Appl Microbiol Biotechnol (1997) 48: 141148

Springer-Verlag 1997

MINI-REVIEW

P. De Wulf E. J. Vandamme

Production of D-ribose by fermentation

Received: 29 January 1997 / Received revision: 13 March 1997 / Accepted: 15 March 1997

Abstract The production of D-ribose by fermentation has received much attention lately, possibly because of the use of this pentose to synthesize antiviral and anticancer drugs. This review briey outlines the methods that have been used to synthesize D-ribose since it was identied in yeast RNA, and focuses in particular on the latest developments in D-ribose fermentation, which have led to D-ribose yields that exceed 90 g/l. Furthermore, the various transketolase-decient D-ribose-producing mutants that are used, and the biochemical and genetic rationales applied to select them or to enhance their D-ribose productivities, are dealt with. Attention is also drawn to the unusual pleiotropic characteristics of the mutant strains, as well as to the industrial and academic applications of D-ribose.

Introduction
It has taken about 80 years, since Kossel (1891) identied D-ribose (C5H10O5, Fig. 1) in hydrolyzed yeast RNA, to establish a bacterial D-ribose fermentation process on an industrial scale (Takeda Chemical Industries Ltd.). In between, many D-ribose synthesis methods had been delineated, ranging from the chemical and enzymatic hydrolysis of yeast RNA (Bredereck and Rothe 1938;

Levene and Clarck 1921), to the chemical synthesis of from D-arabinose (Gehrke and Aichner 1927), D-gluconic acid (Steiger 1936), D-glucose (Sowden 1950; Smith 1955), L-glutamic acid (Taniguchi et al. 1974), and D-xylose (Lacourt-Gadras et al. 1992). Ultimately, only D-ribose production with transketolase and/or D-ribulose-5-phosphate-3-epimerase-decient Bacillus mutant spp. proved commercially feasible. More recently, the introduction of molecular genetics and new fermentation technologies has contributed to further improvements in bacterial D-ribose productivity, leading to D-ribose yields that exceed 90 g/l, starting from 200 g/l D-glucose. Moreover, the fermentation time and the concentration of undesirable by-products has signicantly decreased. The amount of D-ribose produced worldwide by fermentation is estimated to be around 2000 tonnes per year. The companies involved include BASF (Germany), E. Merck (Germany), HomannLa Roche (Switzerland), Pzer (USA) and Takeda Chemical Industries Ltd. (Japan). The market price of D-ribose is about U.S. $ 30/kg, and its production cost an estimated U.S. $ 1520/kg.
D-ribose

Random screening of naturally occurring D-ribose-producing microorganisms

The secretion of D-ribose by a microorganism (Penicillium brevicompactum) was rst observed (but not

P. De Wulf Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA E. J. Vandamme (&) Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Agricultural and Applied Biological Sciences, University of Gent, Coupure links 653, B-9000 Gent, Belgium Fax: + + 32 9 264 62 31 Tel: + + 32 9 264 60 27 e-mail: Erick.Vandamme@rug.ac.be

Fig. 1

D-Ribose

(furanose form)

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quantied) by Simonart and Godin (1951). Twelve years later, an unidentied bacterium was found to produce D-ribose, or D-ribose 5-phosphate (D-ribose-5-P) plus 5-phosphoribosyl 1-pyrophosphate, depending on the respective presence or absence of Mn2+, Fe2+ or Zn2+ (Suzuki et al. 1963). In 1966, Saito and Sugiyama isolated a Pseudomonas reptilivora strain from soil, which produced 0.72 g/l D-ribose from 50 g/l D-glucose. De Wulf et al. (1996a) screened four strains out of 2100 isolates from sugar-rich substrates, which secreted up to 0.36 g/l D-ribose from 30 g/l D-glucose. These included a Staphylococcus hominis strain, 2 Bacillus spp. and a Candida pelliculosa yeast strain.

Mutation and selection of transketolasedecient D-ribose-producing microorganisms

D-Ribose-5-P,

an intermediate of the pentose phosphate pathway The pentose phosphate cycle (PPC) is situated between the glycolysis and various biosynthetic cascades (Fig. 2). This pathway consists of an oxidative part, in which hexoses are decarboxylated into pentoses, and a nonoxidative branch in which transketolase and transaldo-

lase interconvert heptoses, hexoses, pentoses, tetroses and trioses. The importance of the PPC partially lies in these interconversions, which provide intermediates used to synthesize cellular building blocks. The reducing equivalents (NADPH + H+) that are liberated during the oxidative reactions are used to synthesize monomeric (amino acids, fatty acids, ...) and polymeric (proteins, RNA, ...) cell compounds (Mathews and van Holde 1990). Microorganisms that secrete D-ribose are decient in the enzymes transketolase (EC 2.2.1.1) and/or D-ribulose-5-P 3-epimerase (EC 5.1.3.1) (Fig. 2). When cultivated in a glucose-based medium, the strains partially convert this substrate via the oxidative PPC. At the transketolase conversion point, D-ribose-5-P accumulates, and is dephosphorylated and secreted into the medium to overcome feedback inhibitions exerted by this intermediate. Induction and selection of transketolase-decient microorganisms Josephson and Fraenkel (1969) were the rst to isolate transketolase-decient E. coli strains during a study of the regulation of the PPC. Their approach was based on

Fig. 2 The pentose phosphate pathway, glycolysis and some anabolic reactions. r w transketolase deciency. See text for explanation

D-Ribulose-5-phosphate

3-epimerase deciency,

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the fact that organisms that lack transketolase activity cannot utilize pentoses, or convert D-ribose-5-P into aromatic amino acids (Fig. 2). More specically, the (chemically) mutated E. coli culture was transferred to a minimal medium, free of pentoses and aromatic amino acids, but provided with penicillin G. The transketolasepositive strains grew in the medium, and were killed by the antibiotic, while transketolase-decient mutants remained in a non-growing resting mode, and thus survived the selection process. The mutants were isolated by plating culture samples on agar-based medium that contained pentoses and/or aromatic amino acids (Josephson and Fraenkel 1969). By submitting the spores of an inosine-producing Bacillus mutant to UV irradiation followed by an enrichment, as developed by Josephson and Fraenkel (1969), Sasajima et al. (1970) tried to isolate mutant strains that would convert the accumulated D-ribose-5-P into inosine-5-P. Unexpectedly several mutants lost the ability to produce inosine-5-P, and secreted D-ribose and/or D-ribulose instead (Sasajima et al. 1970). The strains lacked transketolase and/or D-ribulose-5-P 3-epimerase activity, and the intracellularly accumulated D-ribose-5-P feedback-inhibited its own conversion into inosine-5-P (Sasajima and Yoneda 1974a, b). By transducing one of these mutants with DNA from a D-ribose-non-producing, adenine- and xanthine-positive B. subtilis strain, a D-ribose-superproducing strain (60 g/l) was isolated (Yokota and Sasajima 1981; Yokota et al. 1979). In addition to D-ribose, D-gluconic acid was also secreted in the growth medium. The D-ribose/ D-gluconic acid ratio depended on the dissolved oxygen concentration during the fermentation; oxygen depletion led to the secretion of D-gluconate rather than D-ribose (Sasajima and Yoneda 1989). An additional mutation to induce an asporogenous phenotype further enhanced the D-ribose yield to 70 g/l (Sasajima 1976; Sasajima and Yoneda 1984). The causal basis of this relationship

remained obscure, until Rhaese and Groscurth (1976, 1979) found that a high concentration of phosphorylated nucleotides initiates spore formation, even in the presence of an excessive amount of carbon, nitrogen and phosphate. Since D-ribose-leaking organisms may not be able to build-up a sucient amount of these D-ribose-5P-based nucleotides, the strains may remain asporogenous. By submitting a screened Candida pelliculosa yeast strain to a mutation and enrichment strategy, as developed by Josephson and Fraenkel (1969), a C. pelliculosa mutant strain was isolated that produced 5 g/l D-ribose from 30 g/l D-glucose (De Wulf et al. 1996a). All transketolase-decient D-ribose-producing mutant strains (all Bacillus spp., Table 1) have been derived via the above or analogous mutation and enrichment processes.

Improved D-ribose productivity by recombinant DNA technology

Several D-ribose-producing bacilli co-secrete D-gluconate in the medium (Asai et al. 1978; Kintaka et al. 1986; Kishimoto et al. 1990; Miyagawa et al. 1992; Sasajima and Yoneda 1989), what decreases the D-ribose yield and interferes with the isolation of D-ribose. Miyagawa et al. (1992) increased the expression level of the bacterial gluconate operon, such that the mutant strain could take up the co-secreted D-gluconic acid and reconvert it into D-ribose. More specically, the operon was isolated from the chromosome of a Bacillus sp. and cloned into an expression vector. The regulator gene of the operon was then deleted and/or the promotor replaced by a stronger promotor sequence. When the D-riboseand D-gluconate-producing B. subtilis IFO 15138 was transformed with this plasmid, the D-ribose yield

Table 1 Batch fermentation data (substrate and product concentrations, process time) found in the literature for various D-riboseproducing Bacillus mutant spp.
Strain B. subtilis ATCC 21360 B. pumilus ATCC 21357 B. subtilis ATCC 21951 B. subtilis ATCC 21951 B. subtilis ATCC 21952 B. subtilis ATCC 31092 B. pumilus ATCC 31094 Bacillus sp. EMP-58 B. subtilis ATCC 21951 B. subtilis ATCC 21951 B. subtilis IFO 1538 B. subtilis ATCC 21951 B. subtilis ATCC 21951 B. subtilis ATCC 21951
a b

D-Glucose

(g/l)

D-Ribose (g/l)

Time of fermentation (h) 68 55 144 90 72 60 60 55 72 72 72 110 156 84

Reference Yoneda & Sasajima (1969) Yoneda & Sasajima (1969) Sasajima & Yoneda (1971) Sasajima & Yoneda (1974) Sasajima & Yoneda (1974) Sasajima et al. (1975) Sasajima et al. (1975) Asai et al. (1978) Kintaka et al. (1986) Kishimoto et al. (1990) Miyagawa et al. (1992) De Wulf et al. (1996b) De Wulf et al. (1997) De Wulf et al. (1997)

100 125 100 150 150 150 150 140 200 200 160 100/100a 200 100/50b

30 31 35 60 56 67 71 64 91 95 62 60 40 45

100 g/l D-glucose plus 50 g/l D-gluconic acid 100 g/l D-glucose plus 100 g/l D-gluconic acid

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obtained from 160 g/l D-glucose increased from 39 g/l to 62 g/l (Miyagawa et al. 1992; Table 1).

Pleiotropic properties of D-ribose-producing Bacillus spp.

In addition to direct eects of transketolase deciency (aromatic amino acid auxotrophy and inability to utilize carbon sources that are catabolized via the oxidative PPC), D-ribose-producing strains display unexpected physiological characteristics, pleiotropically linked to transketolase negativity. Defective phosphoenolpyruvate-dependent phosphotransferase system The decreased ability or inability to utilize D-glucose by transketolase (TKT ) mutants, results from a defective glucose-transporting enzyme II complex of the phosphoenolpyruvate-dependent phosphotransferase system (Sasajima and Kumada 1979; Sasajima et al. 1977). This is rstly because D-glucose still activates the phosphorylation activity of the phosphotransferase system (Sasajima and Kumada 1979), and secondly because D-mannitol and D-fructose, both utilized via the phosphotransferase system by B. subtilis (Marquet et al. 1970; Gay and Delobbe 1977), are metabolized by TKT mutants (Sasajima and Kumada 1979). Since transketolase-negative mutants have been isolated that can utilize D-glucose, transketolase may be related only indirectly to enzyme II activity (Sasajima et al. 1977). Deregulated carbon catabolite repression The synthesis of enzymes that catabolize D-mannitol (the D-mannitol phosphoenolpyruvate-dependent phosphotransferase system, D-mannitol-1-P dehydrogenase) was found to be hypersensitive to repression by D-glucose, D-gluconic acid, D-xylose and L-arabinose in a TKT mutant strain (Sasajima and Kumada 1981a). In contrast, the synthesis of enzymes that convert D-sorbitol (D-sorbitol permease, D-sorbitol dehydrogenase) and glycerol (glycerol permease, glycerol kinase and glycerol dehydrogenase) was insensitive to D-glucose repression (Sasajima and Kumada 1981a). This discrepant sensitivity to repression of enzyme synthesis was linked to a defective cell-surface structure in the TKT mutant. The synthesis of ribitol teichoic acid was probably disturbed, possibly since the latter is derived from D-ribose-5-P (Sasajima and Kumada 1981b). When TKT mutant B. subtilis ATCC 21951 was grown in a medium containing D-glucose plus D-gluconic acid or D-glucose plus D-xylose, L-arabinose, D-xylitol, D-galactose or D-gluronic acid, the strain proved to be insensitive to glucose-based carbon catabolite repression

(De Wulf 1995; De Wulf et al. 1996b). As illustrated by cultivating the transketolase-positive B. subtilis strain ATCC 6051 in the same double-substrate media (De Wulf 1995; De Wulf et al. 1996b), D-fructose 1,6-bisphosphate (glycolytically formed from D-glucose) normally triggers the formation of a serine46-P-HPrCcpa protein complex. The latter then binds to a catabolite-responsive element, which is localized in the operon that encodes the utilization of the second carbon source (Saier et al. 1996). Consequently, the co-substrate is not converted, and diauxic growth occurs. Since B. subtilis ATCC 21951 partially secreted an amount of D-glucose that was converted via the oxidative PPC into D-ribose, the intracellular concentration of D-fructose 1,6-bisphosphate probably was too low to trigger catabolite repression (De Wulf et al. 1996b).

Altered cell membrane and cell wall composition During growth, transketolase-aected Bacillus cells gradually thicken and start to take on a chainwise conguration, whereas the parent strains remain slender and distinct (Sasajima and Kumada 1981b). Cell-envelope analysis showed that TKT mutants were defective in teichoic acid and teichuronic acid formation, or in the synthesis of lipoteichoic acids. The mutants were also more sensitive to phages SP01 and SP10 (Sasajima and Kumada 1981b). TKT mutants are non-motile, asporogenous and undergo cell lysis more slowly than their parent strains (Sasajima and Kumada 1981b; 1983a, b). The isolation of revertant transketolase-positive strains, which did not generate these phenotypic alterations, conrmed a linkage with the TKT locus (Sasajima and Kumada 1979, 1983a,b). Since the phosphorylated nucleotides that initiate bacterial sporogenesis (Rhaese and Groscurth 1976; Rhaese et al. 1977) are synthesized by membrane-bound enzymes, an abnormal membrane consistency may also explain the asporogenous character of the mutant strains (Sasajima et al. 1985).

D-Ribose

production by fermentation with Bacillus spp.

Most information on microbial D-ribose production, which is probably performed only with Bacillus spp., is found in Japanese patents, where it is very vaguely described. These patents claim the use of various D-ribose-producing Bacillus mutant strains (Table 1), derived by UV irradiation or chemical mutagenesis, and sometimes further improved by genetic engineering (Kintaka et al. 1986; Kishimoto et al. 1990; Miyagawa et al. 1992; Sasajima and Yoneda 1974c; Sasajima et al. 1975; Yoneda and Sasajima 1969). The variety of mutants used, and the fact that parameters such as the growth temperature, the agitation speed, the aeration rate, the composition and the pH of the medium are only vaguely

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reported, makes it hard to analyze the fermentation results and to draw general conclusions from them. Cultivation of the inoculum The Bacillus inoculation medium that is mentioned in most patents consists of D-sorbitol (20 g/l), corn steep liquor (20 g/l), K2HPO4 (3 g/l) and KH2PO4 (10 g/l), possibly supplemented with L-tryptophan, L-phenylalanine and L-tyrosine (0.1 g/l each) (Kintaka et al. 1986; Miyagawa et al. 1992; Sasajima et al. 1975; Sasajima et al. 1975; Yoneda and Sasajima 1969). The volumetric ratio between the inoculation and fermentation medium ranges from 1.7% to 10% (Asai et al. 1978; Kintaka et al. 1986; Kishimoto et al. 1990; Sasajima and Yoneda 1971, 1974c; Sasajima et al. 1975; Yoneda and Sasajima 1969). The optimal growth temperature is about 37 C, while an (initial) medium pH between 5.5 and 8.0 has been reported (Kintaka et al. 1986; Miyagawa et al. 1992; Sasajima and Yoneda 1974c; Sasajima et al. 1975; Yoneda and Sasajima 1969). Though the agitation rate, dissolved oxygen tension, etc. have never been reported, these parameters dene the oxygen transfer to the inoculum cells; this determines their physiological status which, in turn, may aect the cellular D-ribose productivity. The subsequent D-ribose production capacity of a post-exponentially grown B. subtilis ATCC 21951 inoculum was shown to be maximal, and exceeded that of an exponentially grown culture by a factor 3 (De Wulf 1995). Since post-exponential cells occur in a chainwise conguration, one can microscopically track the cellmorphological changes over time, and determine the ideal moment to transfer the inoculum to the production fermentor (De Wulf 1995). Composition of the fermentation medium Because Bacillus spp. are able to utilize a wide range of carbon sources, D-glucose, D-fructose, D-mannitol, D-sorbitol, mannose, maltose, lactose, sucrose, glycerol, dextrine, soluble starch, hydrolyzed starch, molasses, acetic acid, D-gluconic acid and ethanol have all been listed as useful carbon substrates in the patents, though D-glucose probably is the best carbon source for economic and productivity reasons (Asai et al. 1978; De Wulf 1995; De Wulf et al. 1996b; Kintaka et al. 1986; Kishimoto et al. 1990; Sasajima and Yoneda 1971, 1974c; Sasajima et al. 1975; Yoneda and Sasajima 1969). Similarly, various nitrogen sources, including dried yeast, yeast extract, peptone, corn steep liquor, meat extract, sh meal, casein hydrolysate, amino acid mixtures and urea, and inorganic nitrogen sources such as ammonia, ammonium sulfate and ammonium nitrate, may be present in the medium (Asai et al. 1978; De Wulf 1995; De Wulf et al. 1996b; Kintaka et al. 1986; Kishi-

moto et al. 1990; Sasajima and Yoneda 1971, 1974c; Sasajima et al. 1975; Yoneda and Sasajima 1969). According to Sasajima (1976), when corn steep liquor or an inorganic nitrogen source, such as (NH4)2SO4 or NH4Cl, is used, aromatic amino acids should be supplemented, to allow sucient growth of the mutant strains. A medium composed of D-glucose (125 g/l or 150 g/l), (NH4)2SO4, CaHPO4, Ca3(PO4)2, CaCO3 and dried yeast (the individual component concentrations were not mentioned; Sasajima and Yoneda 1989) yielded 24 g/l D-ribose. With glycerol as the carbon substrate, only 3.4 g/l D-ribose was synthesized. When the glucosebased medium described above was used, but with yeast extract (15 g/l) added instead of dried yeast, 30 g/l D-ribose was obtained. When corn steep liquor (25 g/l) was used, only 9.3 g/l D-ribose was produced (Sasajima and Yoneda 1989). Despite this, Kintaka et al. (1986), Kishimoto et al. (1990), and De Wulf et al. (1996b, 1997) achieved high D-ribose titers (Table 1), using corn steep liquor as the main source of nitrogen. The signicant variations found in the D-ribose yields obtained with a certain mutant strain (e.g. B. subtilis ATCC 21951, Table 1), may be due to dierences in the process conditions. On the other hand, it may not be excluded that the industrially applied D-ribose-superproducing mutant strains, mentioned in the patents, dier from those that were submitted to the respective culture collections. This may be illustrated by the fact that B. subtilis ATCC 21951, described as co-secreting D-gluconic acid (Kintaka et al. 1986; Kishimoto et al. 1990), did not produce D-gluconic acid at all, but actively secreted acetoin and 2,3-butanediol instead (De Wulf et al. 1996b; 1997). To limit the co-secretion of D-gluconic acid by transketolase-decient Bacillus mutant spp., Sasajima and Yoneda (1974c) patented the addition of dicarboxylic acids (e.g. glutaric acid, oxalic acid, etc.) to the medium. Furthermore, the D-ribose productivity increased by 40%. De Wulf (1995) found that by-product (acetoin and 2,3-butanediol) formation by B. subtilis ATCC 21951 decreased 90% in the presence of 2 g/l sorbic acid, thereby enhancing the D-ribose yield by 40%. This was explained by an energetic exhaustion of the cells, since they need to secrete the carboxylic protons liberated from the intracellularly diused acid. Because ATP, derived from D-ribose-5-P, is used in this process, D-glucose may be more actively ushed into the oxidative PPC to provide sucient levels of ribose-derived ATP. Consequently, a higher D-ribose-5-P concentration may accumulate at the transketolase conversion point, resulting in an increased D-ribose yield (De Wulf 1995). Fermentation conditions
D-Ribose production is usually performed batchwise. Large-scale synthesis of D-ribose by continuous culture, fed-batch techniques or immobilized cell systems, has not been reported.

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In the literature, the agitation and aeration rates have been mentioned only vaguely. Since D-ribose is formed via the oxidative PPC, aerobic process conditions ought to be preferred. This was recently conrmed by a series of batch processes, performed with B. subtilis ATCC 21951, in which the aeration and agitation rates were varied. The more oxidative the process, the faster was the substrate (D-glucose) utilized, the lower the amounts of by-products (acetoin and 2,3-butanediol) formed, and the higher the D-ribose concentration (De Wulf et al. 1997). The best temperature for D-ribose synthesis is about 37 C (Asai et al. 1978; Kintaka et al. 1986; Kishimoto et al. 1990; Sasajima and Yoneda 1971, 1974c; Sasajima et al. 1975; Yoneda and Sasajima 1969). The optimal medium pH, with D-glucose as the carbon source, is pH 6.0 (for B. subtilis ATCC 21951; De Wulf et al. 1997). Lower pH values slow down bacterial growth, while higher pH values stimulate biomass synthesis and glycolytic D-glucose conversion, leading to suboptimal D-ribose titers and high concentrations of glycolytic byproducts (De Wulf et al. 1997). In industry, fermentation processes may be performed without pH control, starting from pH 7.5 (Asai and Kono 1984), pH 7.0 (Asai et al. 1978) or pH 6.0 (Sasajima and Yoneda 1989). The fermentation time span is related to the initial carbon source concentration. The fact that transketolase deciency induces a decreased D-glucose uptake rate may make the D-ribose production process long lasting (De Wulf et al. 1997; Table 1). A typical glucose-based fermentation, performed with B. subtilis ATCC 21951, is shown in Fig. 3. The legend refers to the process parameters. By replacing D-glucose with another carbohydrate (D-fructose, D-sorbitol, D-mannitol), the duration of fermentation decreases, but the D-ribose yield signicantly drops (De Wulf 1995). A successful approach consists of partially replacing D-glucose with a PPC-converted substrate, such as D-gluconic acid or D-xylose (De Wulf et al. 1996b) (Table 1). Under such conditions, a faster substrate conversion is obtained

(optimal pH 6.5), the D-ribose productivity improves (50%), and the synthesis of by-products drastically drops (De Wulf et al. 1997), making D-ribose recovery easier and cheaper. In conclusion, an oxidatively cultured, post-exponentially grown Bacillus inoculum is preferred to start a high-yielding D-ribose fermentation. The composition of the inoculation medium seems not to be as important as the physiological state of the cells (De Wulf 1995). The fermentation process should be performed as oxidatively as possible (using overpressure, oxygen-enriched air and in situ control of the dissolved oxygen concentration around 70% saturation; De Wulf et al. 1997). Regarding the composition of the fermentation medium, yeast extract shifts D-glucose towards glycolysis, leading to low D-ribose yields, and high concentrations of biomass and glycolytic end-products. Corn steep liquor plus (NH4)2SO4 and aromatic amino acids (if inadequate in corn steep liquor), are an excellent alternative (De Wulf 1995). The pH of the medium should preferably be controlled, though pH-free processes, performed in a suciently buered medium, may be appropriate (De Wulf et al. 1997).

Applications of D-ribose
On a large scale, D-ribose is chemically converted into nucleotide avor enhancers (e.g. inosine monophosphate; Atkinson and Mavituna 1991), and riboavin (vitamin B2), which is used in pharmacy, medicine, cosmetics, (health) food, and animal feed (Sasajima and Yoneda 1989). D-Ribose, as such, has been used to treat myocardiac ischemia (Zimmer 1992), to enhance the heart's tolerance to ischemia, and to improve the overall cardiac function (Zimmer 1992; Zimmer et al. 1984). Exercise-induced muscular pain, stiness due to myoadenylate deaminase deciency, and muscle aches resulting from an intracellular shortage of phosphorylase (Mc Ardle's disease), have been treated with D-ribose (Gross et al. 1991). Bicyclo[3.1.0]hexanes have been prepared from D-ribose for conversion into steroids, prostanoids, vitamin D analogues, terpenoids and modied amino acids (Gallos et al. 1994b). (2S,3R,4S)-2-Amino-1cyclohexyl-6-methylheptane-3,4-diol, an isostere of (antihypertensive) renin inhibitors, has been regio- and stereospecically synthesized from D-ribose (Chan and Hsiao 1992). fMethylinosine monophosphate, a purine derivative prepared from D-ribose, was shown to attenuate HIVinduced immunosuppression (Hadden et al. 1992). Modied nucleoside antiviral and anticancer agents, such as neplanocin-A (Hill et al. 1994), formycin, pyrazofurin (Rycroft et al. 1995), and analoges of their key intermediates (Gallos et al. 1994a), have all been enantioselectively synthesized from D-ribose. A homochiral pentacyclic molecule, prepared from D-ribose, was used to synthesize halochondrins, that are highly eective

Fig. 3 Typical D-glucose(200 g/l)-based batch fermentation prole, obtained with the D-ribose-producing Bacillus subtilis strain ATCC 21951. The agitation speed was 1000 rpm; the aeration rate 3 vvm (De Wulf et al. 1997)

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antitumor (melanoma and leukemia) agents (Cooper and Salomon 1990). Enantiomerically pure Z vinyl iodide was derived from D-ribose for conversion into taxol and its congeners, compounds eective against refractory ovarian cancer (Paquette and Bailey 1995). The use of D-ribose as a chiral synthon to produce molecules with therapeutic utility, has thus become wellestablished. This concept may be further stimulated by an increasingly ecient and economic D-ribose production by fermentation.

Future perspectives
During the last decades, high D-ribose levels have been obtained with TKT mutant Bacillus spp., primarily through optimization of the fermentation process. Higher yields may be achieved by engineering mutant strains that have an increased oxidative pentose phosphate pathway activity, and which are totally decient in by-product formation. This approach, combined with already available or new process technology (e.g. fedbach fermentations), may lead to even shorter fermentation times, higher D-ribose yields and a more economic product recovery. Consequently, the market price of D-ribose might decrease which, in turn, may stimulate its use in biomedical and organo-chemical research, leading to new applications for D-ribose and its derivatives.
Acknowledgements Pfeifer und Langen (Dormagen, Germany) is sincerely acknowledged for nancial support of this research. Dr. Wim Soetaert and Dr. Dieter Schwengers are thanked for their helpful discussions on the subject.

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