Two new native ß-glucosidases from Clavispora NRRL Y-50464 confer its dual function as cellobiose fermenting ethanologenic yeast

Authors: WANG, XU - Sichuan Agricultural University; Liu, Zonglin; WEBER, SCOTT - Former ARS Employee; ZHANG, XIAOPING - Sichuan Agricultural University

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/25/2016
Publication Date: 3/24/2016
Citation: Wang, X., Liu, Z.L., Weber, S.A., Zhang, X. 2016. Two new native ß-glucosidases from Clavispora NRRL Y-50464 confer its dual function as cellobiose fermenting ethanologenic yeast. PLoS One. 11(3):e0151293. doi: 10.1371/journal.pone.0151293.

Interpretive Summary: For renewable cellulosic ethanol production from lignocellulosic materials, it is necessary to supply an external source of the enzyme ß-glucosidase in order to break down cellulose into fermentable sugar since most ethanologenic agents are unable to perform this terminal cellulolytic function in an economic simulteneous saccharification and fermentation (SSF) process. ARS scientists previously reported a yeast strain Clavispora NRRL Y-50464 that produces a ß-glucosidase for cellulose-to-ethanol conversion without addition of the external enzyme. In this research, ARS scientists reported new findings on the cloning and characterization of two additional ß-glucosidases from this strain. Thus, a ß-glucosidase gene family was defined with at least three members in this group for Clavispora NRRL Y-50464. The enzymes were tolerant to higher temperatures and the fermentation inhibitors that facilitate lower-cost SSF and near consolidated bioprocess (CBP). In addition to cellobiose, they were also able to hydrolyze another 13 oligosaccharides as a substrate. One enzyme also displayed tolerance to ethanol up to 16% but both enzymes were inhibited by copper. New knowledge obtained from this research aids understanding mechanisms of the unique functions of strain NRRL Y-50464 that benefit research on the next-generation biocatalyst development for advanced biofuels production.

Technical Abstract: Clavispora NRRL Y-50464, a dual functional cellobiose fermenting and ethanologenic yeast strain, is a candidate biocatalyst for lower cost lignocellulose-to-ethanol production using simultaneous saccharification and fermentation. A ß-glucosidase BGL1 protein from this strain was recently reported and confirmed its function in the hydrolysis and utilization of cellobiose. Here, we report cloning and characterization of two additional ß-glucosidase genes encoding enzymes designated as BGL2 and BGL3 from strain Y-50464. The function of BGL2 and BGL3 was confirmed by heterologous expression in a Pichia strain using cellobiose as a sole carbon source and quantitative mRNA transcript analysis. A partially purified protein from each gene clone was obtained and a molecular weight was estimated at approximately 88.3 and 92.5 kD for BGL2 and BGL3, respectively. Both proteins showed the highest specific activity at pH 5. BGL2 displayed tolerance to temperatures from 40-60°C and BGL3 preferred a temperature at 55°C. Both proteins were able to hydrolyze 14 oligosaccharides evaluated in this study. They also showed little inhibition by glucose. While BGL2 tolerated ethanol up to 16%, BGL3 was highly sensitive to ethanol at 4%. BGL2 and BGL3 were active in the present of the fermentation inhibitors furfural and HMF, but inhibited by copper. Our results suggest that a ß-glucosidase gene family exists in Clavispora NRRL Y-50464 with at least three members in this group. This research aids understanding mechanisms of this cellobiose fermenting and ethanologenic yeast for cellulosic ethanol production, and development of the next-generation biocatalyst and consolidated bioprocess toward potential industrial applications.