The first attempt to introduce an L-arabinose utilization pathway in S. cere­visiae by heterologous expression of the complete E. coli L-arabinose pathway
did not result in appreciable arabinose utilization [70], most likely due to the absence of functional expression of the L-arabinose isomerase. It was only when the E. coli araA gene encoding the L-arabinose isomerase was substi­tuted by the corresponding Bacillus subtilis gene that a functional arabinose pathway was established in S. cerevisiae [71]. Similar to the use of the het­erologous XI pathway, other genetic modifications in addition to the new L-arabinose isomerase were required for the recombinant strain to grow on L-arabinose as sole carbon source [71]: an additional copy of the galactose permease (Gal2), which also transports arabinose [72], and an unspecified adaptation for growth on arabinose [71].

The fungal L-arabinose utilization pathway has also been introduced in S. cerevisiae, combining enzymes from P. stipitis and from the filamentous fungus Trichoderma reesei. The enzymes were actively expressed; however, neither appreciable growth on L-arabinose nor significant ethanolic fermen­tation was observed [73]. The dysfunction of the fungal arabinose pathway with respect to ethanolic fermentation parallels the inability of the naturally arabinose-growing yeasts to ferment L-arabinose to ethanol [50,69]. Instead, these yeasts often produce L-arabitol from L-arabinose (Fig. 2) [65,66,69]. Minute ethanolic fermentation has been observed for six yeast species, C. arabinofermentans, P. guilliermondii, C. auringiensis, C. succiphila, Ambro- siozyma monospora, and Candida sp. YB-2248, but only in rich medium [65, 69]. Rich media may contain other fermentable sugars as well as undefined electron acceptors that serve to regenerate reduced cofactors [32,74-76], which appears necessary for ethanolic arabinose fermentation to occur via the fungal pathway. Also, the presence of low amounts of oxygen aids cofactor regeneration [50,77].

4

Functional expression in S. cerevisiae of a highly active fungal XI has paved the way for metabolic engineering of this yeast towards high-yield, rapid production of ethanol from D-xylose under fully anaerobic conditions. On theoretical grounds, this XI-based approach is superior to the extensively studied xylose reductase/xylitol dehydrogenase strategy. While considerable experimental proof to substantiate this statement has been obtained under “academic” conditions, a next important challenge is to do the same under industrial conditions. While the first experiments in real-life plant biomass hydrolysates are quite promising, there remains plenty of scope for integrat­ing the D-xylose-fermentation genotype with other metabolic and process­engineering strategies for further increased robustness under process condi­tions.

In addition to D-xylose, plant biomass hydrolysates contain several other potentially fermentable substrates that cannot be converted by wild-type S. cerevisiae strains [69]. While these compounds often represent only a few percent of the potentially fermentable carbon, they can have a decisive impact on economical competitiveness and sustainability of high-yield, high-volume processes such as fuel ethanol production. Functional integration of a highly efficient D-xylose fermentation pathway with pathways that are under devel­opment (e. g. arabinose [9,36]) or under consideration (e. g. rhamnose [69]) therefore presents an additional challenge in metabolic engineering for ef­ficient fermentation of plant biomass hydrolysates. We are convinced that creative integration of metabolic engineering, evolutionary engineering and process design can result in rapid breakthroughs in these areas.

An early attempt was made to construct a xylose-utilizing strain of Z. mo — bilis by Liu et al. [45,46] involving expression of genes for xylose isomerase (XI), xylulokinase (XK) and the xylose transport protein from X. albilineans XA1-1. Although the recombinant strain was shown to possess both XI and XK activity, it was unable to grow on xylose as the sole carbon source. Subse­quently, Feldmann et al. [47] constructed a recombinant strain of Z. mobilis ZM4 (pZY228) that expressed the xylA and xylB genes from Klebsiella pneu­moniae for XI and XK, respectively, and the tktA gene for transketolase (TKT) activity from Escherischia coli. However, this recombinant strain was also un­able to grow on xylose.

On the basis of these earlier studies, Zhang et al. [10] constructed a recom­binant strain that successfully converted xylose to ethanol by expression of a transaldolase (talB) gene from E. coli in addition to those expressing XI, XK and TKT activity. This recombinant strain encoded genes for enzymes both for xylose assimilation (XI, XK) and for completion of the pentose phosphate pathway (TKT, TAL) in Z. mobilis. The transformation of wild-type strains of Z. mobilis with the 14.4 kb expression vector (pZB5) was then shown to fa­cilitate the efficient conversion of xylose to ethanol via a completed pentose phosphate pathway (Fig. 1).

This research at NREL was continued further by Deanda et al. [11] who successfully developed a strain capable of arabinose utilization. This recombi­nant strain harbored a plasmid (pZB206) expressing five heterologous genes from E. coli encoding L-arabinose isomerase (araA), L-ribulokinase (araB), L-ribulose-5-phosphate-4-epimerase (araD), transaldolase (talB) and trans- ketolase (tktA).

In related studies on the development of a xylose utilizing strain of Z. mo­bilis, De Graaf et al. [48] built on the earlier research by Feldmann et al. [47] and introduced a further plasmid (pZY228) into strain ZM4 (pXY228). This former plasmid harbored talB from E. coli thereby facilitating expression of all the requisite additional enzymes in Z. mobilis for xylose assimilation and metabolism.

Although there were differences in their construction of these two recom­binant strains, the metabolic pathway for both recombinant strains resulting from expression of xylA, xylB, tktA and talB was the same as shown pre-

viously in Fig. 1. Xylose enters the Entner-Doudoroff pathway via fructose — 6-phosphate and glyceraldehyde-3-phosphate and is converted into ethanol. The following balance equations represent the metabolism of glucose and xy­lose by these recombinant xylose-metabolizing strains of Z. mobilis.

Glucose + ADP + Pi ^ 2Ethanol + 2CO2 + ATP,

3Xylose + 3ADP + 3Pi ^ 5Ethanol + 5CO2 + 3ATP,

Theoretical ethanol yield = 0.51 gethanol/g sugar (glucose or xylose).

Further studies on recombinant strains created at NREL have involved the construction of integrant xylose-utilizing strains [36-38] and additionally an
integrant xylose/arabinose-utilizing strain designated AX101 [39]. This strain was produced using random insertion and site-specific insertion via homolo­gous recombination.

The specific enzyme activities of the various xylose-utilizing recombinant strains have been determined as a means of identifying possible rate limita­tions. For the strains developed at NREL, the specific activity associated with XI was the lowest [49,50]. However, based on calculation of the metabolic fluxes associated with the each enzyme introduced for xylose metabolism, De Graaf et al. [48] concluded for their strain that the flux associated with XK was significantly lower than that of others. This suggested that a metabolic bot­tleneck may exist in their strain, ZM4 (pZY228) (pZY557 tal), due to the low expression of xylulokinase.

Subsequent kinetic studies involving the over-expression of XK (Fig. 2) in an acetate-resistant mutant of the NREL-derived strain ZM4 (pZB5) showed no increase in the maximum specific growth rate or specific rate of xylose metabolism, although there was evidence of a small increase (0.4 gL-1) in production of xylitol for the over-expressing strain [51]. Further research on

these recombinant strains is likely to focus on potential rate-limiting sites, as well as expression of heterologous enzymes from other microbial sources for increased ethanol tolerance.

2.3

For the member states of the European Union, the primary policy tool behind the development of a bioethanol industry is the Directive on the promotion of the use of biofuels for transport (Directive 2003/30/EC) [31]. The motiva­tions behind this Directive include improving the security of energy supply, and reducing the environmental impact of the transportation sector [32]. The Directive mandates an increasing share of biofuels from 2% of total fuel sup­ply in 2005 to 5.75% of total fuel supply in 2010 (based on energy content) in order to meet these priorities. Due to relatively slow growth in the industry, it is currently anticipated that renewable fuels will occupy about 4.8% of the market by 2010, which is significantly less than the existing policy target.

The overriding priorities of the European Commission will impact the be­havior of each member nation in setting national policies relating to biofuels. It can be expected that, while economic factors are not the political prior­ity of the EU, the member nations will have a strong interest in utilizing the proposed Directive to meet national goals of employment and economic di­versification. From an economic standpoint, it is anticipated that a biofuel contribution of 1% of the total EU fossil consumption will create between 45 000 and 75 000 new jobs [32].

At the time of writing, many member states have passed the biofuels Di­rective into national law, including Belgium [33], the Czech Republic [34], France [35], Germany [36], Greece [37], Latvia [38], Lithuania [39], and Swe­den [9]. Some countries have announced indicative targets that are below that of the Biofuels Directive, including Malta (target value for 2005 of 0.3%) [40], Hungary (0.4-0.6%) [41], Poland (0.5%) [42], Spain (0.55-0.65%) [43], and Cyprus (1%) [44]. Each of these countries still plan to achieve national targets of 5.75% for the end of 2010. Slovenia follows a slightly different set of tar­gets, ranging from 1.2% in 2006 to at least 5% in 2010 [45]. The Netherlands has set a target percentage of 2% biofuels for 2006, which will be followed in 2007 by requiring suppliers to ensure that these blends are achieved [46]. The UK has announced a Renewable Transport Fuel Obligation, which will place a legal requirement on transport fuel providers to ensure that a specific per­centage of their fuel sales is renewable, ranging from 2.5% in 2008/09 to 5% in 2010/11 [47].

In implementing the biofuels Directive, some countries have set slightly more aggressive targets, including Austria (revised Fuels Ordinance, 4 Novem­ber 2004: BGBI. II, No 417/2004), which mandates that all petrol and diesel marketers blend at least 2.5% biofuels on an energy content basis in all fuels sold within the country [48]. Sweden has set their national target of at least 3% biofuels after 2005, and has mandated that renewable fuels be made avail­able at petrol stations, starting with the largest stations (> 3000 m3 year-1) in 2006, and progressing to smaller stations (> 1000 m3 year-1) by 2009 [9]. Swe­den also has a very aggressive long-term target of 40-50% reduction of fossil fuel use, which should engender significant increases in biofuel use over the next 13 years [49].

Another important piece of legislation is the Directive restructuring the community framework for the taxation of energy products and electricity (Directive 2003/96/EC), which allows excise-tax exemptions for biofuels pro­duced or blended within European countries [50]. This legislation is very important within European nations due to the high level of excise tax that is currently levied on petroleum and diesel in these countries, particularly when compared to North America. Within these countries, a reduction of even a few percent can mean cents per liter, which translates into significant cost savings. For instance, in Austria, a 10% reduction in excise taxes on biodiesel reduces the cost by US $ 0.028 L-1 [51]. This sum is almost equivalent to the federal ex­cise taxes paid for diesel fuel in Canada. A similar percentage reduction in the US federal excise tax for diesel would result in a selling price of US $ 0.058 L-1 and a savings of only US $ 0.006 L-1 [32,52].

Before the release of the second Biofuel Directive, European governments did not always utilize excise tax exemptions to the same extent as their Cana­dian and US counterparts. This was because national controls over excise tax rates were complicated by the rules of the European Economic Community (EEC). A Directive issued by the EEC on 16 October 1992 was intended to harmonize the structures of excise duties among all member nations [52,53]. When France decided to create an aid scheme for biofuels that would ex­empt these fuels from national excise taxes, objections were raised and an appeal to the Commission of the European Communities was made by BP Chemicals [54]. Ultimately, however, the Commission decided to validate the French decision, allowing an exemption amounting to US $ 0.06 L-1 to be extended through 31 December 2003 [13,55,56]. This move created the prece­dent within the EU to allow excise tax exemptions for biofuels, freeing a pow­erful policy tool for decision-makers within the nations of the Union. The second Directive Regarding Tax Relief Applied to Biofuels (2003/96/EC) was issued in 2003, permitting other countries to make the decision to grant ex­cise tax exemptions as biofuel production becomes more widespread within Europe.

Today, most EC member states, including Austria, Belgium, Cyprus, Den­mark, Estonia, France, Germany, Hungary, Italy, Latvia, Lithuania, Luxem­bourg, Malta, Poland, Slovakia, Slovenia, Spain, Sweden and the UK have introduced exemptions at various levels up to 100%, using the precepts laid down in Directive 2003/93/EC. These exemptions are summarized in Table 1.

In implementing tax exemptions, Germany was careful to include a measure that allowed for adjustments to be made in the case of overcompensation. Per­ceived overcompensation has recently been observed in regards to vegetable — oil based fuels, and accordingly, the German government has introduced an Energy Tax Act, which from 1 August 2006 places a tax on these fuels [36].

Currency exchange rates (December 2006): US$ 1.0000 = € 0.7567 = CDN$ 1.1545 “Canadian Federal excise tax rate is shown. Provincial rates are variable, ranging from US 4.5 /L-1 (Yukon Territory) to US 12.1 /L-1 (Newfoundland and Labrador). Provin­cial excise tax exemptions range from US 0.7 /L-1 (Alberta) to US 1.8 /L-1 (Manitoba) b Mexican and Russian rates are ad valorem and vary on a monthly basis, depending on world petroleum prices

c US Federal excise tax rate is shown. State rates are variable, ranging from US 2.0 /L-1 (Georgia) to US 7.7 /L-1 (Rhode Island). State excise tax exemptions range from US 0.1 /L-1 (Florida) to US 0.7 /L-1 (Idaho)

Italy also incorporated measures to adjust in the case of overcompensation; that country currently provides tax exemptions for an annual quota of200 000 t of biodiesel for the period 2005-2010, as well as reduced excise duties on bioethanol and related bio-derived additives [57]. Several countries have ex­perimented with pilot excise tax exemptions on a project-by-project basis, including Finland [58], Ireland [59], and the Netherlands [46]. Latest reports indicate that Greece [37] is also considering tax exemptions for biofuels.

As of late 2005, only one country exceeded the goals set out in the Direc­tive. German biofuel use (primarily biodiesel) accounted for 3.75% of total fuel consumption in 2005 [36]. Swedish biofuel use (primarily bioethanol) accounted for 2.2% of the total in the same year [9], which came closest to achieving the goal; however, since most cars in Sweden are now running at E5 bioethanol blends, the country has encountered a constraint in the form of the EU Directive on Fuel Quality, which limits renewable fuel blends to 5%. Other countries, including the UK, have identified this Directive as a barrier to achieving the goals of the Directive on Biofuel Use [47]. In France, about 1.2% of fuel sales consisted of renewable fuels in 2005, mostly in the form of bio-ETBE or bioethanol [35]. In Austria, biodiesel production had reached almost 100 million L, which is approximately 1.1% of national fuel consump­tion [13,48]. Spain used significant amounts of both bioethanol (1.49% of total petrol) and biodiesel (0.10% of total diesel) [43].

Most EU members had not yet reached their biofuel use goals under the biofuel Directive in 2005, although the situation is changing rapidly as new capacity comes on-line. Lithuania’s use of biofuels has grown, rising to 0.72% in 2005 [39]. Both Italy and Malta report increasing biofuel produc­tion characterized by significant amounts of biodiesel, achieving about 0.57% and 0.52% biofuel use, respectively, in 2005 [40,57]. Other growing biofuel producers include Poland (0.48%) [42] and Latvia (0.33%) [38]. Countries with less than 0.2% biofuel use in 2005 include Greece (0.18%) [37], the UK (0.18%) [47], and Finland (0.1%) [58]. In the case of Finland, it should be noted that a new biodiesel plant designed to be online in 2007 will produce about 200 million L annually, raising this percentage significantly.

Countries reporting less than 0.1% biofuel use in 2005 include Hun­gary (0.07%) [41], the Czech Republic (0.046%) [34], and Luxembourg (0.021%) [60]. Countries with no appreciable biofuel use include Cyprus [44], Ireland [59], the Netherlands [46] and Slovakia [61]. In Denmark, a limited number of Statoil stations began selling 5% bioethanol blends in 2005, but total sales are as yet unknown and unlikely to meet 2% of total transporta­tion fuel sales. Biodiesel is produced in Denmark but exported, primarily for use in Germany [10]. Estonia has some biofuel production, but this volume is completely exported to other EC member countries [62].

Direct funding mechanisms have been implemented in a number of EU member states. In Belgium, the Federal Public Service of Finance has issued a call for tenders to market increasing amounts of biofuels, beginning in November 2006 for biodiesel and in October 2007 for bioethanol. Some re­search funding has also been made available [33]. In Cyprus, legislation to comply with the biofuel Directive includes a grant scheme for energy conser­vation and renewable energy utilization. Under this legislation, four applica­tions for biodiesel plants have been submitted [44]. In the Czech Republic, state aid for biodiesel production has been introduced at a level of about US$ 39 million (CZK 821 million) [34]. In Estonia, about US$ 5000 (EEK 57 600) was granted as support to draw up business plans for the production of liquid biofuel in 2005 [62]. Ireland announced a renewable energy grant aid package in 2005 which provides up to US $ 86 million (€ 65 million) annually to a range of projects, including biofuel initiatives [59]. Latvia provided about US $ 680 000 (LVL 358 980) for bioethanol and US $ 380 000 (LVL 201 770) for biodiesel production [38]. Lithuanian producers of biofuels may claim re­funds on every tonne of feedstock used in biofuel production [39]. Poland has provided approximately US $ 550 000 (PLN 1 601 700) in funding to research projects related to biofuels, and an additional US$ 95 000 (PLN 271 500) to­wards two production start-up projects [42]. Sweden has provided an invest­ment of approximately US$ 120 million (SEK 815 million year-1) for energy research, which includes research into transportation fuels [9]. The UK has created grant programmes to help upgrade infrastructure and to provide dir­ect support for the development of a biofuels industry [47].

Some countries have also implemented other measures to promote bio­fuels. The Czech Republic has introduced policies that provide resources to support biomass production for non-transport energy purposes [34], and Es­tonia has set aside resources to support the expansion of energy crops [62], as has Slovenia [45]. Ireland has established a number of initiatives, including tax exemptions for corporate fleets and for flex-fuel vehicle sales [59]. The UK created a fleet biofuel mandate for its Government Car and Dispatch Agency in 2005, which specifies 5% biodiesel use in the fleet [47]. Sweden has created a number of progressive measures, including a provision that state-owned vehicles be environmentally sound (which includes power by biofuels), and introduction of a congestion charge for Stockholm to which biofuel-powered vehicles are exempt [9]. Sweden has also released a report entitled “Making Sweden an oil-free society”, which has among its goals the reduction of petrol and diesel in transportation fuels of 40-50% by 2020 [49].

2.4

In addition to the transport flux and the flux through the initial pentose­converting enzymes, the “pulling” effect [55] of the flux through enzymatic reactions downstream of xylitol, as well as through glycolysis, appears to be equally important for ethanolic pentose fermentation. It was early recognized that the presence of glucose during xylose fermentation enhanced the gly­colytic activity [122-124]. Furthermore, it was recently shown that no xylitol was formed in the glucose-xylose coconsumption phase during xylose fer­mentation with recombinant S. cerevisiae in mineral medium [54], nor in lignocellulose hydrolysates which contain hexose sugars [6,12,14].

4.7

Other Modifications

Transcription factors involved in glucose repression have also been modi­fied in order to affect ethanolic xylose fermentation. The gene MIG1, or both MIG1 and MIG2, were deleted in an XR-XDH-XK-carrying strain of S. cere­visiae [125] to generate strains which were constantly glucose de-repressed during glucose-xylose cofermentation. This engineering strategy had little ef­fect on ethanol formation. It rather led to increased xylitol formation [125] (strains CPB. CR2 and CPB. MBH2, Table 3). Similarly, when truncated ver­sions of the MIG1 gene were expressed in xylose-utilizing strains of S. cere — visiae, growth and ethanol formation were only marginally affected [126]. The bacterial phosphoketolase pathway, which converts xylulose-5-phosphate directly to glyceraldehyde-3-phosphate and acetyl-P, has also been introduced in S. cerevisiae to enhance ethanolic xylose fermentation [127,128]. The xyl­itol yield decreased without any increase in the ethanol yield [128] (strain TMB3001c-p6XFP/p4PTA/p5EHADH2, Table 2). In contrast, heterologous ex­pression of a bacterial hemoglobin gene to render the cells a more oxidized state in oxygen-limited conditions was successful [129]. Improved ethanolic xylose fermentation was observed. This strategy is, however, only applicable in oxygenated cultures [129].

4.8

The utility of KO11 for production of ethanol from biomass has been demon­strated with multiple substrates including, but not limited to, rice hulls [19], sugar cane bagasse [20], agricultural residues [20], Pinus sp. hydrolysate [21], corn cobs, hulls and AFEX-pretreated fibers [22,23], orange peel [12], wil­low [24], pectin-rich beet pulp [25], sweet whey [26], brewery waste [27], and cotton gin waste [28]. The final ethanol titers and fermentation times for these substrates are presented in Table 1. Consistent with the robustness of the parental E. coli W, KO11 is relatively robust to changes in temperature and pH [29]. KO11 has also been the subject of an empirical kinetic model [24].

While similar ethanol yields are obtained from glucose and xylose, differ­ences in transport mechanisms result in a lower ATP yield for xylose. Both KO11 and LY01 grow approximately 50% faster and produce three times as much ATP from glucose relative to xylose [30]. As expected, the expression

of xylose metabolic genes is increased during xylose growth relative to glu­cose growth. However, genes contributing to metabolism of other pentose sugars, such as arabinose, ribose and lyxose, also have increased expression during xylose growth, consistent with a relaxation of the cAMP-CRP control system [30].

2.1.3

As outlined in the earlier reviews and summarized in Table 3, wild-type strains of Z. mobilis (and their mutants) can convert simple sugars to ethanol at faster rates and higher yields compared to yeasts. However, the ethanol industry has traditionally used yeasts, and despite the apparent advantages of Z. mobilis, there appears to be little incentive for change with sugar and

Table 3 Characteristics of Z. mobilis for production of fuel ethanol and higher value products

1. Considerably faster specific rates of sugar uptake and ethanol production (specific rates 2-3 times faster than yeasts).

2. Higher ethanol and lower biomass yields compared to yeasts due to different carbo­hydrate metabolism (Entner-Doudoroff vs. glycolytic pathway).

3. Higher reported productivities (120-200 gL-1 h-1) in continuous processes with cell recycle (maximum reported values for yeasts are 30-40 gL-1 h-1).

4. Simpler growth conditions. Z. mobilis grows anaerobically (not strict anaerobe) and does not require the controlled addition of oxygen to maintain cell viability at high ethanol concentrations.

5. Ethanol tolerance comparable if not better than yeasts. Ethanol concentrations of 85gL-1 (11% v/v) reported for continuous culture and up to 127gL-1 (16% v/v) in batch culture.

6. Laboratory scale studies with strains of Z. mobilis over many years in controlled fer­mentations (pH = 5.0, T = 30 ° C) have not revealed any significant contamination or bacteriophage infection problems.

7. The wide range of techniques developed for the genetic manipulation of bacteria (such as Escherichia coli) can be applied to developing recombinant strains of Z. mo — bilis and/or their metabolic engineering.

8. Integrant rec strains of Z. mobilis available for efficient ethanol production from glu­cose, xylose and arabinose. Ethanol concentrations above 60 g L-1 in 48 h reported for medium containing 65 g L-1 glucose, 65 g L-1 xylose.

9. Sequencing of ZM4 genome now provides information for its metabolic engineering for additional higher value products (e. g., succinic acid).

10. Potential for use of its enzymes for fine chemical biotransformations.

starch-based raw materials. Some of the reasons lie in the concerns that Z. mobilis may be less robust than yeast and more susceptible to contamina­tion in large-scale processes, as well as the lack of ethanol industry experience with large-scale bacterial fermentations. In addition, an established feed mar­ket exists for the high protein yeast by-product (as dried distiller’s grains) and any new market for a high protein by-product from a Zymomonas process would need to be established. The key issues and alternative capabilities are summarized in Table 4.

The construction of recombinant strains of Z. mobilis able to use the addi­tional C5 sugars xylose and arabinose have now opened up new opportunities as illustrated by the recently announced Dupont/Broin partnership to develop a Zymomonas-based process for conversion of corn stover to ethanol [43]. In an Integrated Corn Biorefinery (ICBR), this would be associated with conver­sion also of the corn starch to higher value products (e. g. to 1,3-propandiol using recombinant strains of Escherichia coli). Experience with large-scale re­combinant bacterial fermentations could provide a future platform as well for an increased range of higher value products generated via the metabolic en­gineering of micro-organisms such as Z. mobilis which are capable of both rapid and highly efficient sugar metabolism.

The activities of commercial reference preparations were first measured at higher temperatures in order to evaluate their general performance and to es­timate the role of the background activities originating from the production strain. The hydrolysis of the pretreated spruce substrate by the commercial preparations (with and without added в-glucosidase, BG) at various tempera­tures from 50 to 70 °C was estimated during the first 24 h of the hydrolysis. The native Trichoderma cellulases and the Aspergillus BG were rapidly inac­tivated during the first 2 h of hydrolysis of the pretreated spruce substrate at temperatures above 60 °C (Fig. 2). The hydrolysis ceased after 24 h at 60 °C and after 48 h at 55 °C (results not shown). As expected, the effect of the added BG on the sugar yield was significant. The relative inactivation of BG was more pronounced even at 60 °C (Fig. 2b). The hydrolytic effect of the rather high loading (about 20 FPU g-1 cellulose) of T. reesei and Aspergillus enzymes was obviously due to the initial stage of hydrolysis during which the enzymes remained active. The hydrolysis yield of sugars from spruce dur­ing the first 2 h was 15% of the theoretical maximum at 70 °C, 22% at 65 °C and 33% at 60 °C. There were indications that the temperature optimum of the commercial T. reesei enzymes in the hydrolysis of the pretreated spruce substrate was about 5 °C lower than on pure cellulose (results not shown).

Fig. 2 Hydrolysis of washed, steam pretreated spruce substrate (cellulose content 18.3 gL-1) with Celluclast 1.5 L FG alone (A) or supplemented with Novozym 188 (B) at various temperatures at pH 5. The dosage of Celluclast was 22 FPU g-1 cellulose and the Novozym 188 P-glucosidase 550 nkatg-1 cellulose. И50 °C, Ш55 °C, ♦бО °C, <>65 °C and • 70 °C

8

In view of the intrinsic redox restrictions associated with the combined in­troduction of xylose reductase and xylitol dehydrogenase into S. cerevisiae, it is relevant to explore alternative metabolic engineering strategies. As will be discussed below, expression of heterologous genes for xylose isomerase (an enzyme that does not naturally occur in S. cerevisiae) offers such an alterna­tive [14]. In the following sections, we will briefly discuss the properties and taxonomic distribution of xylose isomerases. This will be followed by a brief overview of previous attempts at functional expression of xylose isomerases in S. cerevisiae. We will then discuss how, in the past few years, fast progress has been made due to the discovery of a new, fungal xylose isomerase gene. Finally, we will discuss the status of the xylose isomerase strategy with regard to full-scale industrial application.

2

Cellulose is organized into insoluble crystalline ribbons with extensive hydro­gen bonds between strands [98,99]. This structure is not easily hydrated and the fungal cellulase enzymes used for hydrolysis have low catalytic rates in comparison to other glycosidases. Thus, the cost of these enzymes is a ma­jor consideration in cellulose utilization [100]. An additional challenge is the feedback inhibition of cellulose hydrolysis by glucose and cellobiose, the products of the hydrolysis process. Simultaneous saccharification and fer­mentation (SSF), developed by Gulf Oil Company in 1976, combines cellulose saccharification and fermentation of the resultant glucose by Saccharoymyces in a single vessel [ 101,102]. Recent work in this area has focused on reducing the supplemental cellulase demand by engineering the biocatalysts to produce recombinant cellulase enzymes.

Erwinia chysanthemi contains two endoglucanases, CelZ and CelY, which work synergistically to degrade amorphous cellulose and carboxymethyl cel­lulose [103]. In order to effectively reduce the demand for cellulase supple­mentation, CelZ and CelY need to be expressed at high levels and secreted by the biocatalyst. The use of a surrogate Z. mobilis promoter and addition of the E. chrysanthemi out secretion system resulted in high levels of CelZ expres­sion in E. coli and K. oxytoca P2, with active glycan hydrolase representing approximately 5% of total cellular protein in both organisms [104,105]. High endoglucanase activity from recombinantly expressed CelZ and CelY enabled K. oxytoca M5A1 to produce ethanol from amorphous cellulose without the addition of supplemental cellulase enzymes [106,107].

As described above, the primary product of cellulose digestion by en — doglucanase and cellobiohydrolase is cellobiose. Unfortunately, cellobiose is a potent inhibitor of these enzymes [108]. The ability to metabolize cel — lobiose is widespread in prokaryotes [108] and is desirable for biomass­utilizing strains. Ethanologenic K. oxytoca P2 has the native ability to trans­port and metabolize cellobiose, reducing the initial demand for supplemental P-glucosidase [50]. The K. oxytoca cellobiose-utilization operon casAB has been functionally expressed in E. coli KO11, enabling production of ethanol from cellobiose or, with the aid of commercial cellulase, from mixed-waste office paper [50,109].

In the pursuit of a decreased supplemental cellulase demand, an alterna­tive approach to biocatalyst engineering is the use of non-biological processes to improve cellulose hydrolysis. For example, the use of ultrasound during SSF resulted in a 20% increase in ethanol production from mixed-waste office paper by K. oxytoca P2 [110]. Additionally, fungal cellulase demand dur­ing mixed waste office paper fermentation by P2 was reduced by recycling cellulase [54].

5