Xylitol was a significant biochemical feature of the metabolic routes for the xylose presented to ethanologenic cultures in hydrolysates of the hemicelluloses from ligno — cellulosic biomass (chapter 2, section 2.3, and chapter 3, section 3.2). With an organ­oleptic sweetness to human taste approximately equivalent to sucrose, however, it is a fine chemical product in its own right as a low-calorie sweetener — xylose sugars
are not metabolized by the human consumers of xylitol-containing chewing gums (figure 3.2). Other envisaged uses include15

• Production of anhydrosugars (as chemical intermediates) and unsaturated polyester resins

• Manufacture of propylene and ethylene glycols as antifreeze agents and unsaturated polyester resins

• Oxidation to xylonic and xylaric acids to produce novel polymers (polyes­ters and nylon-type structures)

The production of xylitol for use as a building block for derivatives essentially requires no technical development, and if the xylose feedstock is inexpensive (as a product of biomass processing), then the production of xylitol could be done for very low cost.

The accumulation of xylitol during ethanologenesis from lignocellulosic sub­strates is, of course, unwanted and quite undesirable — for process as well as for eco­nomic reason (chapter 3, section 3.2). Viewed as an economically valuable product, xylitol formation and production acquire a different biotechnological perspective, and patenting activity has recently been intense (table 8.2). Biochemical efforts also continue to locate and exploit enzymes for bioprocessing hemicelluloses and hemi — cellulosic waste streams, for example:

•

An L-xylulose reductase identified from the genome sequence of the fil­amentous mold Neurospora crassa has been heterologously produced in E. coli for the production of xylitol.49

• A P-xylosidase from Taloromyces emersonii has been shown to be superior to the enzyme from the industrial fungus Hypocrea jecorina in releasing xylose from vinasse, the solid-waste material from ethanol fermentations.50

• Xylan residues in hemicelluloses can be variably esterified with acetyl, fer — uloyl, and p-coumaryl residues; hemicellulose deacetylating esterases have been characterized from fungal species and shown to only be highly effec­tive when mixed in multiplexes of enzymes capable of using all the possible structures as substrates for enzyme action.51

• Feruloyl esterases have recently been designed as novel chimeric forms with cellulose and hemicellulose binding proteins to improve their efficien­cies with plant polymeric substrates.52,53

• Ferulic acid has a number of potential commercial applications as an anti­oxidant, food preservative, anti-inflammatory agent, photoprotectant, and food flavor precursor; major sources — brewer’s spent grain, wheat bran, sugarbeet pulp, and corn cobs — make up 1-2% of the daily output of the global food industry, and ferulic acid can be released from brewer’s grain and wheat bran by feruloyl esterases from the thermophilic fungus Humi — cola insolens.54

Once acquired, a hemicellulose hydrolysate contains a variety of hexoses and pentoses; yeast highly suitable for the production of xylitol from the xylose present in the mix may, however, preferentially utilize glucose. One solution to this problem is to remove the rapidly utilized hexoses before removing the cells and replacing them with a purposefully xylose-grown cell batch, as was demonstrated with dilute acid hydrolysates of corn fiber.55 With Saccharomyces cerevisiae expressing the Pichia stipitis gene for xylose reductase, the presence of glucose inhibited xylose uptake and biased the culture toward ethanol production; controlling the glucose concentra­tion by feeding the fermentation to maintain a high xylose:glucose ratio resulted in a near-quantitative conversion of xylose to xylitol, reaching a titer of 105 g/l xylitol.56 With a double-recombinant strain of S. cerevisiae carrying the xylose reductase genes from both P. stipitis and Candida shehatae, quite different microbial biochem­istry occurred in a more process-friendly formation (at close to theoretical levels) of xylitol from a mixture of xylose (the major carbon source) and glucose, galactose, or mannose as the cosubstrate — indeed, the presence of the cosubstrate was manda­tory for continued metabolism of the pentose sugar.57 In a gene-disrupted mutant of C. tropicalis, with no measurable xylitol dehydrogenase activity, glycerol proved to be the best cosubstrate, allowing cofactor regeneration and redox balancing, with a xylitol yield that was 98% of the maximum possible.58

Bioprocess engineering for xylitol production appears straightforward; aerated cultures with pH regulation and operated at 30°C appear common. With Brazilian sugarcane bagasse as the source of the hemicellulosic sugars, xylitol from the har­vested culture broth was recovered at up to 94% purity crystallization from a clari­fied and concentrated broth.59

Xylitol dehydrogenase enzymes catalyze reversible reactions. The catabolism of xylitol proceeds via the formation of D-xylulose (figure 3.2). Fungal pathways of L-arabinose utilization can include L-xylulose as an intermediate, a much less common pentulose sugar.60-63 Such “rare” sugars are of increasing interest to metabolic bio­chemists because it has long been appreciated that many naturally occurring antibi­otics and other bioactives contain highly unusual sugar residues, sometimes highly modified hexoses derived by lengthy biosynthetic pathways (e. g., erythromycins); many secondary metabolites elaborated by microbes may only exhibit weak anti­biotic activity but are or can easily be converted into chemicals with a wide range of biologically important effects: antitumor, antiviral, immunosuppressive, anti- cholesterolemic, cytotoxic, insecticidal, or herbicidal.64 To the synthetic chemist, therefore, the availability of such novel carbohydrates in large quantities offers new horizons in development novel therapeutic agents: for example, L-xylulose offers a promising route to inhibiting the glycosylation of proteins, including those of viruses.65

Equally, such unconventional chemicals may have very easily exploited proper­ties as novel fine chemicals. The hexose D-tagatose is an isomeric form of the com­monly occurring sugar D-galactose (a hexose present in hemicelluloses, figure 1.23) and has attracted interest for commercial development.66 Because of its very rare occurrence in the natural world, a structure such as d-tagatose presents a “tooth — friendly” metabolically intractable sugar to human biochemistry. Fortuitously, the enzyme L-arabinose isomerase includes among its spectrum of possible substrates d-galactose; the enzyme can be found in common bacteria with advanced molecular genetics and biotechnologies, including E. coli, Bacillus subtilis, and Salmonella typhimurium and, when expressed in suitable hosts, can convert the hexose into d — tagatose with a 95% yield.67 Even more promising for industrial use is the efficient bioconversion of d-galactose to d-tagatose using the immobilized enzyme, more active than free L-arabinose isomerase and stable for at least 7 days.68 Both the enzyme and recombinant L-arabinose isomerase-expressing cells can be used in packed-bed bioreactors, the cells being particular adaptable to long production cycles.69,70

Synthetic chemistry offers only expensive and low-yielding routes to the rare sugars, but uncommon tetroses, pentoses, and hexoses can all be manufactured with whole cells or extracted enzymes acting on cheap and plentiful carbon sources, including hemicellulose sugars; bioproduction strategies use an expanding toolkit of enzymes, including D-tagatose 4-epimerase, aldose isomerase, and aldose reduc­tase.71-73 Together, the rare sugars offer new markets for sugars and sugar derivatives of at least the same magnitude as that for high-fructose syrups manufactured with xylose (glucose) isomerase.