Construction of a trifunctional cellulase and expression in Saccharomyces cerevisiae using a fusion protein.

BACKGROUND: Cellulose is the most important component of lignocellulose, and its degradation requires three different types of enzymes to act synergistically. There have been reports of single gene duality, but no gene has been described to have more than two functions. Cloning and expression of fusion cellulases containing more than two kinds of catalytic domains has not been reported thus far. RESULTS: We synthesized three different cellulase genes and linked the three catalytic domains with a (G4S)3 flexible linker. The trifunctional cellulase gene (BCE) containing three types of cellulase functions was constructed and expressed in S. cerevisiae successfully. The ß-glucosidase, the exoglucanase and the endoglucanase activity of the trifunctional cellulase BCE reached 16.80 IU/mg, 2.26 IU/mg and 20.67 IU/mg, which was 46.27, 6.73 and 46.20% higher than the activities of the ß-glucosidase BG, the endoglucanase CBH and the endoglucanase EG. The filter paper enzyme activity of BCE was higher than those of BG, CBH and EG, reached 2.04 IU/mg. CONCLUSIONS: The trifunctional cellulase BCE was designed based on ß-glucosidase BG, endoglucanase EG and exoglucanase CBH, and it possessed ß-glucosidase activity, endoglucanase activity and exoglucanase activity simultaneously. The BCE has better filter paper activity, it means the potential practical application.

Construction of recombinant Yarrowia lipolytica and its application in bio-transformation of lignocellulose.

Lignocellulose is a polysaccharide and an abundant biomass resource that widely exists in grains, beans, rice, and their by-products. Over 10 million tons of lignocellulose resources and processing products are produced every year in China. Three recombinant Y. lipolytica strains with cellulase (ß-glucosidase, endoglucanase and cellobiohydrolase) were constructed. The enzymatic activities of these enzymes were 14.181 U/mL, 16.307 U/mL, and 17.391 U/mL, respectively. The whole cell cellulases were used for a stover bio-transformation. The celluloses in the stover were partly degraded by the cellulases, and the degradation products were transformed into single cell protein (SCP) by the Y. lipolytica cells. After 15 d of fermentation with the whole cell cellulases, the protein content of the maize stover and the rice straw reached 16.23% and 14.75%, which increased by 168.26% and 161.52% compared with the control, respectively. This study provides a new stage for the efficient utilization of stover in the feed industry.

Synergistic effect of cellulase and xylanase during hydrolysis of natural lignocellulosic substrates.

Synergistic combination of cellulase and xylanase has been performed on pre-treated substrates in many previous studies, while few on natural substrates. In this study, three unpretreated lignocellulosic substrates were studied, including corncob, corn stover, and rice straw. The results indicated that when the mixed cellulase and xylanase were applied, reducing sugar concentrations were calculated as 19.53, 15.56, and 17.35mg/ml, respectively, based on the 3,5 dinitrosalicylic acid (DNS) method. Compared to the treatment with only cellulose, the hydrolysis yields caused by mixed cellulase and xylanase were improved by 133%, 164%, and 545%, respectively. In addition, the conversion yield of corncob, corn stover, and rice straw by cellulase-xylanase co-treatment reached 43.9%, 48.5%, and 40.2%, respectively, based on HPLC analysis, which confirmed the synergistic effect of cellulase-xylanase that was much higher than either of the single enzyme treatment. The substrate morphology was also evaluated to explore the synergistic mechanism of cellulase-xylanase.

Simultaneous saccharification and fermentation of corncobs with genetically modified Saccharomyces cerevisiae and characterization of their microstructure during hydrolysis.

Cellulose is an abundant natural polysaccharide that is universally distributed. It can be extracted from corncobs, which are inexpensive, easily accessible, renewable, and environmentally friendly. A common strategy for effectively utilizing cellulose is efficient heterogeneous expression of cellulase genes in Saccharomyces cerevisiae. However, the improvement of cellulose utilization is a relevant issue. Based on our previous findings, we constructed an integrated secretion expression vector, pHBM368-pgk, containing a constitutive promoter sequence. Three genetically modified S. cerevisiae strains containing heterologous ß-glucosidase, exoglucanase, and endoglucanase genes were constructed. The results of a 1-L bioreactor fermentation process revealed that the mixed recombinant S. cerevisiae could efficiently carry out simultaneous saccharification and fermentation (SSF) by using corncobs as the sole carbon source. The ethanol concentration reached 6.37 g/L after 96 hours of fermentation, which was about 3 times higher than that produced by genetically modified S. cerevisiae with the inducible promoter sequence. To investigate the microstructure characteristics of hydrolyzed corncobs during the fermentation process, corncob residues were detected by using a scanning electron microscope. This study provides a feasible method to improve the effect of SSF using corncobs as the sole carbon source.