Abstract

BackgroundYarrowia 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.ResultsInitially, 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.ConclusionsTaken 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0148-0) contains supplementary material, which is available to authorized users.

Highlights

  • Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which accumulate intracellularly

  • Morphological observation of Y. lipolytica culture on YPD agar plate The morphology of Y. lipolytica Po1g growing on the surface of yeast peptone dextrose (YPD) medium is illustrated in Figure S1

  • For the transformant Y. lipolytica[CBHII], the cells were tested on carboxymethyl cellulose (CMC) mineral medium, and the results indicated that it generated clear zones on CMC mineral plates, suggesting that the expressed CBHII likely hydrolyzes CMC into soluble sugars

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Summary

Introduction

Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which accumulate intracellularly. It lacks the suite of cellulolytic enzymes required to break down biomass cellulose and cannot utilize biomass directly as a carbon source. Yarrowia lipolytica has become one of the model oleaginous yeasts for the development of biofuels [1,2,3]. Y. lipolytica is known as an oleaginous microorganism that intracellularly accumulates lipids, which could serve as an alternative to plant oils for biodiesel production [4]. Wild-type strains can secrete 1 to 2 g/L of alkaline extracellular protease (XPR2) under optimal physiological conditions [17]

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