Enhanced cellobiose fermentation by engineered Saccharomyces cerevisiae expressing a mutant cellodextrin facilitator and cellobiose phosphorylase

• Published in: Journal of biotechnology Vol. 275; pp. 53 - 59
• Main Authors: Kim, Heejin, Oh, Eun Joong, Lane, Stephan Thomas, Lee, Won-Heong, Cate, Jamie H.D., Jin, Yong-Su
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 10.06.2018
• Subjects: Cellobiose - chemistry; Cellobiose fermentation; Cellobiose phosphorylase; Cellodextrin transporter; Cellulose - analogs & derivatives; Cellulose - metabolism; Cerevisiae; Dextrins - metabolism; Fermentation; Glucosyltransferases - genetics; Glucosyltransferases - metabolism; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Metabolic Engineering; Recombinant Proteins; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - metabolism; Cellobiose fermentation; Cellobiose phosphorylase; Cellodextrin transporter; Cerevisiae
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2018.04.008

•Laboratory evolution was used to improve cellobiose facilitator.•A mutation near predicted substrate binding site improved cellobiose fermentation.•Mutant transporter with phosphorylase allow efficient cellobiose utilization.•Energy-saving cellobiose pathway is advantageous for cellobiose fermentation. To efficiently ferment intermediate cellodextrins released during cellulose hydrolysis, Saccharomyces cerevisiae has been engineered by introduction of a heterologous cellodextrin utilizing pathway consisting of a cellodextrin transporter and either an intracellular β-glucosidase or a cellobiose phosphorylase. Among two types of cellodextrin transporters, the passive facilitator CDT-2 has not enabled better cellobiose fermentation than the active transporter CDT-1, which suggests that the CDT-2 might be engineered to provide energetic benefits over the active transporter in cellobiose fermentation. We attempted to improve cellobiose transporting activity of CDT-2 through laboratory evolution. Nine rounds of a serial subculture of S. cerevisiae expressing CDT-2 and cellobiose phosphorylase on cellobiose led to the isolation of an evolved strain capable of fermenting cellobiose to ethanol 10-fold faster than the original strain. After sequence analysis of the isolated CDT-2, a single point mutation on CDT-2 (N306I) was revealed to be responsible for enhanced cellobiose fermentation. Also, the engineered strain expressing the mutant CDT-2 with cellobiose phosphorylase showed a higher ethanol yield than the engineered strain expressing CDT-1 and intracellular β-glucosidase under anaerobic conditions, suggesting that CDT-2 coupled with cellobiose phosphorylase may be better choices for efficient production of cellulosic ethanol with the engineered yeast.