Engineering yeasts for xylose metabolism
Introduction
Rising concerns over the cost of petroleum and the prospect of global warming are driving the development of technologies for the production of alternative fuels such as ethanol [1]. The long-term source of ethanol is from plant matter (biomass) through the fermentation of biomass carbohydrates to ethanol. The conversion of biomass to useable energy is not economical, however, unless hemicellulose is used in addition to the cellulose [2]. Xylose is the second most abundant carbohydrate in nature and its commercial fermentation to ethanol could provide an alternative fuel source for the future.
Microbes such as yeasts and bacteria are essential for the fermentation of xylose [3, 4]. The larger sizes, thicker cell walls, better growth at low pH, less stringent nutritional requirements, and greater resistance to contamination give yeasts advantages over bacteria for commercial fermentations. Saccharomyces cerevisiae does not naturally use xylose as a substrate, however, and must be engineered to both transport and ferment xylose. Engineering can also improve the fermentative activities of some native xylose-metabolizing yeasts such as Pichia stipitis. A number of different approaches have been used to engineer yeasts for this purpose, including modeling, flux analysis and expression analysis followed by the targeted deletion or altered expression of key genes. In this review we consider some of the different approaches used to engineer yeast for xylose metabolism and discuss recent advances in this area.
The pentose phosphate pathway (PPP), which is the biochemical route for xylose metabolism, is found in virtually all cellular organisms where it provides d-ribose for nucleic acid biosynthesis, d-erythrose 4-phosphate for the synthesis of aromatic amino acids and NADPH for anabolic reactions. The PPP is thought of as having two phases. The oxidative phase converts the hexose, d-glucose 6P, into the pentose, d-ribulose 5P, plus CO2 and NADPH. The non-oxidative phase converts d-ribulose 5P into d-ribose 5P, d-xylulose 5P, d-sedoheptulose 7P, d-erythrose 4P, d-fructose 6P and d-glyceraldehyde 3P. d-Xylose and l-arabinose enter the PPP through d-xylulose (Figure 1). In bacteria the conversion of d-xylose to d-xylulose goes by way of xylose isomerase (xylA). In yeasts, filamentous fungi and other eukaryotes, this proceeds via a two-step reduction and oxidation, which are mediated by xylose reductase (XYL1, Xyl1p) and xylitol dehydrogenase (XYL2, Xyl2p), respectively. The cofactor requirements of these two reactions affect cellular demands for oxygen, as explained in the text.
Section snippets
Modeling
Metabolic engineering is most effective when guided by systematic biochemical models to integrate the intrinsic variables and extrinsic changes. Models basically consist of biochemical pathways and information about cell physiology. In one example, models were used to identify the reactions that could be targeted to alleviate the problems associated with excess NADH accumulation during growth on xylose. Recombinant S. cerevisiae engineered to express the xylose reductase (PsXYL1) and xylitol
Flux analysis
Flux balances can be used to calculate the partitioning of metabolites among the various pathways using stoichiometric metabolic matrices based on known reactions. Flux balances show the fraction of each metabolite flowing into various branches through the pathway and the rates at each branch point are derived from the overall rates of substrate uptake, cell growth and product formation [9]. Fractional 13C labeling has been employed to measure the steady-state concentrations of intermediate
Expression analysis
The main challenge of global expression analysis using microarray technologies is analyzing the results. S. cerevisiae YSX3 grown on glucose or xylose under aerobic or oxygen-limited conditions showed significant changes in levels of more than 600 transcripts [13••]. Following gene classification by function [14], changes were most apparent in the 165 genes associated with energy production. Transcripts associated with the tricarboxylic acid (TCA) cycle and respiration were often at their
Xylose transport
S. cerevisiae takes up xylose through its glucose transporters even though their affinity for this sugar is very low. Moreover, competition with glucose restricts xylose assimilation [17], so heterologous expression of a specific xylose transporter could be very useful. Nobre and Lucas deposited the sequence of a putative xylose permease from Debaryomyces hansenii in the Protein Data Bank (PDB) in 2003, but it was not characterized. Candida intermedia possesses glucose-repressible,
Other approaches to alleviate redox imbalance
To alleviate redox imbalance, Jeppsson et al. [21•] expressed a mutated Xyl1p that has a higher Km for NADPH than the wild-type enzyme. This increased ethanol yield to 0.4 g/g xylose and decreased xylitol production. The endogenous xylose reductase of S. cerevisiae (GRE3) increased xylitol production from xylose in the presence of Xyl1p; in the absence of Xyl1p it increased xylose uptake [22]. Watanabe et al. [23•] increased the thermostability and changed the coenzyme specificity of Xyl1p from
Evolutionary adaptation and anaerobic growth
The anaerobic rumen fungus Piromyces sp. E2 reportedly uses xylose isomerase (XylA) for the assimilation of xylose [26]. The cloned gene from Piromyces sp. ATCC 76762 shows very close identity to xylA of the intestinal bacteria Bacteroides thetaiotamicron and Bacteroides fragilis. When the clone was expressed in S. cerevisiae, transformants grew and produced ethanol slowly (specific growth rate 0.005 h−1). After strain selection and engineering the growth rate on xylose increased to 0.09 h−1, and
Other PPP enzymes
Jin et al. [35] transformed a P. stipitis gene library into a recombinant S. cerevisiae strain carrying P. stipitis XYL1 and XYL2. Of the 16 transformants recovered that grew on xylose, 10 carried vectors with XYL3, thereby showing that this activity is essential for xylose utilisation. A second round of transformation obtained 15 fast-growing transformants, all of which carried P. stipitis TAL1. Unlike overexpression of S. cerevisiae TAL1, P. stipitis TAL1 did not cause growth inhibition on
Xylanase and cellulase expression
Most xylanases produce xylobiose and xylotriose as the major oligosaccharides, and exocellobiohydrolases produce cellobiose. Simultaneous saccharification and fermentation must therefore use yeasts that assimilate these oligomers. The native xylose-fermenting yeast P. stipitis has genes for seven β-glucosidases and one endoxylanase, facilitating oligosaccharide utilization for this organism (see the JGI P. stipitis genome portal: www.jgi.doe.gov). Amino acid supplements enhance the expression
Novel xylose-fermenting yeasts
P. stipitis, Pichia segobiensis, Candida shehatae, Pachysolen tannophilus and a few other yeasts constitute a small group that will ferment xylose directly to ethanol. Although they are still poorly understood, work by Suh, Blackwell and others has greatly improved knowledge of their natural origins by isolating more than 650 yeasts from microflora in the hindgut of beetles [45]. Of these, at least 200 were previously characterised, which is equivalent to almost 30% of all the currently
Conclusions
Interest in the use of yeasts for the commercial fermentation of xylose to ethanol is steadily increasing in light of rising petrol prices and concern over global warming. Several approaches have been successfully employed to engineer xylose metabolism. Modeling is a powerful tool for targeting metabolic changes, and many more model-driven attempts to alter the NADH/NADPH balance can be expected. Likewise, expression analysis is a powerful tool, but its retrospective use often reveals more
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
This research was supported in part by NIH grant GM067933-03 to TWJ.
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