Simultaneous saccharification and fermentation of cellulose in ionic liquid for efficient production of α-ketoglutaric acid by Yarrowia lipolytica

Abstract

Ionic liquids (ILs) are benign solvents that are highly effective for biomass pretreatment. However, their applications for scale-up biorefinery are limited due to multiple expensive IL recovery and separation steps that are required. To overcome this limitation, it is very critical to develop a compatible enzymatic and microbial biocatalyst system to carry the simultaneous saccharification and fermentation in IL environments (SSF-IL). While enzymatic biocatalysts have been demonstrated to be compatible with various IL environments, it is challenging to develop microbial biocatalysts that can thrive and perform efficient biotransformation under the same conditions (pH and temperature). In this study, we harnessed the robust metabolism of Yarrowia lipolytica as a microbial platform highly compatible with the IL environments such as 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]). We optimized the enzymatic and microbial biocatalyst system using commercial cellulases and demonstrated the capability of Y. lipolytica to convert cellulose into high-value organics such as α-ketoglutaric acid (KGA) in the SSF-IL process at relatively low temperature 28 °C and high pH 6.3. We showed that SSF-IL not only enhanced the enzymatic saccharification but also produced KGA up to 92% of the maximum theoretical yield.

Activating and Elucidating Metabolism of Complex Sugars in Yarrowia lipolytica

“…Plasmid pSL16-CEN1-1-227, containing the CYC1 terminator (T CYC1 ) and leucine selection marker, was kindly provided by M. Matsuoka (Sojo University, Japan) (35). Plasmid pSR001 was created by inserting the Y. lipolytica TEF promoter (P TEF ) (36) in front of T CYC1 of pSL16-CEN1-1-227 as described elsewhere (31). The list of primers used in this study is shown in Table S2.…”

“…Y. lipolytica not only can be harnessed to produce large amounts of intracellular neutral lipids (Ͼ90% of dry cell weight [DCW]) (23,24), oleochemicals (25), food supplements (e.g., omega-3 eicosapentaenoic acid) (26), high-value organics (e.g., citric, ␣-ketoglutaric, succinic, and pyruvic acids), and proteins (e.g., proteases and lipases) (27) but also is capable of assimilating complex substrates (e.g., organic acids, alcohols, triglycerides, and hydrocarbons) (27) as well as of thriving in a wide pH range (pH 2 to 11) (28) and in the presence of inhibitory acid-pretreated biomass hydrolysates (29) or high (Ͼ12% NaCl) salt concentrations (30) or even high (10% [vol/ vol]) concentrations of ionic liquids (31). While native Y. lipolytica has been known for decades to use only some C 6 sugars such as glucose, mannose, and fructose (32), its capability of assimilating other sugars such as xylose and cellobiose and their mixtures with glucose is poorly understood.…”

Exceptional Solvent Tolerance inYarrowia lipolyticaIs Enhanced by Sterols

“…Since wildtype Y. lipolytica can grow in at least 10% (v/v) [EMIM][OAc] (7), we hypothesize that it has a novel endogenous metabolism conferring exceptional IL robustness. To test this, we performed ALE to generate Y. lipolytica mutants with enhanced tolerance to high concentrations of the benchmark IL, [EMIM][OAc] (Fig.…”

“…These solvents are inhibitory because they severely disrupt cell membranes and intracellular processes (21–23). For applications such as simultaneous saccharification and fermentation of IL-pretreated lignocellulosic biomass, microbes that are active in high IL-containing media are desirable because expensive IL washing and recycling steps prior to fermentation are not needed (7, 24). Unfortunately, most industrially-relevant platform organisms, Escherichia coli and Saccharomyces cerevisiae , are severely inhibited in IL-containing media even at low concentrations, for instance, 1% (v/v) [EMIM][OAc] (25, 26).…”

“…Among the non-conventional yeasts, Y. lipolytica is attractive for fundamental study and industrial application due to its oleaginous nature (32, 33) and robustness in extreme environments (20, 34–36). Remarkably, wildtype Y. lipolytica (ATCC MYA-2613) exhibited robust growth in media containing at least 10% (v/v) [EMIM][OAc] while producing 92% maximum theoretical yield of alpha-ketoglutarate from IL-pretreated cellulose (7). Currently, mechanisms of IL toxicity and superior tolerance in microorganisms are not completely elucidated (37).…”