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Effect of sugars on D-arabitol production and glucose metabolism in Saccharomyces rouxii.

The effect of sugars on the production of d-arabitol and on the glucose catabolic pathways was investigated in the osmotrophic yeast Saccharomyces rouxii. The activity of d-arabitol dehydrogenase, which served as a measure of total d-arabitol production, increased when cells were grown in the presence of increasing glucose concentrations. Growth in sucrose had no effect on the enzyme activity. A high intracellular concentration of d-arabitol could be demonstrated when the cells were grown in a 60% glucose medium and could be eliminated by anaerobic growth or growth in the presence of 4 mg of chloramphenicol per ml. A mutant was isolated that would not grow in 60% glucose; although the regulation of d-arabitol dehydrogenase was altered in this strain, the production of d-arabitol was not eliminated. The activity of d-arabitol dehydrogenase followed the growth phases of the parent strain when the cells were preadapted to 30% glucose. If the cells were adapting from 1 to 30% glucose, a large increase in enzyme activity was detected before growth occurred. Protein synthesis was found to be involved in this increase in activity. There was an increased participation of the pentose phosphate pathway when the cells were grown in the presence of increasing glucose concentrations. The mutant strain had only an 11% pentose phosphate pathway participation compared with 20% for the parent strain in glucose. The results suggest that the active pentose phosphate pathway is involved in glucose tolerance by providing a plentiful supply of reduced nicotinamide adenine dinucleotide phosphate which is necessary for cell survival.

Adaptation, Physiological

Purification and properties of Klebsiella aerogenes D-arabitol dehydrogenase.

An Escherichia coli K12 strain was constructed that synthesized elevated quantities of Klebsiella aerogenes D-arabitol dehydrogenase; the enzyme accounted for about 5% of the soluble protein in this strain. Some 280 mg of enzyme was purified from 180 g of cell paste. The purified enzyme was active as a monomer of 46,000 mol.wt. The amino acid composition and kinetic constants of the enzyme for D-arabitol and D-mannitol are reported. The apparent Km for D-mannitol was more than 3-fold that for D-arabitol, whereas the maximum velocities with both substrates were indistinguishable. The enzyme purified from the E. coli K12 construct was indistinguishable by the criteria of molecular weight, electrophoretic mobility in native polyacrylamide gel and D-mannitol/D-arabitol activity ratio from D-arabitol dehydrogenase synthesized in wild-type K. aerogenes. Purified D-arabitol dehydrogenase showed no immunological cross-reaction with K. aerogenes ribitol dehydrogenase. During electrophoresis in native polyacrylamide gels, oxidation by persulphate catalysed the formation of inactive polymeric forms of the enzyme. Dithiothreitol and pre-electrophoresis protected against this polymerization.

Amino Acids

Growth on D-arabitol of a mutant strain of Escherichia coli K12 using a novel dehydrogenase and enzymes related to L-1,2-propanediol and D-xylose metabolism.

Escherichia coli K12 cannot grow on D-arabitol, L-arabitol, ribitol or xylitol (Reiner, 1975). Using a mutant of E. coli K12 (strain 3; Sridhara et al., 1969) that can grow on L-1,2-propanediol, a second-stage mutant was isolated which can utilize D-arabitol as sole source of carbon and energy for growth. D-Arabitol is probably transported into the bacteria by the same system as that used for the transport of L-1,2-propanediol. The second-stage mutant constitutively synthesizes a new dehydrogenase, which is not present in the parent strain 3. This enzyme, whose native substrate may be D-galactose, apparently dehydrogenates D-arabitol to D-xylulose, and its structural gene is located at 68.5 +/- 1 min on the E. coli genetic map. D-Xylulose is subsequently catabolized by the enzymes of the D-xylose metabolic pathway.

Carbohydrate Epimerases

Xylitol and D-arabitol toxicities due to derepressed fructose, galactitol, and sorbitol phosphotransferases of Escherichia coli.

d-Arabitol was observed to be toxic to many laboratory strains of Escherichia coli K-12, and xylitol was found to be toxic to an existing E. coli C mutant strain. Fructose-specific components of the phosphoenolpyruvate:sugar phosphotransferase system are required for xylitol toxicity. Selection for xylitol resistance results in Fru(-) strains blocked in fructose phosphotransferase. Introduction of the ptsF or ptsI mutation into a xylitol-sensitive strain eliminates sensitivity. [(14)C]fructose uptake experiments imply that the mutation to xylitol sensitivity, which is co-transducible with ara and leu, results in derepression of normally inducible fructose phosphotransferase. Wild-type strains also become xylitol sensitive if induced by (and then removed from) fructose. Xylitol toxicity is prevented by fructose in both wild-type and mutant strains. Circumstances causing xylitol, a new food additive, to become toxic to an otherwise insensitive wild-type organism have not been reported previously. The d-arabitol-sensitive laboratory strains are galactitol (dulcitol) utilizers, although most other strains are not. Selection for d-arabitol resistance results in Gat(-) strains blocked in a constitutive galactitol-specific component of the phosphotransferase system. A mutation causing d-arabitol sensitivity occurred many years ago in AB284, the parent of AB311, AB312, AB313, and many other strains. d-Arabitol sensitivity also occurs in sorbitol-constitutive strains and is shown, like the previous two instances of pentitol toxicities, to result from a constitutive phosphotransferase, which is blocked in mutants selected for resistance.

Arabinose

Ribitol and D-arabitol catabolism in Escherichia coli.

In Escherichia coli C, the catabolism of the pentitols ribitol and D-arabitol proceeds through separate, inducible operons, each consisting of a dehydrogenase and a kinase. The ribitol operon is induced in response to ribulose, and the D-arabitol operon is induced in response to D-arabitol. Each operon is under negative control. The genes of the ribitol and D-arabitol operons are very closely linked and lie in a mirror image arrangement, rtlB-rtlA-rtlC-atlC-atlA-atlB, between metG and his on the E. coli chromosome.

Chromosome Mapping

Engineering of xylose metabolic pathways in Rhodotorula toruloides for sustainable biomanufacturing.

The oleaginous yeast Rhodotorula toruloides is a promising microbial cell factory for the sustainable production of biofuels and value-added chemicals from renewable carbon sources. Unlike the conventional yeast Saccharomyces cerevisiae, R. toruloides can naturally metabolize xylose, the second most abundant sugar in lignocellulosic hydrolysates. However, its native xylose metabolism is inefficient, characterized by slow xylose uptake and accumulation of D-arabitol. Moreover, despite its phenotype, research on the enzymes involved in xylose metabolism has yet to reach a consensus. Therefore, this review provides a comprehensive analysis of the non-canonical xylose metabolism in R. toruloides, focusing on the properties of key enzymes involved in xylose metabolism. Native xylose reductase and xylitol dehydrogenase exhibit broad substrate promiscuity compared to their counterparts in the xylose-fermenting Scheffersomyces stipitis. Additionally, the absence of xylulokinase expression under xylose-utilizing conditions redirects metabolism toward D-arabitol accumulation. Consequently, D-arabitol dehydrogenases and ribulokinase play essential roles in the xylose metabolism of R. toruloides. These findings highlight the fundamental differences between R. toruloides xylose metabolism and the oxidoreductase pathways observed in other xylose-fermenting yeast, providing insights for metabolic engineering strategies to improve xylose utilization and enhance bioconversion of cellulosic hydrolysates to different bioproducts by R. toruloides.

Xylose

A unique pattern of toxic synthesis in pentitol catabolism: implications for evolution.

All of our Escherichia coli C mutants blocked in the first step of D-arabitol catabolism (D-arabitol dehydrogenase) became unable to grow in the presence of D-arabitol. We have shown that this sensitivity is eliminated by a defect in the second enzyme of the pathway (D-xylulokinase), leading to a pattern of toxicity and its relief which has not been previously reported. We have found a similar pattern of toxicity and its relief in the closely related ribitol pathway. The evolutionary significance of these findings is discussed.

Alcohol Oxidoreductases

Unusual Enterobacteriaceae. "Proteus rettgeri" that "change" into Providencia stuartii.

A blood culture bottle from a patient with bacteremia contained both Proteus rettgeri biogroup 5 and Providencia stuartii (described in Bergey's Manual of Determinative Bactiology [8th ed., 1974] as Proteus inconstans), which had the same unusual antibiotic resistance pattern. Single colonies of this P. rettgeri biogroup 5 isolate were shown to produce urea- clones. If current taxonomy is used, the strain changed from P. rettgeri to P. stuartii in the laboratory and probably also in the patient. Urea- clones were also found in three of six other strains of P. rettgeri biogroup 5. No urea-negative clones were found in two isolates each of P. rettgeri biogroups 1 and 3. Previous data from deoxyribonucleic acid-deoxyribonucleic acid hybridization, biochemical reactions, and serological cross-reactions all have indicated that the taxon now called P. rettgeri biogroup 5 should be classified as P. stuartii urea+. We propose that this taxonomic change be made. Urease production is probably plasmid mediated in P. stuartii urea+ and can easily be lost, as shown in our case report and in three stock cultures. Urea hydrolysis will no longer be the key test for differentiating P. rettgeri from P. stuartii. Rather, acid production from trehalose, D-arabitol, adonitol, and D-mannitol will be the key tests. Whereas P. rettgeri is usually trehalose-, D-arabitol+, adonitol+, and D-mannitol+, P. stuartii has the opposite reactions.

Aged

Acquisition of ability to utilize Xylitol: disadvantages of a constitutive catabolic pathway in Escherichia coli.

Ribitol+ strains of Escherichia coli acquire the ability to utilize xylitol by mutating to constitutive production of the coordinately controlled ribitol catabolic enzymes ribitol dehydrogenase (RDH) and D-ribulokinase (DRK). Such strains concomitantly acquire toxicity to galacitol and L-arabitol, and to D-arabitol if they are unable to utilize it for growth. Strains selected for resistance to these polyols have DRK structural gene mutations or other mutations that eliminate the constitutive production of DRK, consistent with the view that DRK phosphorylates those polyols to toxic substances. Ribitol+ strains selected for growth on 8 mM xylitol fail to grow on 30 mM xylitol. A product of ribitol and xylitol catabolism represses synthesis of RDH, an enzyme required for growth on xylitol. At 30 mM xylitol, greater than 99% of RDH synthesis is repressed. Strains that grow on 8 mM xylitol can mutate to grow on 30 mM xylitol. Such mutants, relieved of this repression, overproduce RDH, resulting in good growth on the poor substrate, xylitol, but poor growth on the normal substrate, ribitol.

Escherichia coli

Selective inhibition of Klebsiella aerogenes growth on pentoses by pentitols.

Selective inhibition of growth by pentitols was observed when Klebsiella aerogenes M-7 which could not utilize pentitols was grown on pentoses. D-Arabitol inhibited the growth on D-arabinose as a sole carbon source, but had no effect on the growth on L-arabinose, D-xylose, and D-ribose. Similarly, L-arabitol inhibited the growth on D-arabinose and L-arabinose, ribitol inhibited the growth on D-arabinose and L-arabinose, and xylitol inhibited the growth on D-xylose. From the following reasons, we postulated that the selective growth inhibition by pentitols was due to the competitive inhibition of pentose isomerase reaction by the cell by pentitols. (i) D-Arabinose transport activity was not inhibited by pentitols. (ii) Induction of D-arabinose and L-arabinose isomerases was not inhibited by D- and L-arabitol, respectively. (iii) The specificity of growth inhibition by pentitols was the same as that of competitive inhibition of pentose isomerases by pentitols.

Arabinose