title: Engineering towards a complete heterologous cellulase secretome in Yarrowia lipolytica reveals its potential for consolidated bioprocessing.
abstract: BACKGROUND
  Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which then accumulate intracellularly. However, it lacks the suite of cellulolytic enzymes required to break down biomass cellulose and cannot therefore utilize biomass directly as a carbon source. Toward the development of a direct microbial conversion platform for the production of hydrocarbon fuels from cellulosic biomass, the potential for Y. lipolytica to function as a consolidated bioprocessing strain was investigated by first conducting a genomic search and functional testing of its endogenous glycoside hydrolases. Once the range of endogenous enzymes was determined, the critical cellulases from Trichoderma reesei were cloned into Yarrowia.
  
  
  RESULTS
  Initially, work to express T. reesei endoglucanase II (EGII) and cellobiohydrolase (CBH) II in Y. lipolytica resulted in the successful secretion of active enzymes. However, a critical cellulase, T. reesei CBHI, while successfully expressed in and secreted from Yarrowia, showed less than expected enzymatic activity, suggesting an incompatibility (probably at the post-translational level) for its expression in Yarrowia. This result prompted us to evaluate alternative or modified CBHI enzymes. Our subsequent expression of a T. reesei-Talaromyces emersonii (Tr-Te) chimeric CBHI, Chaetomium thermophilum CBHI, and Humicola grisea CBHI demonstrated remarkably improved enzymatic activities. Specifically, the purified chimeric Tr-Te CBHI showed a specific activity on Avicel that is comparable to that of the native T. reesei CBHI. Furthermore, the chimeric Tr-Te CBHI also showed significant synergism with EGII and CBHII in degrading cellulosic substrates, using either mixed supernatants or co-cultures of the corresponding Y. lipolytica transformants. The consortia system approach also allows rational volume mixing of the transformant cultures in accordance with the optimal ratio of cellulases required for efficient degradation of cellulosic substrates.
  
  
  CONCLUSIONS
  Taken together, this work demonstrates the first case of successful expression of a chimeric CBHI with essentially full native activity in Y. lipolytica, and supports the notion that Y. lipolytica strains can be genetically engineered, ultimately by heterologous expression of fungal cellulases and other enzymes, to directly convert lignocellulosic substrates to biofuels.
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Wei
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
authors.forename: Markus
authors.initials: M
authors.lastname: Alahuhta
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
authors.forename: Todd
authors.initials: T
authors.lastname: Vander Wall
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
authors.forename: John O
authors.initials: JO
authors.lastname: Baker
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
authors.forename: Larry E
authors.initials: LE
authors.lastname: Taylor
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Decker
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Himmel
authors.affiliation: National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
keywords: Advanced biofuels; Cell consortia; Cellobiohydrolase I; Cellobiohydrolase II; Cellulase; Endoglucanase II; Heterologous expression; Oleaginous yeast; Yarrowia lipolytica