Engineering Cellulolytic Ability into Process Organisms

The yeast Saccharomyces cerevisiae has long been employed for the industrial production of ethanol (Kuyper et al. 2005; Van Dijken et al. 2000). Attributes that make it suitable for industrial ethanol production include a high rate of ethanol production from glucose (3.3 g/L/h), high ethanol tolerance, and its GRAS status. However, this yeast species has a number of shortcomings in terms of a CBP pro­cessing organism such as its inability to hydrolyze cellulose and hemicellulose or utilize pentose sugars available in lignocellulosic biomass. A number of research groups have been working on improving the substrate range of S. cerevisiae through genetic engineering to include the monomeric forms of sugars contained in plant biomass including xylose (Hahn-Hagerdal et al. 2007; Kuyper et al. 2005), arabinose (Karhumaa et al. 2006) and cellobiose (van Rooyen et al. 2005). There have been many reports detailing the expression of one or more cellulase or hemicellulose encoding gene(s) in S. cerevisiae (Van Zyl et al. 2007). Strains of S. cerevisiae were created that could grow on and ferment cellobiose, the main product of the action of cellobiohydrolases, at roughly the same rate as on glucose in anaerobic conditions (van Rooyen et al. 2005). Recently the high affinity cellodextrin transport system of the model cellulolytic fungus Neurospora crassa was reconstituted into S. cerevisiae (Galazka et al. 2010) leading to growth of a recombinant strain also producing an intracellular b-glucosidase on cellodextrins up to cellotetraose. Subsequently, strains of S. cerevisiae were engineered to co-ferment mixtures of xylose and cellobiose (Ha et al. 2011). A xylose fermenting strain was engineered to also produce a high — affinity cellodextrin transporter and an intracellular b-glucosidase to hydrolyze cellobiose. It was shown that intracellular hydrolysis of cellobiose minimized glu­cose repression of xylose fermentation allowing co-consumption of cellobiose and xylose that improved ethanol yields. This was partly due to circumventing the competition between xylose and glucose for transport into the cell. Sadie et al. (2011) recently showed that expression of the gene encoding lactose permease of Kluy — veromyces lactis (lac 12) also facilitated transport of cellobiose into a recombinant S. cerevisiae strain. This report further showed the successful expression of a Clostridium stercorarium cellobiose phosphorylase (cepA) and that strains co-pro­ducing the heterologous CepA and Lac12 were able to grow on cellobiose as sole carbohydrate source.

There have also been reports showing production of cellulases in S. cerevisiae specifically with the aim of enabling the organism to grow on a polymeric sub­strate. Cho et al. (1999) showed that for SSF experiments with a strain co-pro­ducing a b-glucosidase and an exo/endocellulase activity, loadings of externally added cellulase could be reduced. Fujita et al. (2002, 2004) reported co-expression and surface display of cellulases in S. cerevisiae and high cell density suspensions of a strain displaying the T. reesei endoglucanase II, cellobiohydrolase II, and the Aspergillus aculeatus b-glucosidase were able to directly convert 10 g/L phos­phoric acid swollen cellulose (PASC) into approximately 3 g/L ethanol. An S. cerevisiae strain co-expressing the T. reesei endoglucanase 1 (cel7B) and the S. fibuligera b-glucosidase 1 (cel3A) was able to grow on and convert 10 g/L PASC into ethanol up to 1.0 g/L (Den Haan et al. 2007). Jeon et al. (2009) constructed a similar strain that produced significantly more endoglucanase activity than the strain reported by Den Haan et al. (2007) and notably improved conversion of PASC into ethanol was achieved. When the processive endoglu- canase Cel9A of the moderately thermophilic actinomycete Thermobifida fusca was functionally produced in S. cerevisiae growth of the strain expressing only this one cellulase encoding gene could be demonstrated on media containing PASC due to a sufficient amount of glucose cleaved from the cellulose chain (van Wyk et al. 2010). It was shown that the enzyme released cellobiose and glucose from cellulosic substrates in a ratio of approximately 2.5:1. In an effort to construct an engineered yeast with efficient cellulose degradation, Yamada et al. (2010) developed a method, cocktail delta(d)-integration to optimize cellulase expression levels. Different cellulase expression cassettes encoding b-glucosidase, endoglu — canase, or cellobiohydrolase, were integrated into yeast chromosomes in one step, and strains expressing an optimum ratio of these cellulases were selected for by growth on media containing PASC as carbon source. Although the total integrated gene copy numbers of an efficient cocktail d-integrant strain was about half that of a conventional d-integrant strain, the PASC degradation activity (64.9 mU/g-wet cell) was higher than that of a conventional strain (57.6 mU/g-wet cell) suggesting that optimization of the cellulase expression ratio improved PASC degradation activity more than overexpression. Matano et al. (2012) enhanced cellulase activities on a recombinant S. cerevisiae yeast cell surface displaying T. reesei

EG2 and CBH2 and A. aculeatus BGL1 by additionally integrating eg2 and cbh2 genes into the recombinant strain. As a result, a high ethanol titer (43.1 g/L) was produced from high-solid (200 g-dry weight/L) pretreated rice straw by per­forming a 2-h liquefaction and subsequent 72-h fermentation in the presence of 10 FPU/g-biomass added cellulase. Ethanol yield from the cellulosic material by the recombinant strain reached 89 % of the theoretical yield, which was 1.4-fold higher than the strain without additional gene copies.

As exoglucanase activity is required for the successful hydrolysis of crystalline cellulose, the addition of successful, high level expression of a cellobiohydrolases to these strains should enable conversion of crystalline cellulose into ethanol. While there have been reports of successful expression of cellobiohydrolase encoding genes in S. cerevisiae the titers achieved were generally low (Ilmen et al.

2011) . Recently the expression of relatively high levels of exoglucanases in S. cerevisiae was reported for the first time (Ilmen et al. 2011; Mcbride et al.

2012) . Ilmen et al. (2011) reported a large increase in the maximum titer achieved for two critical exocellulases: Cel6A (CBH1) and Cel7A (CBH2). The cellulase expression levels achieved in this study meets the calculated levels for growth on cellulose at rates required for an industrial process (Olson et al. 2012). Using these exoglucanases, a yeast strain was constructed that was able to convert most of the glucan available in paper sludge into ethanol (Mcbride et al. 2012). The strain was also able to displace 60 % of the enzymes required to convert the sugars available in pretreated hardwood into ethanol in an SSF configuration. A similar strain expressing three alternative cellulases produced ethanol in one step from pre­treated corn stover without the addition of exogenously produced enzymes fer­menting 63 % of the cellulose in 96 h to 2.6 % (v/v) ethanol (Khramtsov et al.

2011). These results demonstrate that cellulolytic S. cerevisiae strains can be used as a platform for developing an economical advanced biofuel process.

As it has been shown that the close proximity of multiple enzymes on the cell surface enables synergistic hydrolysis of lignocellulosic materials, several groups have attempted to reconstruct a minicellulosome on the S. cerevisiae cell surface (Ito et al. 2009; Lilly et al. 2009; Tsai et al. 2009; Wen et al. 2010). Ito et al. (2009) constructed a chimeric scaffoldin to allow cell surface display of both T. reesei EG2 and A. aculeatus BGL1, yielding yeast strains capable of hydrolyzing b-glucan. S. cerevisiae strains were also engineered to display a trifunctional minicellulosome consisting of a mini-scaffoldin containing a cellulose binding domain and three cohesin modules, anchored to the cell surface and three types of cellulases, EG2 and CBH2 originating from T. reesei, and BGL1 from A. acule­atus, each bearing a C-terminal dockerin (Wen et al. 2010). This strain was able to break down and ferment PASC to ethanol with a titer of 1.8 g/L. Tsai et al. (2010) engineered yeast strains capable of displaying a trifunctional scaffoldin carrying three divergent cohesin domains originating from C. thermocellum, C. cellulo — lyticum and Ruminococcus flavefaciens. In addition, strains were constructed that secreted one of the three corresponding dockerin-tagged cellulases namely an EG from C. thermocellum, an exoglucanase from C. cellulolyticum, or a BGL from R. flavefaciens. Using a yeast consortium composed of one strain displaying the mini-scaffoldin and three strains secreting the dockerin-tagged cellulases, the secreted cellulases were docked onto the displayed mini-scaffoldin in a predictably organized manner. By adjusting the ratio of different populations in the consor­tium, cellulose hydrolysis and ethanol production was successfully fine-tuned and *30 % of 10 g/L PASC was solubilized in 73 h. Displaying cellulosomal com­ponents on the yeast cell surface was also recently employed to create strains that could convert xylan into ethanol (Sun et al. 2012). These strains displayed mini — hemicellulosomes that consisted of a mini-scaffoldin originating from C. ther- mocellum tethered to the cell surface through the S. cerevisiae a-agglutinin adhesion receptor and up to three enzymes. Up to three types of hemicellulases, an endoxylanase (T. reesei Xyn2), an arabinofuranosidase (Aspergillus niger AbfB), and a b-xylosidase (A. niger XlnD), each with a C-terminal dockerin, were assembled onto the mini-scaffoldin via cohesin-dockerin interactions. The result­ing quaternary trifunctional complexes exhibited an enhanced hydrolysis rate of arabinoxylan over the other configurations. Furthermore, in strains with an inte­grated xylose utilizing pathway, the recombinant yeast displaying a mini-hemi — cellulosome containing the xylanase and xylosidase could simultaneously hydrolyze and ferment birchwood xylan to ethanol although less than 1 g/L eth­anol was produced.

Zymomonas mobilis is a well-known Gram-negative fermenting bacterium that produces ethanol at very high rates and is used to produce some traditional alco­holic beverages (Zhang et al. 1997). However, Z. mobilis cannot ferment or utilize the pentose sugar xylose and it cannot hydrolyze polysaccharides. Zhang et al. (1997) engineered a Z. mobilis strain capable of fermenting the major pentose sugars present in plant material, namely xylose and arabinose. Co-fermentation of 100 g/L sugar (glucose:xylose:arabinose—40:40:20) yielded an ethanol concen­tration of 42 g/L in 48 h. Brestic-Goachet et al. (1989) expressed the Erwinia chrysanthemi cel5Z in Z. mobilis. The maximum endoglucanase activity obtained was 1,000 IU/L with 89 % of the enzyme secreted to the extracellular medium. Expression of the Ruminococcus albus b-glucosidase enabled Z. mobilis to ferment cellobiose to ethanol very efficiently in 2 days and most of the recombinant enzyme was secreted (Yanase et al. 2005). Recently, numerous strains of Z. mobilis were shown to possess native extracellular activities against carboxymethyl cellulose (Linger et al. 2010). Furthermore, two cellulolytic enzymes, E1 and GH12 from Acidothermus cellulolyticus, were produced heterologously as active, soluble enzymes in Z. mobilis. While the E1 enzyme was less abundant, the GH12 enzyme comprised as much as 4.6 % of the total cell protein. Additionally, fusing predicted secretion signals native to Z. mobilis to the N-termini of these enzymes was shown to direct secretion of significant levels of active E1 and GH12 enzymes, though a significant portion of both still resided in the periplasmic space.

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