Metabolic Engineering - T. Scheper and Jens Nielsen

procedure. In general, hardwood hydrolysates contain a high proportion of xylose, while softwood hydrolysates are rich in mannose [57, 58].

In hardwood, the xylan, or glucuronoxylan, consists mainly of D-xylose residues, most of which are acetylated, and 4-O-methyl-D-glucuronic acid residues (Fig. 2). Minor amounts of other constituents, such as L-rhamnose and D-galacturonic acid, can also be found. The molar ratio of 4-O-methyl-a-D-glu- curonic acid, acetyl groups, and xylose has been estimated to be 1:7:10 [59]. Hardwood also contains small amounts (2–5% dry weight (DW)) of glucomannan, composed of D-glucose and D-mannose.

In softwood, mannan is the dominating type of hemicellulose and can account for ~20% of the DW. The typical softwood mannan is a glucomannan with varying contents of D-galactose. A mannan with high galactose content is referred to as a galactogluco mannan. Softwood also contains arabinoglucuronoxylan, which is composed of L-arabinose, 4-O-methyl-a-D-glucuronic acid, and D-xylose in the molar ratio 1.3:2:10 [59].

Agricultural residues, such as wheat straw and sugarcane bagasse, contain large amounts of xylan (Table 1), some arabinan, and only very small amounts of mannan. Acid hydrolysis of wheat straw and sugarcane bagasse has been found to result in hydrolysates in which glucose and xylose together make up 95% or more of the recovered monosaccharides [57]. Corn fiber hemicellulose is basically an arabinoglucuronoxylan containing a very high proportion of pentose residues (xylose and arabinose) and, in addition, some hexoses and uronic acids, such as galactose and 4-O-methylglucuronic acid. The sugars obtained from corn fiber are mostly glucose (25–38%), xylose (30–41%), and arabinose (21–28%) [60].

2.2

Hydrolysis of Lignocellulose Polysaccharides

Monosaccharides are formed from the lignocellulose polysaccharides, hemicellulose and cellulose, by using either acid or enzymatic hydrolysis. Acid hydrolysis of lignocellulosic materials can be performed as a pretreatment stage, using sulfur dioxide or sulfuric acid [61, 62], resulting in a hemicellulose hydrolysate. In the second step, the solid residue is hydrolyzed using acid or cellulolytic enzymes in order to release glucose from cellulose. When cellulolytic enzymes are employed, the hydrolysis of the cellulose may be performed as a simultaneous saccharification and fermentation (SSF) process [63, 64].

Enzymatic saccharification steps usually involve enzymes obtained from an external source, such as cellulolytic enzymes from the filamentous fungus Trichoderma reesei [65]. The yeast S. cerevisiae is not known to hydrolyze efficiently either cellulose or hemicellulose. Much research has been conducted during the past two decades on the heterologous expression of genes encoding cellulolytic and hemicellulolytic enzymes in S. cerevisiae, working towards the potential consolidation of lignocellulosic hydrolysis and fermentation by a single microorganism, such as S. cerevisiae.

The interwoven nature of cellulose and hemicellulose in lignocellulosic raw material necessitates partial hydrolysis of cellulose to expose the hemicellulose

fraction (mannans and xylans) to enzymatic hydrolysis. The enzymatic hydrolysis of crystalline cellulose requires the synergistic action of three different types of cellulolytic enzymes: endo-b-1,4-D-glucanases (endoglucanases), cellobiohydrolases, and cellobiases (b-D-glucosidases). Endoglucanase genes of both bacterial and fungal origin have been expressed in S. cerevisiae, generating recombinant yeasts that efficiently degrade glucans and amorphous cellulosic materials [66–71]. Cellobiohydrolase genes have been expressed with only limited success. Recombinant S. cerevisiae strains producing fungal cellobiohydrolases catalyzed only partial solubilization of microcrystalline cellulose [71–74]. However, various recombinant b-glucosidase S. cerevisiae strains have been constructed that allow the effective conversion of cellobiose and shorter oligosaccharides, released by endoglucanases and cellobiohydrolases from cellulose, to fermentable D-glucose [71, 74–79].

The enzymatic hydrolysis of galacto(gluco)mannans, present particularly in softwoods, is accomplished through the action of endo-b-1,4-mannanases which randomly cleave the b-mannosidic linkages within the main chain, together with b-1,4-mannosidases and a-1,6-galactosidases. Genes for mannandegrading enzymes have been expressed and characterized in S. cerevisiae [80–83]; however, these genes have not yet been co-expressed in S. cerevisiae to develop a mannan-utilizing recombinant yeast. The effective enzymatic release of D-xylose from xylan requires the simultaneous production of several hemicellulases: b-1,4-xylanases (xylanases) and side-chain-splitting enzymes such as a-l-arabinofuranosidases, a-glucuronidases, and acetyl and phenolic esterases. The final hydrolysis of xylobiose and small xylo-oligosaccharides to D- xylose requires the action of b-xylosidases. Numerous xylanases and side- chain-splitting enzymes have been successfully produced in S. cerevisiae [84–93]. Recombinant S. cerevisiae producing b-xylanase II from T. reesei yielded both D-xylose and xylobiose as end products from birchwood xylan, with xylobiose as the major product [93]. However, S. cerevisiae producing both T. reesei b-xylanase II and Aspergillus niger b-xylosidase released substantially more D-xylose than xylobiose as end-products, representing a conversion of xylan to D-xylose of more than 40%. The introduction of the genes for these xylanolytic enzymes into xylose-utilizing recombinant S. cerevisiae strains [94] could pave the way for the bioconversion of the hemicellulose fraction of lignocellulosic materials to ethanol, as a microbial phenomenon by a single fermentative microorganism, S. cerevisiae.

2.3

Fermentation Inhibitors in Lignocellulose Hydrolysates

Only recently has the fermentative performance of recombinant xylose-utiliz- ing Saccharomyces strains been investigated in lignocellulose hydrolysates [95–97]. In addition to dealing with varying proportions and concentrations of the monosaccharides, the metabolically engineered strains will be affected by a variety of low molecular weight compounds present in the hydrolysates. Individually, and in synergy, these may both stimulate and inhibit fermentation, which makes it difficult to predict the fermentability of a particular hydrolysate.

with good affinity towards xylose [115] had no effect on the xylose fermentation rate in a similar recombinant strain [116]. However, these strains produced low XDH activities [117] and XK was not overproduced, thus the xylose-me- tabolizing pathway had severe limitations (see Sects. 4 and 5). In this context, it is worth noting that XR, the first enzyme in the xylose-utilizing pathway, has a low affinity towards xylose (KM is 68 mmol l–1 or 97 mmol l–1, depending on the cofactor [118]), which means that high intracellular concentrations of xylose are necessary for efficient utilization. Calculations based on the zero trans-in- flux measurements do not account for the significant efflux of xylose under physiological conditions due to facilitated diffusion working in both directions. For glucose-derepressed cells the net glucose influx is only half of the uptake rate determined in vitro, because of the build-up of intracellular glucose [113].

3.2

Xylose Transport in Natural Xylose-Utilizing Yeasts

Most of the natural xylose-utilizing yeasts have at least two kinetically distinct xylose transport systems (Table 2) [119–125]. The low-affinity transporter is generally shared with the structural analog glucose, while the high-affinity transporter is specific for xylose. High-affinity systems symport xylose together with a proton, using the proton motive force (Fig. 3). The low-affinity systems, on the other hand, are generally thought to transport xylose by a facilitated diffusion process driven by the concentration gradient [119, 121]. A low-affinity proton symporter reported from P. stipitis displayed a KM of 2–3 mmol l–1 [123], whereas another investigation [122] reported a markedly different low-affinity system (KM = 380 mmol l–1). Since the low-affinity xylose-proton symporter

was strongly inhibited by glucose and starvation changed its kinetic constants similarly to the glucose-proton symporter, it may be an unspecific glucose-pro- ton symporter. In Candida utilis, the low-affinity xylose transport was inhibited by D2O, protonophores, and ATPase inhibitors, suggesting that it might be a proton symport [120]. On the other hand, proton movement was not observed and diethylstilbestrol, a potent ATPase inhibitor, had no effect on the xylose transport rate.

Recently, three genes from P. stipitis coding for low-affinity glucose transporters have been cloned and sequenced [124]. The glucose-induced SUT1 has a KM = 1.5 mmol l–1 for glucose and KM = 149 mmol l–1 for xylose. It seems to be the major contributor to the low-affinity component of the glucose and xylose transport, which is evident from the lack of a low-affinity component in the sut1 disruption strain grown on glucose. SUT2 and SUT3 have somewhat higher affinities for xylose (KM = 49 mmol l–1 and 103 mmol l–1, respectively), but they are only expressed under fully aerobic conditions, and have a substrate-con- centration-modulated affinity for glucose. Such phenomena have previously been observed for the S. cerevisiae HXT2 [126].

It is noteworthy that the Michaelis constant for the low-affinity xylose transport in P. stipitis is different from that for the SUT1 determined in an hxt1–7 deletion strain of S. cerevisiae [124]. This suggests that the apparent low affinity component is in fact the superposition of several individual transporters. In S. cerevisiae three or more hexose transporter genes are transcribed at the same time [127], yet only two components of the glucose uptake system can be kinetically distinguished [128]. It is, therefore, expected that the kinetic constants for xylose transporters from various species will be refined, once the corresponding genes are cloned and expressed in a model system such as the hxt1–7 S. cerevisiae strain.

3.3

Engineering Xylose Transport in S. cerevisiae

The low affinity of S. cerevisiae transporters for xylose and the inhibition by glucose underlines the necessity of engineering this metabolic step. However, there are no known nucleotide sequences of yeast or fungal origin coding for a xylose transporter with suitable properties. Cloning and characterization of yeast xylose transporters may be greatly facilitated by the use of hexose transporter deleted S. cerevisiae strains. The cloning of the low-affinity glucose/xylose transporters from P. stipitis was accomplished by functional complementation [124] of the glucose uptake by an hxt1–7 deletion strain [129]. A similar approach was used for cloning of an unspecific monosaccharide transporter from the filamentous fungus T. reesei [130]. Recently, an S. cerevisiae strain, EBY.VW4000, has been developed with all hexose transporters deleted (hxt1–17, gal2, stl1, agt1, ydl247w, yjr160c) [131]. This strain is expected to become a highly useful tool for cloning of specific high-affinity xylose transporter genes from natural xylose-utilizing organisms.

studied in strains with ratios of XR:XDH enzyme activities ranging from 17.5 to 0.06. The strains were cultivated in shake flasks with minimal medium under oxygen-limited conditions. The strain with the highest XR:XDH ratio produced xylitol with a yield of 0.82 g xylitol g xylose–1, whereas no xylitol was formed by the strain with the lowest ratio.

In a theoretical approach to optimizing the levels of XR and XDH and also XK, the enzyme phosphorylating xylulose to xylulose 5-phosphate, a kinetic model including the three enzymes was constructed [143]. Based on reported kinetic data for the three enzymes, the optimal XR:XDH:XK ratio was determined to be 1:10:4 for minimal xylitol formation. Experiments confirmed that a decreasing XR:XDH ratio decreased xylitol and acetate formation, whereas the formation of ethanol increased. Overproduction of XK enhanced the specific xylose consumption [143].

4.1.2

Protein Engineering – Fusion Protein

Xylitol formation would decrease if recycling of NADH/NAD+ could take place in a single enzyme where NADH was oxidized at the XR site and reduced at the XDH site. Xylitol would then remain an enzyme-bound intermediate and the high microenvironmental concentration of NADH around the XR site would favor the utilization of NADH. A series of XR and XDH fusion proteins were constructed [144]. The specific activities of XR and XDH depended on the order in which the two polypeptides were coupled in the hybrid protein, as well as on the length and composition of the connecting peptide. To obtain both XR and XDH activity, XDH had to be at the N-terminus and XR at the C-terminus of the fusion protein. Constructs with the opposite order lacked XR activity. The specific XDH activity increased threefold in the construct containing a linker consisting of 12 amino-acid residues, compared with a 7-residue linker, while the XR activity remained constant.

The fusion protein exhibited only one tenth of the XR and XDH activity exhibited by the two enzymes when expressed from separate genes. When the fusion protein was co-expressed with the individual XR and XDH enzymes, aggregates composed of the fusion protein and the separate XR and XDH subunits were confirmed by gel chromatography. This construct had specific XR and XDH activities similar to the individually expressed enzymes. Recombinant S. cerevisiae strains harboring the fusion protein aggregate utilized xylose under oxygen-limited conditions in a defined medium and produced less xylitol than a strain expressing the enzymes separately.

4.1.3

Protein Engineering – Site-Specific Mutagenesis

Protein engineering has also been used to alter the co-factor preferences of XR and XDH. Inhibition studies of P. stipitis XR suggested that histidine and cysteine residues might be involved in co-factor binding [145]. Using site-directed mutagenesis, the three cysteine residues were individually changed into serine

residues [146]. The three mutant forms of XR showed activity when expressed in E. coli, but only at levels 50–70% lower than that of the wild type. The affinities for xylose, NADPH and NADH did not vary significantly and it was concluded that none of the cysteine residues directly participates in the binding of co-factor.

Yeast xylose reductases as well as mammalian aldo-keto reductases contain a strictly conserved binding motif for NADPH (Ile-Pro-Lys-Ser). It has been suggested that the 2¢-phosphate group of NADPH binds to the lysine residue in human aldose reductase. When this group was changed into a methionine residue, using site-specific mutagenesis, the resulting enzyme lost 80–90% of its specific activity and the affinity for xylose decreased by more than tenfold [147]. The affinity for NADPH decreased, but remained constant for NADH. There are, to date, no reports of the expression of any of the mutated forms of XR in S. cerevisiae.

Attempts have also been made to alter the co-factor specificity of XDH towards NADP+ instead of NAD+. Through sequence analysis of XDH, a coen- zyme-binding domain, conserved in most examined NAD+-dependent dehydrogenases was localized [148]. In the coenzyme-binding domain of horse liver alcohol dehydrogenase, an aspartate residue and a lysine/arginine residue are responsible for the interaction with the adenine ribose of NAD+. The steric properties of the aspartate residue and the repulsion between the negatively charged groups of the phosphate of NADP+ and the carboxyl group of aspartate prevent binding of NADP+. The specificity for NAD+ decreased when the aspartate residue was changed to a glycine residue. Although steric and electrostatic hindrances were avoided through this substitution, the affinity for NADP+ remained unchanged. Furthermore, the specific activity of the mutated XDH decreased to half of that of the original enzyme.

The putative binding motif of an NADP+-dependent alcohol dehydrogenase of Thermoanaerobium brockii was introduced into XDH from P. stipitis [148]. The resulting enzyme showed a specific activity of 31% of that of the unaltered enzyme and, as above, the affinity for NAD+ decreased ninefold whereas the affinity for NADP+ remained unchanged. When the altered enzyme was expressed together with a xylose reductase in S. cerevisiae, growth was observed on xylose minimal medium plates.

4.1.4

Xylitol Transport

The equilibrium constant for the conversion of xylitol to xylulose favors xylitol formation. The equilibrium would shift towards xylulose formation if the intracellular concentration of xylitol were increased. This could be achieved by limiting the xylitol excretion. The S. cerevisiae gene FPS1 encodes a channel protein, Fps1p, responsible for the facilitated diffusion of glycerol [149]. Its main role is to control the cellular osmoregulation by the accumulation and release of glycerol [150]. However, it has recently been demonstrated that xylitol inhibited the glycerol transport by this protein, suggesting that Fps1p also transports xylitol [151]. When the FPS1 gene was deleted in an S. cerevisiae strain harboring

the P. stipitis genes for XR and XDH, the excretion of xylitol decreased and ethanol production increased compared to the parental strain (B. HahnHägerdal, unpublished work). Furthermore, it was confirmed that the intracellular xylitol concentration increased.

4.1.5

Oxygen Utilization

When present, oxygen is used as an electron acceptor in the electron transport chain regenerating NAD+ for xylitol oxidation. In P. stipitis, the yield of ethanol from xylose increased with decreasing oxygen flux [33]. Recently, the impact of oxygen on xylose utilization by a recombinant S. cerevisiae strain harboring the P. stipitis genes for XR and XDH, as well as an overexpressed XK gene, was investigated in a series of continuous cultivation experiments on mixtures of 15 g l–1 xylose and 5 g l–1 glucose [143]. With increasing oxygenation, the ethanol yield, calculated, as grams of ethanol per gram of total carbohydrate uptake, remained approximately constant at around 0.34, whereas the yields of glycerol and xylitol decreased and more carbon was used instead for biomass synthesis.

4.2

Xylose Isomerase

The co-factor imbalance generated by the first two steps in xylose metabolism could be entirely circumvented if the conversion of xylose to xylulose were to be catalyzed by the prokaryotic enzyme xylose isomerase (XI, Fig. 1). D-Xylose (glucose) isomerase EC 5.3.1.5 catalyses the reversible isomerization of D-xylose and D-glucose to D-xylulose and D-fructose, respectively. XI does not require redox cofactors and cannot generate cofactor imbalance during anaerobic xylose utilization.

The different bacterial XIs fall into two distinct groups (Fig. 4) based on their physical properties and their sequence homology (reviewed in [152]). Enzymes from the high G+C Gram-positive bacteria (Actinoplanes, Streptomyces, Arthrobacter species) and Thermus species belong to group I. These enzymes have a molecular mass of approximately 45 kDa and exhibit alkaline pH optima. Group II enzymes comprise all the other XIs (for example E. coli, Bacillus, Clostridium, and Thermotoga species), including the only characterized eukaryotic XI from Hordeum vulgare [153]. The group II XIs have an extended N- terminal region and a molecular mass of approximately 50 kDa. Their pH optima are close to neutral.

4.2.1

Expression of Xylose Isomerase in S. cerevisiae

Early attempts to produce XI in S. cerevisiae have failed. Transformation with

Actinoplanes missouriensis [53] and Clostridium thermosulfurogenes [55] xylA did not result in the expression of XI, although the specific mRNA was present. The heterologous expression of the E. coli [51, 52] and the Bacillus subtilis [53]