Yeast metabolic engineering for hemicellulosic ethanol production
Introduction
Bioconversion of lignocellulose to ethanol must occur at high rate, in good yield, and to concentrations that are economically recoverable. While readily achieved with starch, these goals are much more difficult with cellulose and hemicellulose. For cellulose, the major barrier is enzymatic saccharification. For hemicellulose, it is the use of glucose, xylose, mannose, galactose, arabinose, and rhamnose, in the presence of acetic and ferulic acids along with various degradation products from thermochemical pretreatment. While most hexoses are readily phosphorylated as soon as they enter the cell, hemicellulosic sugars must go through several biochemical steps before phosphorylation (Figure 1). Eukarya and bacteria use two distinct pathways each for the assimilation of d-xylose and l-arabinose. Most yeast metabolic engineering for ethanol production from xylose has focused on improving sugar uptake and the initial assimilation steps.
Section snippets
Xylose transport
Saccharomyces cerevisiae takes up xylose poorly owing to low affinity of its native nonspecific hexose-transport system for xylose, which are around 130–880 mm, or about 10–100 times higher than for glucose (Table 1) [1•]. Moreover, native transporters in S. cerevisiae are not properly regulated to facilitate xylose uptake [2•]. A recent metabolic model that combines induction of sugar transporters with the kinetic characteristics of the various proteins predicts that xylose transport by S.
Engineering xylose isomerase
The eukaryotic pathways for xylose and arabinose metabolism use oxidoreductases coupled to different cofactor requirements while the corresponding bacterial pathways use isomerases [4•]. XI avoids the cofactor imbalance that could impede metabolite flux, but at equilibrium xylose is clearly favored over xylulose. Homologs of bacterial XI from the anaerobic fungi, Orpinomyces and Piromyces have been successfully expressed in S. cerevisiae after 25 years of attempts in various laboratories [4•,
Engineering XR and XDH
Different requirements for XR and XDH activities (NADPH versus NAD) can lead to cofactor imbalances if routes are not available for their regeneration [10••, 13, 22••, 23••]. Xylitol accumulation clearly results from lower XK, XI, and alcohol dehydrogenase (ADH) activities. All three of these changes impede flux, and the latter two also affect redox balances, but none directly affect the cofactor imbalance between NADPH demand and NADH supply. Altering the cofactor specificity for XR and/or XDH
Other modifications
Overexpression of TAL1 or downregulation of PHO13 enables growth of S. cerevisiae that has been over engineered for XK expression [20]. Deleting pho13 dramatically increases the capacities of engineered S. cerevisiae to grow on and ferment xylose [21]. Optimal expression of genes downstream of XK likewise increases ethanol production. In an XI strain, overexpressing several PPP genes significantly improved the growth rate [4•, 10••]. Protoplast fusion between a thermotolerant S. cerevisiae and
‘Omic’ approaches to strain improvement
Transcriptome analysis of recombinant S. cerevisiae strains showing enhanced growth on xylose were examined for genes with altered expression levels in four yeasts. Out of 13 genes showing common changes, 5 proved advantageous. Upregulation of SOL3 and TAL1 and downregulation of YLR042C, MNI1, and RPA49 each increased growth on xylose [34••]. The transcriptomes and proteomes of recombinant S. cerevisiae cultivated on xylose are not identical to those of cells grown on glucose or to those from
Pichia stipitis and other yeasts
Completion of the P. stipitis genome has revealed the presence of seven β-glucosidases, three endoglucanases, mannanases, xylanases, and numerous sugar transporters in this native xylose fermenting yeast [37••]. A cluster of genes coding for a novel l-rhamnose dehydrogenase degradation pathway was demonstrated [38, 39, 40], along with gene clusters for cellulose, maltose, galactose, urea, and iron metabolism [41]. Resequencing of the P. stipitis FPL-SHI21 (cyc1) strain, which was isolated after
Conclusions
Despite the efforts of several excellent research groups, development of yeast strains with sufficient activity and resilience to ferment hemicellulose hydrolysates remains elusive, but we have seen rapid progress resulting from biochemical and metabolic engineering guided by genomic and transcriptomic studies.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgement
The authors acknowledge funding in support of JHV from the USDA, CSREES NRI project #2006-355-04-17436 and from the Department of Energy sponsored Great Lakes Bioenergy Research Center (GLBRC).
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