0.2 g/L). The engineered strains may provide a valuable material for the development of lignocellulosic ethanol.
Highlights
Bioethanol, as a promising alternative to petroleum resources, can be produced from lignocellulosic biomass and starch-rich plants (Kou et al, 2017)
Nearly no glucose was released from the orange peel, as determined by the High-performance liquid chromatography (HPLC) method
The fermentation mixture containing 15 OD600 of the recombinant S. cerevisiae cells, 20 g/L glucose, residual lignocellulose, and culture medium was treated at 30◦C under anaerobic conditions (Figure 5)
Summary
Bioethanol, as a promising alternative to petroleum resources, can be produced from lignocellulosic biomass and starch-rich plants (Kou et al, 2017). The development of lignocellulosic ethanol could be more promising in the future than food crop ethanol considering its sustainable, renewable, and environmentally friendly features (Cai et al, 2016). The conversion of lignocellulosic materials requires three key steps, namely, biomass pretreatment, saccharification, and fermentation (Chang et al, 2013). Pretreatment changes the physical and chemical properties of the raw materials. Saccharification produces fermentable sugars from cellulosic materials via enzymatic degradation, acidolysis, and ionic hydrolysis (Zhao et al, 2018). A highly efficient transformation of lignocellulosic materials requires the integration of these three processing technologies (Koppram et al, 2014)
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