Analysis of cellodextrin transporters from Neurospora crassa in Saccharomyces cerevisiae for cellobiose fermentation

• Published in: Applied microbiology and biotechnology 2014-02, Vol.98 (3), p.1087-1094
• Main Authors: Kim, Heejin, Lee, Won-Heong, Galazka, Jonathan M, Cate, Jamie H. D, Jin, Yong-Su
• Format: Article
• Language: English
• Subjects: aeration; Amino acids; beta-glucosidase; Biomass energy; Biomedical and Life Sciences; Biotechnological Products and Process Engineering; Biotechnology; Brewer's yeast; cellobiose; Cellobiose - metabolism; Cellulase; Cellulose; Cellulose - analogs & derivatives; Cellulose - metabolism; Dextrins; Dextrins - metabolism; endo-1,4-beta-glucanase; engineering; Enzymes; Ethanol; Experiments; Fermentation; fungi; Genetic aspects; Glucose; hydrolysis; Life Sciences; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Microbial Genetics and Genomics; Microbiology; Neurospora; Neurospora crassa; Neurospora crassa - enzymology; Neurospora crassa - genetics; Plasmids; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Studies; transporters; Yeast
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-013-5339-2

Saccharomyces cerevisiae can be engineered to ferment cellodextrins produced by cellulases as a product of cellulose hydrolysis. Direct fermentation of cellodextrins instead of glucose is advantageous because glucose inhibits cellulase activity and represses the fermentation of non-glucose sugars present in cellulosic hydrolyzates. To facilitate cellodextrin utilization by S. cerevisiae, a fungal cellodextrin-utilizing pathway from Neurospora crassa consisting of a cellodextrin transporter and a cellodextrin hydrolase has been introduced into S. cerevisiae. Two cellodextrin transporters (CDT-1 and CDT-2) were previously identified in N. crassa, but their kinetic properties and efficiency for cellobiose fermentation have not been studied in detail. In this study, CDT-1 and CDT-2, which are hypothesized to transport cellodextrin with distinct mechanisms, were introduced into S. cerevisiae along with an intracellular β-glucosidase (GH1-1). Cellobiose transport assays with the resulting strains indicated that CDT-1 is a proton symporter while CDT-2 is a simple facilitator. A strain expressing CDT-1 and GH1-1 (DCDT-1G) showed faster cellobiose fermentation than the strain expressing CDT-2 and GH1-1 (DCDT-2G) under various culture conditions with different medium compositions and aeration levels. While CDT-2 is expected to have energetic benefits, the expression levels and kinetic properties of CDT-1 in S. cerevisiae appears to be optimum for cellobiose fermentation. These results suggest CDT-1 is a more effective cellobiose transporter than CDT-2 for engineering S. cerevisiae to ferment cellobiose.