Series Editor: T. Scheper

Abstract Industrial biotechnology is the conversion of biomass via biocatalysis, microbial fermentation, or cell culture to produce chemicals, materials, and/or energy. Industrial biotechnology processes aim to be cost-competitive, environmentally favorable, and self­sustaining compared to their petrochemical equivalents. Common to all processes for the production of energy, commodity, added value, or fine chemicals is that raw ma­terials comprise the most significant cost fraction, particularly as operating efficiencies increase through practice and improving technologies. Today, crude petroleum represents the dominant raw material for the energy and chemical sectors worldwide. Within the last 5 years petroleum prices, stability, and supply have increased, decreased, and been threatened, respectively, driving a renewed interest across academic, government, and corporate centers to utilize biomass as an alternative raw material. Specifically, bio-based ethanol as an alternative biofuel has emerged as the single largest biotechnology com­modity, with close to 46 billion L produced worldwide in 2005. Bioethanol is a leading example of how systems biology tools have significantly enhanced metabolic engineer­ing, inverse metabolic engineering, and protein and enzyme engineering strategies. This enhancement stems from method development for measurement, analysis, and data in­tegration of functional genomics, including the transcriptome, proteome, metabolome,

and fluxome. This review will show that future industrial biotechnology process devel­opment will benefit tremendously from the precedent set by bioethanol — that enabling technologies (e. g., systems biology tools) coupled with favorable economic and socio­political driving forces do yield profitable, sustainable, and environmentally responsible processes. Biofuel will continue to be the keystone of any industrial biotechnology-based economy whereby biorefineries leverage common raw materials and unit operations to integrate diverse processes to produce demand-driven product portfolios.

Keywords Bioethanol • Biofuels • Biorefinery • Metabolic engineering • Systems biology

1

Introduction

1.1

The lignin content and type of lignin has a significant effect on the hydro­lysis of various cellulosic substrates as lignin acts as both a physical barrier, restricting access of cellulases to cellulose [68], and as an attractant to cel­lulases, resulting in non-productive binding [69,70]. It has been shown that the chemical and physical structure of lignin plays a significant role in de­termining the magnitude of inhibition it contributes to hydrolysis, and the structure of lignin is heavily dependent on the conditions of the substrate pre­treatment. However, some general observations can be made for substrates treated by specific pretreatments. The main chemical bond linking lignin sub­units is the P-O-4 aryl ether bond [53,54]. As a result, previous work that has examined the structure of lignin in pretreated substrates has mainly observed changes in P-O-4 aryl ethers and the resulting increase in free phenolic groups that occur after P-O-4 cleavage. During SO2-catalyzed steam pretreatment, lignin tends to undergo decreases in both P-5 and P-O-4 aryl ethers [71,72]. Due to the addition of SO2, acid-catalyzed condensation reactions also occur, which are manifested by an increase in the number of aromatics substi­tuted at the C6 [73-75]. It has also been shown that steam pretreatment performed at higher severity results in greater reductions in P-O-4 struc­tures, resulting in more depolymerized lignin and a higher amount of free phenolic groups. Organosolv lignin from a mixed hardwood exhibited sig­nificantly lower amounts of P-O-4 structures than did steam-pretreated lignin from both yellow poplar and aspen [75,76], which is indicative of the greater degree of delignification that occurs during the organosolv process.

Early work showed that exposure of cellulases to soluble lignin obtained from an alkaline organosolv process resulted in reduced hydrolytic activ­ity [77]. Converse et al. [74] reported that the adsorption of cellulases to lignin resulted in decreases in the rate of enzymatic hydrolysis. There have been limited studies investigating the effects of specific lignin functionalities on cellulase activity, however, these studies have concluded that the likelihood of lignin binding cellulases can be linked directly to the presence of specific functional groups. This work is complicated by the fact that subtle changes in pretreatment conditions can result in significant changes in lignin struc­ture [75,76]. Sewalt et al. [78] added powdered lignins to ideal substrates in order to study the effects of lignin structure on cellulose hydrolysis. In the pres­ence of a filter paper substrate and 1.5 mg/mL of lignin, cellulases exhibited reductions in activity of up to 60%. The inhibition by lignin was only moder­ately remedied by increasing the cellulase loading from 5 to 50 FPU/g cellulose, thus indicating that the inhibition resulted from a binding of cellulase to the substrate. It should be noted that these studies added insoluble lignin to the reaction with filter paper, thus the particle sizes of the added lignin should also be considered. The authors concluded that the 6.3% phenolic group content measured in the organosolv lignin compared to 4.3% obtained for the steam- exploded lignin was most likely responsible for the increase in the inhibitory effect of the organosolv lignin. To test this further, the phenolic groups on the added lignin were blocked by hydroxypropylation, which resulted in a virtual elimination of the inhibitory effect of the added lignin.

Sewalt et al. [78] also incubated cellulases with lignin in the absence of substrate, which resulted in a 10-30% decrease in the protein content in the liquid phase, indicating a precipitation with lignin. The authors surmised that the enzyme was bound to lignin, resulting in its deactivation. However, the binding was strictly due to the presence of phenolic groups that mediated the addition of the enzyme to quinone methide groups on lignin. In a recent study, Berlin et al. [18] compared organosolv lignin isolated in the ethanol — soluble stream to the pulp residual lignin isolated by digest by cellulases. Both lignin preparations contained low amounts of P-O-4 and P-5 linkages, in­dicative of their extensive delignification. The dissolved lignin contained 19% more phenolic hydroxyl groups than the isolated residual lignin; however, the residual lignin contained 46% more aliphatic hydroxyl groups and 67% more carboxylic groups. Not surprisingly, the residual lignin was also found to be more condensed than the ethanol-soluble lignin. The incubation of cellulases with the ethanol-soluble lignin, with its higher phenolic content, resulted in a decrease in hydrolytic activity to a greater degree than the enzymatically liberated residual lignin sample. Unlike Sewalt et al, Berlin et al. attributed the difference in the inhibitory effects between the two lignin preparations to the lower amount of carboxylic groups and aliphatic hydroxyl groups of the ethanol-soluble lignin. This may have resulted in a more hydrophobic lignin preparation that was more amenable to hydrophobic interactions with cellu- lases. Unlike previous studies, the particle size of the lignin preparations was considered; however, the amount of cellulases that may have bound to the lignin preparations was not measured. The most likely explanation for the re­sults is that a combination of increased lignin phenolic groups and increased hydrophobicity was responsible for the inhibition of cellulases by the various lignin preparations.

There has also been strong evidence [70, 79] supporting the role of hy­drophobic interactions in the non-productive binding of cellulases to lignin. Multiple studies [70,80,81] have shown that the addition of the surfactant Tween, to cellulolytic hydrolysis improved hydrolysis yields. Similarly, the addition of agents such as BSA (bovine serum albumin) [69,78], gelatin, and PEG (polyethylene glycol) [78] have also reduced the inhibition of cellulases by lignin. It seems safe to assume that during the hydrolysis of lignocellu — losic substrates, the addition of a hydrophobic compound or surfactant to compete with the cellulase proteins for adsorption sites on lignin would re­suit in a reduction in non-productive binding and an increase in hydrolysis performance [79,80]. The surfactant may also facilitate the desorption of cel — lulases that have bound to lignin, similar to the enhancement in cellulase desorption observed during the hydrolysis of pure cellulose substrates in the presence of non-ionic surfactants. Overall, it is apparent that the surfactant, added protein or compound possessing both a hydrophobic and hydrophilic component, aids in reducing the adsorption of cellulases to lignin thereby improving the hydrolysis performance.

Considering the detrimental effect of lignin-enzyme interactions on hydrolysis performance, Berlin et al. [82] introduced a novel approach to enzyme improvement involving a reduction in the affinity of enzymes for lignin rather than an alteration of the substrate. It was shown that natu­rally occurring enzymes with similar catalytic activities tested on “model” substrates such as Avicel and Sigmacell may differ in their interaction with lignin, which may therefore affect performance on the native substrate [82]. As mentioned earlier, Berlin et al. [18] investigated enzyme-lignin interac­tions, and isolated and characterized two lignin preparations from softwood using ethanol organosolv pretreatment. After testing seven different cellulase preparations, three different xylanase preparations and one P-glucosidase preparation, it was shown that the various cellulases differed by up to 3.5- fold in their inhibition by lignin, while the xylanases showed less variability. Moreover, в-glucosidase was least affected by lignin. The authors concluded that the selection or engineering of so-called “weak-lignin-binding enzymes” in the future will offer an alternative means of enzyme improvement [82]. Overall, it has been demonstrated that the presence of lignin presents a sig­nificant obstacle during hydrolysis. However, early work [83] has also shown that hemicellulose removal during pretreatment also results in significant improvements in hydrolysis performance. Hemicelluloses differ significantly from lignin, since their recovery is quite sensitive to changes in processing conditions, and their hydrolysis can potentially be used to fortify recovered sugars to increase ethanol yields in subsequent fermentation [59].

4

The performance of the thermostable enzymes at a lower temperature, the 35 °C commonly used in SSF, was compared. The T. reesei deletion strains produced only low amounts of background cellulase activities, mainly due to the presence of native EGIII (Cel12A) and EGV (Cel45A). However, the deletion strains used for the production of thermoenzymes produced some hemicellulases. For practical use, any mesophilic background activity en­hancing the hydrolysis can be considered useful, but in order to evaluate the performance of the thermophilic enzymes, the level of remaining background activities was evaluated. The FPU activity in the background was negligible and the endoglucanase activity was very low as compared to the commercial preparations. Most of the endoglucanase activity, 85-90%, was inactivated during the thermal treatment at 60 °C, pH 6.5 for 2 h. Obviously, the EGV ac­tivity was the most stable remaining activity. Thus, the background activities originating from the T. reesei deletion strains had only a minor contribution to the total hydrolysis above 65 °C.

The actual hydrolysis performance of the new thermostable enzyme mix­tures on various pretreated lignocellulose substrates (spruce and corn stover) at 35 °C showed some variations as compared with the T. reesei enzymes: on

A

100 і

Fig. 7 Hydrolysis of steam pretreated washed spruce (a) and unwashed corn stover (b) by Celluclast (■) and (□) the thermostable enzymes (TM 1 for spruce and TM 2 for corn stover) at 35 °C. Enzyme dosages: Celluclast 5 FPU g-1 substrate, supplemented with 100 nkatNovozym 188 g-1 substrate; thermostable enzymes 5 FPU g-1 substrate, substrate concentration 10 gL-1, hydrolysis time 72 h at pH 5, triplicates with mixing
spruce, the sugar yield obtained by the thermophilic enzymes was generally lower and on corn stover higher than with the commercial T. reesei enzymes (Fig. 7). The result was the same, irrespective of the presence of the thermoxy — lanase in the preparation (TM 1 in Fig. 7 or TM 3 in Fig. 5). Thus, with this substrate the relatively lower cellulase activity at 35 °C is obviously the rea­son for the poorer hydrolysis at the lower temperature. In contrast, on the xylan-containing substrate, corn stover, the additional xylanase activity in the thermostable enzyme mixture had a more profound effect. The total xylanase activity was somewhat higher in the thermostable preparation, emphasising the importance of hemicellulases in the hydrolysis of substrates containing residual xylans. Further research would be needed to study in detail the struc­tural differences of both cellulose and hemicellulose in the two substrates and their impact on the performance of the enzyme patterns used.

10

The expression of a cofactor-independent, heterologous XI is the solution for bypassing the intrinsic redox constraints of the XR/XDH approach. Suc­cessful implementation, however, requires an in vivo activity of XI similar to that of key glycolytic enzymes such as hexokinase and phosphofructokinase. In practice, this corresponds to an activity, under physiological conditions, of 0.5-1.0 ^mol D-xylose converted per milligram soluble cell protein per minute [68]. The apparent simplicity of this objective turned out to be decep­tive. In fact, studies on the functional expression of heterologous structural genes for XI in S. cerevisiae now spans roughly two decades.

Expression in S. cerevisiae of the E. coli xylA gene (which clusters with the XI genes from other Proteobacteria, Fig. 3), resulted in no [13] or very low in vitro XI activities [59]. Sarthy et al. (1987) showed that, while the E. coli XylA protein was produced in S. cerevisiae, its specific activity was three orders of magnitude below that of XylA protein produced in E. coli [59]. Improper protein folding, sub-optimal intracellular pH, post-translational modification, inter — or intramolecular disulfide bridge formation and a lack of specific cofactors or metal ions in S. cerevisiae were mentioned as possible causes [59]. However, no single factor was identified that could explain the low activity, and attempts to increase E. coli XI expression levels in S. cere­visiae were unsuccessful [59]. Subsequently, attempts were made to express XI-encoding genes from other prokaryotic phyla. Attempts to express XI genes from Clostridium thermosulfurogenes [48], Bacillus subtilis or Actino — planes missouriensis [1], which originate from different prokaryotic phyla (Fig. 3), also failed to result in the production of an active XI enzyme in S. cerevisiae.

The first study that achieved significant activities of a heterologous XI enzyme in S. cerevisiae was based on expression of the XI gene from the thermophile Thermus thermophilus [70]. Indeed, an enzyme activity of up to 1.0 ^mol(mg protein)-1 min-1 was found in cell extracts of the engineered S. cerevisiae strain. However, this activity was assayed at the optimum tem­perature for activity of the T. thermophilus XI of 85 °C, which is not com­patible with yeast growth or survival. At 30 °C, the optimum temperature for growth of S. cerevisiae, activity was only 0.04 ^mol (mg protein)-1 min-1 [70]. Although subsequent random mutagenesis resulted in variants of the T. ther­mophilus XI with improved temperature characteristics [26,47], in vivo en­zyme activities of the T. thermophilus XI in S. cerevisiae strains remained too low to sustain rapid anaerobic growth on D-xylose ( [35], see Sect. 5).

A breakthrough came with the discovery of a XI in an unicellular eu­karyote, the anaerobic fungus Piromyces sp. E2 [28]. Expression of this Piromyces xylA gene in S. cerevisiae resulted in high enzyme activities (up to

1.1 ^mol(mg protein)-1 min-1 at 30 °C [42].

The molecular basis for the high functional expression levels obtained with the Piromyces xylA gene remains unclear. We have recently expressed the XI sequence from Bacteroides thetaiotaomicron into S. cerevisiae. This prokary­otic sequence is 83% identical and 88% similar to the Piromyces xylA gene. S. cerevisiae strains expressing this prokaryotic XI can utilise D-xylose, albeit

at a somewhat lower rate than similar strains expressing the Piromyces xylA gene (A. A. Winkler et al. unpublished). This indicates that its probable evolu­tionary history (horizontal gene transfer followed by evolutionary adaptation to a eukaryotic host) may not be the sole factor in the successful expression of the Piromyces enzyme.

In terms of GC content and codon usage, the Piromyces xylA gene appears to have favourable characteristics for expression in S. cerevisiae. At 45%, its GC content is much closer to that of S. cerevisiae (39%), than that of, for ex­ample, the T. thermophilus gene (little over 64% GC). Also the high codon bias index of the Piromyces gene for expression in S. cerevisiae (0.642 versus — 0.018 for the T. thermophilus gene) may contribute to its efficient expression. Future structure-function studies will likely identify critical factors for high- level functional expression in yeast, in the S. cerevisiae genome as well as in the sequence of heterologous XI genes. However, while of great scientific interest, innovation in D-xylose fermentation is no longer dependent on such research, as the availability of the Piromyces xylA gene has paved the way for metabolic engineering of S. cerevisiae for anaerobic fermentation of D-xylose to ethanol. Recent progress in this area will be discussed in the following paragraphs.

4

In the post-genomic era, increasing efforts have been made to quantitatively describe the relationship between the genome and phenotype of cells. At the interface between the environment and DNA-encoded processes, metabolite levels are quantitative phenotypic indicators that provide an important com­plement to the measurements of mRNA and proteins when studying cellular function. In the same way as for proteomics, where mRNA expression is often assumed to correlate linearly with protein expression and further correlate with protein activity, the false pretence of a one-to-one relationship between all gene expression and metabolite formation exists. In fact, metabolite lev­els may be viewed as the final result of a complex integration of gene ex­pression, RNA translation, post-translational modification, enzyme activity, and pathway regulation [117]. Metabolomics is a burgeoning field produc­ing volumes of data that, like other x-omic data, brings together analytical technology, genomics, bioinformatics, and model construction, and lies at the core of the systems biology agenda [118]. The general idea of metabolomics was first defined several years ago in the field of microbiology [119], and its importance in plant science was pointed out soon thereafter [120]. To­day, metabolomics is also a powerful tool in drug discovery and develop­ment, especially for the identification of drug metabolites and biomarkers for organ-specific toxicities [121,122]. Industrial biotechnology has also be­gun to benefit from integration of metabolomics into the systems biology framework. In metabolic engineering, quantitative metabolomics, by assign­ing function and confirming in silico pathways, could provide a measure of changes in regulatory driving forces and elucidate the impact of changes in enzyme activities on fluxes [123].

Panagiotou and colleagues performed a thorough examination of the metabolome (amino and non-amino acids of the pyruvate, glycine, serine,

threonine, phenylalanine, tyrosine, tryptophan, histidine, glutamine, gluta­mate and dibasic acid metabolism, and the TCA cycle) of Fusarium oxys — porum, a promising microorganism for bioethanol production, in different physiological states [124-127]. They demonstrated that in spite of the di­versity of mechanisms in fungi that regulate the flux of intracellular amino acids, the production of amino acids was closely correlated with the oxy­gen supply and growth medium, while less so to the cultivation phase [126]. By investigating the profile of several intracellular metabolites during culti­vations on glucose and cellulose, metabolic limitations in F. oxysporum that determine the reduced growth rate of this organism compared to other fila­mentous fungi could be pinpointed [125,127]. For example, one of the major drawbacks on the glucose-to-ethanol conversion by F. oxysporum is the for­mation of significant amounts of acetic acid as a by-product. A systematic metabolome analysis suggested that the y-aminobutyric acid (GABA) shunt is active under anaerobic conditions [125]. This led to the suggestion that the high production of acetic acid, which indicates NAD(P)H regeneration, may be associated with a GABA shunt activation since such pathways act as sinks for excess NAD(P)H, e. g., when the TCA cycle is inhibited [128]. Also, a determination of the sugar phosphorylated profiles under aerobic and anaerobic cultivations in order to improve the understanding of slow arabi- nose fermentation by F. oxysporum [126] was performed. The identification of key metabolites in F. oxysporum cultivations uncovered the activation of novel pathways and possible bottlenecks of others, offering specific genetic targets for improved fermentation performance (overexpression of phospho — glucomutase, transaldolase/transketolase).

Metabolomics has not only been used as a tool for identification of targets for metabolic engineering as described above, but also as an all — encompassing approach to understanding total, yet fundamental, changes occurring after subtle genetic perturbations. For example, key intermediates in the pentose phosphate pathway (PPP) and the Entner-Doudoroff pathway (EMP) pathway were analyzed to gain further insight into the metabolism of laboratory and industrial S. cerevisiae strains [129]. The results verified that the profiles of metabolites are indicative of the reference genetic background of the strains and engineered genotype. Devantier et al. (2005) have investi­gated the influence of very high gravity simultaneous saccharification and fer­mentation process conditions on yeast cellular metabolism [130]. Laboratory and industrial S. cerevisiae strains were cultured mimicking fermentation conditions commonly found in the fuel ethanol industry. Concurrently, GC — MS metabolite profiling was performed to determine if there was a metabolic stress response under defined conditions. Metabolite profiling and multivari­ate data analysis was demonstrated by the ability to distinguish strains and fermentation conditions based on intra — and extracellular metabolites. Fur­thermore, the increased energy consumption of stressed cells was reflected in increased intracellular concentrations of pyruvate and related metabolites.

Consequently Villas-Boas and coworkers (2005) used the metabolite profile of S. cerevisiae during very high gravity ethanol fermentation [130] to elucidate un-described metabolic pathways [131]. They proposed a de novo pathway for glycine catabolism and glyoxylate biosynthesis in recombinant S. cerevisiae strains, demonstrating the great potential of coupling metabolomics and iso­tope labeling analysis for pathway reconstructions.

A recent literature review of the applications of metabolome data from mi­croorganisms was summarized by Wang et al. (2006), and included compara­tive metabolite studies, fermentation control, metabolic control analysis and flux studies, and integration of metabolomics for strain improvement [132]. Clearly, metabolomics will have a strong impact on industrial biotechnology in the coming years as one of the corner stones of the systems biology toolbox being applied to metabolic engineering for bioethanol strain improvement.

3.5

The principal components of biomass are: cellulose (~ 30-50%), hemicellu­lose (~ 20-30%) and lignin (~ 20-30%); with minor components of starch, protein and oils. The exact composition of each biomass varies depending both on the plant species and the plant tissue utilized. Table 1 shows a var­iety of substrates in an effort to illustrate the variability of the composition of different substrates. In addition to the variability seen between plant species, work at the US National Renewable Energy Laboratory has demonstrated that even within a single plant species there is considerable variability in compo­sition [6]. Using near infrared spectroscopy, they showed that the total sugar content contributed by cellulose and hemicellulose varied from 45 to 68% of dry mass between 1061 samples of corn stover. Lignin content, which has a direct impact on enzymatic digestibility, varied between 12 and 20%. These differences can be attributed to the genetic background of the corn variety, environmental factors such as weather, location, and pest invasion, and dif­ferences in farming practices.

The substrate characteristics that have been shown to impact the rate of hydrolysis include accessibility, degree of cellulose crystallinity, and the type and distribution of lignin [8]. The presence of lignin in a cellulose-cellulase

reaction is hypothesized to decrease the quantity of the enzyme associated with the cellulose due to nonspecific adsorption of the enzyme to lignin [9] and steric hindrance [10]. Steric hindrance occurs when lignin encapsulates the cellulose and makes it less accessible to enzyme attack [11]. Each of the factors summarized above are known to effect enzyme action and no sin­gle parameter correlates absolutely with enzymatic digestibility. The variation in composition of a given biomass requires some tailoring in the conversion method.

3.2

4.2.1

Cofactor Dependence

The production of xylitol and L-arabitol during pentose consumption by natural as well as recombinant pentose-utilizing yeasts has been rational­ized with the difference in cofactor preferences between the enzymes in the initial pentose utilization pathways. XR from P stipitis preferentially uses NADPH, but can also use NADH as a cofactor [23], whereas XDH exclu­sively uses NAD+ [24,46]. This may result in excess NADH formation and lack of NAD+, since yeasts do not harbor a transhydrogenase enzyme that would allow direct conversion of NADP+ to NAD+ [32]. Numerous investiga­tions have supported this metabolic model; external electron acceptors, which are reduced by NADH-dependent enzymes in S. cerevisiae, reduce xylitol for­mation [32,74-76]. Xylitol formation in recombinant S. cerevisiae was also reduced by changing the kinetic properties of the enzymes involved by ex­pressing a fusion protein of XR and XDH [87], or by expressing mutated XR with altered cofactor affinity [88,89] (strains TMB3270, TMB3271, R267H, Ta­bles 1-3). However, significantly increased ethanolic xylose fermentation as a result of such engineering strategies has been less frequently reported [89].

4.2.2

The composition of plant biomass can vary substantially but all plant biomass is composed of four major polymeric compounds: cellulose (~ 33-51%), hemicellulose (~ 19-34%), pectin (~ 2-20%), and lignin (~ 20-30%) [16, 31]. Upon hydrolysis, plant biomass yields a variety of fermentable hexoses (glucose, 36-50%; mannose, 0.3-12%; galactose, 0.1-2.4%) and pentoses (xy­lose, 3.4-23%; arabinose, 1.1-4.5%). S. cerevisiae can ferment all the hexoses to ethanol, but not the pentoses, which can be a significant portion (25%) of, for example, sugarcane bagasse, a preferred feedstock for bioethanol pro­duction [32]. More than three decades of research have been devoted to the development of yeast for efficient xylose fermentation, initiating with the search for alternative yeasts, such as Pachysolen tannophilus, Pichia stipitis, and Candida shehatae, and in the last two decades focusing on the genetic en­gineering of S. cerevisiae to utilize xylose and arabinose [33]. Please refer to the chapters by Hahn-Hagerdal et al. and van Maris et al. (in this volume) for detailed reviews of this topic [34,35].

After several unsuccessful attempts to produce a functional bacterial xy­lose isomerase in S. cerevisiae, many groups focused for the last decade on efficient expression of fungal xylose utilizing genes and manipulating the pentose phosphate pathway to enhance xylose utilization and fermentation in S. cerevisiae [36]. These research efforts ensured steady but slow progress toward the development of xylose utilizing S. cerevisiae strains, and it was recent successes with the production of a functional Piromyces sp. xylose iso — merase in recombinant S. cerevisiae that opened the way for efficient xylose fermentation by S. cerevisiae at low oxygen levels [37,38]. The main advan­tage of this approach is the circumvention of the redox imbalance problem created by expressing xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) fungal genes in S. cerevisiae. Recognizing the need for S. cerevisiae to ferment all hexoses and pentoses produced during enzymatic hydrolysis of wood, both laboratory and industrial S. cerevisiae platform strains have been developed that can utilize xylose [39] and later co-utilize xylose and arabinose [40].

Apart from monosaccharides, S. cerevisiae can utilize the disaccharides su­crose and maltose, and some Saccharomyces strains can also utilize melibiose and the trisaccharides maltotriose and raffinose [41,42]. However, the major end products of cellulose hydrolysis are cellobiose and cellooligosaccharides, which cannot be utilized by S. cerevisiae. The heterologous expression of four different в-glucosidases in S. cerevisiae was evaluated and the P-glucosidase (BGL1) of Saccharomycopsis fibuligera was found to be produced at the high­est activity levels [43]. Expression of the в-glucosidases encoding genes of Candida wickerhamii, Aspergillus kawachii, and T. reesei yielded activities at least one order of magnitude lower than that of Saccharomycopsis fibuligera. It was shown that multicopy expression of the S. fibuligera BGL1 gene could enable growth on cellobiose as sole carbon source at a rate equivalent to that found on glucose [43,44]. Recently, a S. cerevisiae strain was developed that could utilize both xylose and cellobiose [45].

Even with the introduction of pentose and cellobiose utilizing genes, S. cerevisiae strains preferentially utilize glucose before the other mono — and disaccharides. Deregulation of the strong glucose repression effect in S. cerevisiae would be required to allow cometabolism of sugars derived from plant biomass for high ethanol productivity. Disrupting both the MIG1 and MIG2 genes allowed cometabolism of glucose and sucrose [46], and simi­lar strategies could be used to allow co-utilization of sugars released from plant biomass. Furthermore, simultaneous co-transport of glucose and xylose must be facilitated as the delayed utilization of xylose (in a recombinant xy­lose utilizing strain) is in part an effect of competition for the same glucose transporters in the absence of a xylose-specific transporter [37].

4

There is a vast range of materials suitable for the production of ethanol. They can, somewhat arbitrarily, be put into three categories: agricultural, hard­wood and softwood materials. It must be emphasized that it is not always possible to transfer the results from one type of material to another. During the last three decades, many types of materials using various pretreatment methods have been studied. Some hardwood materials, such as poplar, salix or aspen, have been frequently used in various investigations [48-52]. Other

materials examined have been straw [53-58], sugar cane bagasse [59-61] and olive tree wood [62], to mention a few.

In this summary some lignocellulosic materials were chosen for a more in­depth discussion. The materials that are discussed are an agricultural residue (corn stover) for which the hemicellulose is mainly composed of the pen­tose sugar xylose (about 22% xylose, 5.5% arabinose and 3% galactose; all as anhydro-sugars) and a softwood (spruce) where the hemicellulose mainly consists of the hexose sugar mannose (about 12-13% mannose, 5% xylose, 2% galactose and 2% arabinose; all as anhydro-sugars). Table 1 shows the composition of these materials as well as the theoretical amount of ethanol that can be produced from the hexose and the pentose fractions. It is clear that in some cases it is very important to utilize not only the hexose fraction, but also the pentose part of the material.

4.1

Liisa Viikari1 (И) • Marika Alapuranen2 • Terhi Puranen2 •

Jari Vehmaanpera2 • Matti Siika-aho3

University of Helsinki, P. O. Box 27, 00014 Helsinki, Finland liisa. viikari@helsinki. fi

2ROAL, Valta-akseli, 05200 Rajamaki, Finland

3VTT Technical Research Centre of Finland, P. O. Box 1000, 02044 Espoo, Finland

1 Introduction…………………………………………………………………………………… 122

2 Enzymatic Hydrolysis of Cellulose………………………………………………….. 122

3 Thermostable Cellulases…………………………………………………………………. 123

4 Process Concepts……………………………………………………………………………. 127

5 Evaluation of Novel Thermophilic Enzymes; Materials and Methods… 129

6 Composition of the Thermophilic Enzyme Mixtures………………………….. 131

7 Performance of Commercial Fungal Preparations at Elevated Temperatures 132

8 Evaluation of New Thermostable Enzyme Mixtures………………………….. 133

9 Performance of the Thermostable Enzymes at Lower Temperatures…. 137

10 Discussion…………………………………………………………………………………….. 138

References ……………………………………………………………………………………………………. 141

Abstract Thermostable enzymes offer potential benefits in the hydrolysis of lignocellulosic substrates; higher specific activity decreasing the amount of enzymes, enhanced stability allowing improved hydrolysis performance and increased flexibility with respect to pro­cess configurations, all leading to improvement of the overall economy of the process. New thermostable cellulase mixtures were composed of cloned fungal enzymes for hydrolysis ex­periments. Three thermostable cellulases, identified as the most promising enzymes in their categories (cellobiohydrolase, endoglucanase and в-glucosidase), were cloned and produced in Trichoderma reesei and mixed to compose a novel mixture of thermostable cellulases. Thermostable xylanase was added to enzyme preparations used on substrates containing residual hemicellulose. The new optimised thermostable enzyme mixtures were evaluated in high temperature hydrolysis experiments on technical steam pretreated raw materials: spruce and corn stover. The hydrolysis temperature could be increased by about 10-15 °C, as compared with present commercial Trichoderma enzymes. The same degree of hydro­lysis, about 90% of theoretical, measured as individual sugars, could be obtained with the thermostable enzymes at 60 ° C as with the commercial enzymes at 45 ° C. Clearly more effi­cient hydrolysis per assayed FPU unit or per amount of cellobiohydrolase I protein used was

obtained. The maximum FPU activity of the novel enzyme mixture was about 25% higher at the optimum temperature at 65 ° C, as compared with the highest activity of the com­mercial reference enzyme at 60 °C. The results provide a promising basis to produce and formulate improved enzyme products. These products can have high temperature stability in process conditions in the range of 55-60 ° C (with present industrial products at 45-50 ° C) and clearly improved specific activity, essentially decreasing the protein dosage required for an efficient hydrolysis of lignocellulosic substrates. New types of process configurations based on thermostable enzymes are discussed.

Keywords Thermostable • Cellulases • Cellobiohydrolase • Endoglucanase •

P-Glucosidase • Lignocellulose • Hydrolysis

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Introduction

The present challenge is to substantially increase the production and use of biofuels for the transport sector. In order to reach the future goals of sub­stituting fossil based fuels, it will be necessary to promote the transition towards second generation biofuels. These can be produced from a wider range of feedstock, including lignocellulosic raw materials. Biomass resources can be broadly categorised as agricultural or forestry-based, including secondary sources derived from agro — and wood industries, waste sources and municipal solid wastes. Fuels from lignocellulosic biomass have a higher potential to re­duce greenhouse gas emissions, and hence are an important means to fulfil the CO2 emissions targets, as compared with first generation biofuels. Lignocellu- losic raw materials comprise an abundant source of carbohydrates (cellulose and hemicellulose) for a variety of biofuels, including bioethanol. The conver­sion technologies of lignocellulosic raw materials are, however, more complex and need novel enzyme systems and advanced fermentation technologies. The rate-limiting step in the conversion of cellulose to fuels is hydrolysis, especially the initial attack on the highly ordered, insoluble structure of crystalline cellu­lose. In spite of recent achievements, further developments are still needed to improve the overall economy of the lignocellulose-to-ethanol process. These novel conversion techniques would also be applicable for the production of other sugar platform-based chemicals.

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