Conferring cellulose-degrading ability to Yarrowia lipolytica to facilitate a consolidated bioprocessing approach

Abstract

Background Yarrowia lipolytica, one of the most widely studied “nonconventional” oleaginous yeast species, is unable to grow on cellulose. Recently, we identified and overexpressed two endogenous β-glucosidases in Y. lipolytica, thus enabling this yeast to use cello-oligosaccharides as a carbon source for growth. Using this engineered yeast platform, we have now gone further toward building a fully cellulolytic Y. lipolytica for use in consolidated bioprocessing of cellulose.ResultsInitially, different essential enzyme components of a cellulase cocktail (i.e,. cellobiohydrolases and endoglucanases) were individually expressed in Y. lipolytica in order to ascertain the viability of the strategy. Accordingly, the Trichoderma reesei endoglucanase I (TrEG I) and II (TrEG II) were secreted as active proteins in Y. lipolytica, with the secretion yield of EG II being twice that of EG I. Characterization of the purified His-tagged recombinant EG proteins (rhTrEGs) revealed that rhTrEG I displayed higher specific activity than rhTrEG II on both cellotriose and insoluble cellulosic substrates, such as Avicel, β-1, 3 glucan, β-1, 4 glucan, and PASC. Similarly, cellobiohydrolases, such as T. reesei CBH I and II (TrCBH I and II), and the CBH I from Neurospora crassa (NcCBH I) were successfully expressed in Y. lipolytica. However, the yield of the expressed TrCBH I was low, so work on this was not pursued. Contrastingly, rhNcCBH I was not only well expressed, but also highly active on PASC and more active on Avicel (0.11 U/mg) than wild-type TrCBH I (0.065 U/mg). Therefore, work was pursued using a combination of NcCBH I and TrCBH II. The quantification of enzyme levels in culture supernatants revealed that the use of a hybrid promoter instead of the primarily used TEF promoter procured four and eight times more NcCBH I and TrCBH II expressions, respectively. Finally, the coexpression of the previously described Y. lipolytica β-glucosidases, the CBH II, and EG I and II from T. reesei, and the N. crassa CBH I procured an engineered Y. lipolytica strain that was able to grow both on model cellulose substrates, such as highly crystalline Avicel, and on industrial cellulose pulp, such as that obtained using an organosolv process.ConclusionsA Y. lipolytica strain coexpressing six cellulolytic enzyme components has been successfully developed. In addition, the results presented show how the recombinant strain can be optimized, for example, using artificial promoters to tailor expression levels. Most significantly, this study has provided a demonstration of how the strain can grow on a sample of industrial cellulose as sole carbon source, thus revealing the feasibility of Yarrowia-based consolidated bioprocess for the production of fuel and chemical precursors. Further, enzyme and strain optimization, coupled to appropriate process design, will undoubtedly lead to much better performances in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0819-8) contains supplementary material, ...

Expressing accessory proteins in cellulolytic Yarrowia lipolytica to improve the conversion yield of recalcitrant cellulose

“…To achieve this, we expressed in a Y. lipolytica strain a range of enzymes including BGLs, EGs, and CBHs, taking care to control the relative proportions of each of these in order to obtain a combination that is quantitatively similar to that of the native cellulase system of T. reesei . The engineered cellulolytic Y. lipolytica was shown to grow efficiently on industrial cellulose pulp (CIMV-cellulose, mostly amorphous), but displayed some difficulty to degrade recalcitrant crystalline cellulose [ 30 ]. Therefore, in the present work, we describe how the cellulolytic Y. lipolytica strain has been further manipulated to increase the hydrolysis of crystalline cellulose and complex substrates.…”

The in vivo detection and measurement of the unfolded protein response in recombinant cellulase producing Saccharomyces cerevisiae strains

“…This can be used as a way of readily revealing the presence of xylanase as shown in a previous study by Guo et al. confirming successful transformation.…”

“…When the UPR is active in the cell, the HAC1 and KAR2 gene promoters are able to sense that and express the reporter genes producing either xylanase ( T.r.xyn2 ) or GFP fluorescence ( eGFP ) depending on the relative transformant. Xylanase activity can be detected on AZCL‐xylan containing SC − URA medium plates . AZCL xylan plates provide a good initial plate screen to determine if the UPR is active in the cell by means of indicating the presence of T.r .Xyn2, which can be observed by blue halos around the colonies (Fig.…”

Optimization of Yarrowia lipolytica-based consolidated biocatalyst through synthetic biology approach: transcription units and signal peptides shuffling

“…Due to unique native metabolic properties, like intrinsically high production of lipids [6], polyols [7], organic acids [8], and recently evidenced superior capacity for heterologous proteins production over a typical workhorse in this regard -Komagataella phaffi (P. pastoris) [9], Y. lipolytica stains are frequently subjected to genetic modifications broadening its scope of utilized substrates, with the aim to improve economic feasibility of production processes [10]. For example, Y. lipolytica strains has been endowed with artificial ability to grow on sucrose [11], inulin [12], galactose [13], starch [14] or even cellulose [15,16]. Utilization of renewable substrates, usually required introduction of several heterologous genes to Y. lipolytica cells, to either efficiently decompose biopolymer, or provide a link between new substrate and native metabolism of the host cell.…”