Genetic Modification of Yeasts
As wild-type strains of Saccharomyces cerevisiae are unable to utilize xylose, many efforts have been made to construct recombinant yeast strains to incorporate xylose fermentation ability to yeast. Given that
S. cerevisiae is able to ferment xylulose to ethanol, it can potentially be metabolically engineered to ferment xylose to ethanol by the introduction of genes encoding the enzymes xylose reductase (XR) and xylitol dehydrogenase (XDH), which are present in the natural xylose-utilizing yeast Pichia stipitis [24]. In 1998, Ho and coworkers of Purdue University, USA, were able to produce a recombinant S. cerevisiae strain effective in xylose fermentation, as well as capable of co-fermentation of a xylose and glucose mixture [25]. The recombinant plasmids with XR and XDH genes from P. stipitis and xyluloki — nase (XKS) gene from S. cerevisiae were transformed into S. cerevisiae for the co-fermentation of glucose and xylose. In a contrasting report the overexpression of endogenous XKS from S. cerevisiae was found to inhibit its growth on xylulose [26]. Similarly some other workers have also reported lower consumption of xylose in such strains [27-29]. However, Toivari and coworkers reported successful xylose fermentation to ethanol through over-expression of the endogenous XKS 1 and PsXR and XDH genes [30]. Recently, following similar strategies, improved xylose utilization and high ethanol production have been reported by a number of other research groups [31-33]. Therefore, based on more recent work it can be concluded that the controlled overexpression of the XKS gene in S. cerevisiae enhanced the xylose consumption as well as ethanol production in the recombinant S. cerevisiae. After these initial experimentations in the 1990s a number of research groups have now succeeded in genetic modifications of wild-type yeast strains to produce recombinant yeast strains capable of efficiently co-fermenting mixtures of C-5, C-6 sugars as are found in typical biomass hydrolyzates, and some selected examples of pentose fermenting recombinant yeast strains are shown in Table 8.2.
It is worthwhile to point out that in recent work Ni et al. has identified some interesting spontaneous or chemically-induced mutants of recombinant S. cerevisiae that can overcome the growth inhibition caused by overexpression of ScXKS and PsXKS genes [37]. Improving the intracellular cofactor concentration in S. cerevisiae is another strategy to enhance ethanol yields [38]. Hou and coworkers have studied the impact of over-expression of NADH kinase (encoded by the POS5 gene) on glucose and xylose metabolism in recombinant xylose-utilizing S. cerevisiae [39]. The expression of NADH kinase in cytosol instead of mitochondria redirected the carbon flow from CO2 to ethanol during aerobic growth on glucose, whereas under anaerobic growth the flux directed toward ethanol and acetate fermentation. In this study, Hou and coworkers found
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Table 8.2 Recombinant yeast strains capable of fermenting C-5 xylose sugars and co — fermenting mixtures of C-5, C-6 sugars and their references.
* A — arabinose; G — glucose; Gal — galactose; X — xylose |
that cytosolic NADH kinase appeared to revert these effects during anaerobic metabolism of xylose by channeling carbon flow from ethanol to xylitol [39]. Heterologous expression of a xylose isomer — ase (XI) can also be another approach to enable S. cerevisiae cells to metabolize xylose. In pursuing this approach, Brat and coworkers screened nucleic acid databases for sequences encoding putative xylose isomerases and cloned them to express a highly active xylose isomerase from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae, which resulted in an efficient metabolism of xylose as the only carbon and energy source by recombinant yeast cells [40].
In a real biomass application example, Zho and Xia utilized genetically modified yeast to ferment corn stover hydrolyzates [41]. In this study ethanol production from corn stover hemicellu — losic hydrolyzate was investigated using immobilized recombinant Saccharomyces cerevisiae yeast cells. Detoxification of hemicellulosic hydrolyzate by roto-evaporation and lime neutralization was carried out to remove volatile fermentation inhibitors. All furfural and more than 50% of the acetic acid in the hydrolyzate were removed, meanwhile the xylose concentration was enhanced to 71.8 g/L. The fermentability of the detoxified hydrolyzate was significantly improved using Ca-alginate immobilized cells of recombinant S. cerevisiae (ZU-10). An ethanol concentration of 31.1 g/L and the
corresponding ethanol yield on fermentable sugars of 0.406 g/g were obtained within 72 h in batch fermentation of the detoxified hydrolyzate with immobilized cells; the concentration of ethanol in each batch maintained above 30.1 g/L with the ethanol yield on fermentable sugars over 0.393 g/g. With these experiments Zho and Xia demonstrated the viability of ethanol production from corn stover hydrolyzate using C-5 and C-6 co-fermenting recombinant S. cerevisiae, and the effect of immobilization of this yeast [36].
The time course of ethanol production from detoxified corn stover hemicellulose hydrolyzate by recombinant S. cerevisiae ZU-10 using free and immobilized cells is shown in Figure 8.3. In addition, repeated batch fermentation of immobilized recombinant S. cerevisiae
Glucose
• Xylose
* Ethanol Xylitol
u Glycerol
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Repeated batches Figure 8.4 Repeated batches fermentation of hemicellulose hydrolyzate by immobilized recombinant S. cerevisiae ZU-10 cells: residual xylose (white); ethanol (dashed); ethanol yield (•). Error bars represent the standard deviation. (Reprinted with permission from reference [36]; copyright 2010 Elsevier). |
cells was attempted for ethanol production for five batches, demonstrating the reusability of the immobilized S. cerevisiae ZU-10. The results of the reusability experiment are shown in Figure 8.4.
In another example Carlos Martin et al. used recombinant xyloseutilizing Saccharomyces cerevisiae for ethanol production from enzymatic hydrolyzates of sugarcane bagasse [42]. In their experiments, sugarcane bagasse was first pretreated by steam explosion at 205 and 215°C and hydrolyzed with cellulolytic enzymes. The hydroly — zates were then subjected to enzymatic detoxification by treatment with the phenoloxidase laccase and to chemical detoxification by over-liming. Approximately 80% of the phenolic compounds were specifically removed by the laccase treatment. Over-liming partially removed the phenolic compounds, but also other fermentation inhibitors such as acetic acid, furfural and 5-hydroxymethylfurfu — ral. The resultant hydrolyzates were fermented with the recombinant xylose-utilizing Saccharomyces cerevisiae laboratory strain TMB 3001, a CEN. PK derivative with overexpressed xylulokinase activity and expressing the xylose reductase and xylitol dehydrogenase of Pichia stipitis, and the S. cerevisiae strain ATCC 96581, isolated from spent sulphite liquor from a fermentation plant. They reported that the fermentative performance of the lab strain in undetoxified hydrolyzate was better than the performance of the industrial strain. An almost two-fold increase of the specific productivity of the strain TMB 3001 in the detoxified hydrolyzates compared to the undetoxified hydrolyzates was observed. The ethanol yield in the fermentation of the hydrolyzate detoxified by over-liming was 0.18 g/g dry bagasse, whereas it reached only 0.13 g/g dry bagasse in the undetoxified hydrolyzate. Furthermore, a partial xylose utilization with low xylitol formation was observed with this recombinant yeast strain Saccharomyces cerevisiae TMB 3001 [42].