Production of D- and L-xylulose by mutants of Klebsiella pneumoniae and Erwinia uredovora.

D-Xylulose and L-xylulose were produced biologically by the oxidation of a corresponding pentitol. A Klebsiella pneumoniae mutant was constructed for the oxidation of D-arabitol to D-xylulose. This mutant constitutively synthesized the D-arabitol permease system and D-arabitol dehydrogenase but was unable to produce the D-xylulokinase of the D-arabitol pathway or the D-xylose isomerase and D-xylulokinase of the D-xylose pathway. An Erwinia uredovora mutant which constitutively synthesized a novel xylitol-4-dehydrogenase but could not synthesize L-xylulokinase was used for the oxidation of xylitol to L-xylulose. Washed cell suspensions of either mutant incubated with 0.5% pentitol would oxidize 60 to 65% of the pentitol to the corresponding ketopentose in 18 h and excrete the ketopentose into the medium. Ketopentoses were rapidly purified from the remaining pentitol by hydroxyl affinity chromatography.     

Directed evolution of a second xylitol catabolic pathway in Klebsiella pneumoniae.

Klebsiella pneumoniae PRL-R3 has inducible catabolic pathways for the degradation of ribitol and D-arabitol but cannot utilize xylitol as a growth substrate. A mutation in the rbtB regulatory gene of the ribitol operon permits the constitutive synthesis of the ribitol catabolic enzymes and allows growth on xylitol. The evolved xylitol catabolic pathway consists of an induced D-arabitol permease system that also transports xylitol, a constitutively synthesized ribitol dehydrogenase that oxidizes xylitol at the C-2 position to produce D-xylulose, and an induced D-xylulokinase from either the D-arabitol or D-xylose catabolic pathway. To investigate the potential of K. pneumoniae to evolve a different xylitol catabolic pathway, strains were constructed which were unable to synthesize ribitol dehydrogenase or either type of D-xylulokinase but constitutively synthesized the D-arabitol permease system. These strains had an inducible L-xylulokinase; therefore, the evolution of an enzyme which oxidized xylitol at the C-4 position to L-xylulose would establish a new xylitol catabolic pathway. Four independent xylitol-utilizing mutants were isolated, each of which had evolved a xylitol-4-dehydrogenase activity. The four dehydrogenases appeared to be identical because they comigrated during nondenaturing polyacrylamide gel electrophoresis. This novel xylitol dehydrogenase was constitutively synthesized, whereas L-xylulokinase remained inducible. Transductional analysis showed that the evolved dehydrogenase was not an altered ribitol or D-arabitol dehydrogenase and that the evolved dehydrogenase structural gene was not linked to the pentitol gene cluster. This evolved dehydrogenase had the highest activity with xylitol as a substrate, a Km for xylitol of 1.4 M, and a molecular weight of 43,000.     

The determination of lactate turnover in vivo with 3H- and 14C-labelled lactate. The significance of sites of tracer administration and sampling.

The D-ribulokinase and D-xylulokinase of Klebsiella aerogenes were purified to homogeneity from Escherichia coli K12 construct strains that synthesized these enzymes constitutively. The D-ribulokinase, which is encoded in the ribitol operon, is active as a dimer of 60 000 subunit mol.wt., whereas the D-xylulokinase, which is encoded in the D-arabitol operon, is active as a dimer of 54 000 subunit mol.wt. The amino acid compositions and N-terminal sequences of both pentulokinases are reported. The Kapp. values of the enzymes for their D-pentulose substrates were determined, and the D-ribulokinase was shown to have a low-affinity side-specificity for ribitol and D-arabitol. These results are discussed in the context of the evolution of the Klebsiella aerogenes pentitol operons.     

D-arabitol metabolism in Candida albicans: construction and analysis of mutants lacking D-arabitol dehydrogenase.

Candida albicans produces large amounts of the acyclic pentitol D-arabitol in culture and in infected animals and humans, and most strains also grow on minimal D-arabitol medium. An earlier study showed that the major metabolic precursor of D-arabitol in C. albicans was D-ribulose-5-PO4 from the pentose pathway, that C. albicans contained an NAD-dependent D-arabitol dehydrogenase (ArDH), and that the ArDH structural gene (ARD) encoded a 31-kDa short-chain dehydrogenase that catalyzed the reaction D-arabitol + NAD <=> D-ribulose + NADH. In the present study, we disrupted both ARD chromosomal alleles in C. albicans and analyzed the resulting mutants. The ard null mutation was verified by Southern hybridization, and the null mutant's inability to produce ArDH was verified by Western immunoblotting. The ard null mutant grew well on minimal glucose medium, but it was unable to grow on minimal D-arabitol or D-arabinose medium. Thus, ArDH catalyzes the first step in D-arabitol utilization and a necessary intermediate step in D-arabinose utilization. Unexpectedly, the ard null mutant synthesized D-arabitol from glucose. Moreover, 13C nuclear magnetic resonance studies showed that the ard null mutant and its wild-type parent synthesized D-arabitol via the same pathway. These results imply that C. albicans synthesizes and utilizes D-arabitol via separate metabolic pathways, which was not previously suspected for fungi.     

Membrane-bound sugar alcohol dehydrogenase in acetic acid bacteria catalyzes L-ribulose formation and NAD-dependent ribitol dehydrogenase is independent of the oxidative fermentation

To identify the enzyme responsible for pentitol oxidation by acetic acid bacteria, two different ribitol oxidizing enzymes, one in the cytosolic fraction of NAD(P)-dependent and the other in the membrane fraction of NAD(P)-independent enzymes, were examined with respect to oxidative fermentation. The cytoplasmic NAD-dependent ribitol dehydrogenase (EC 1.1.1.56) was crystallized from Gluconobacter suboxydans IFO 12528 and found to be an enzyme having 100 kDa of molecular mass and 5 s as the sedimentation constant, composed of four identical subunits of 25 kDa. The enzyme catalyzed a shuttle reversible oxidoreduction between ribitol and D-ribulose in the presence of NAD and NADH, respectively. Xylitol and L-arabitol were well oxidized by the enzyme with reaction rates comparable to ribitol oxidation. D-Ribulose, L-ribulose, and L-xylulose were well reduced by the enzyme in the presence of NADH as cosubstrates. The optimum pH of pentitol oxidation was found at alkaline pH such as 9.5-10.5 and ketopentose reduction was found at pH 6.0. NAD-Dependent ribitol dehydrogenase seemed to be specific to oxidoreduction between pentitols and ketopentoses and D-sorbitol and D-mannitol were not oxidized by this enzyme. However, no D-ribulose accumulation was observed outside the cells during the growth of the organism on ribitol. L-Ribulose was accumulated in the culture medium instead, as the direct oxidation product catalyzed by a membrane-bound NAD(P)-independent ribitol dehydrogenase. Thus, the physiological role of NAD-dependent ribitol dehydrogenase was accounted to catalyze ribitol oxidation to D-ribulose in cytoplasm, taking D-ribulose to the pentose phosphate pathway after being phosphorylated. L-Ribulose outside the cells would be incorporated into the cytoplasm in several ways when need for carbon and energy sources made it necessary to use L-ribulose for their survival. From a series of simple experiments, membrane-bound sugar alcohol dehydrogenase was concluded to be the enzyme responsible for L-ribulose production in oxidative fermentation by acetic acid bacteria.     

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.     

Identification of a salvage pathway for D-arabinose in Mycobacterium smegmatis

Extracts of Mycobacterium smegmatis, which was adapted to growth in synthetic medium containing D-arabinose as sole carbon source, catalyzed the NADPH-mediated reduction of D-arabinose to D-arabitol. When arabinose-adapted bacteria were transferred to glycerol medium, resumption of growth was accompanied by a sharp drop in the specific activity of this enzyme. Moreover, extracts of cells grown in D-arabinose medium contained large amounts of an NAD+-linked pentitol dehydrogenase, as compared to bacteria multiplying in glycerol medium. The specific activity of mycobacterial extracts was ten-fold higher for D-arabitol than for its L-isomer, and eight-fold higher than for xylitol (it was more than forty-fold lower in the case of glycerol-grown cells). The product of the pentitol dehydrogenase reaction was identified as D-xylulose by three different procedures. On the basis of these data, it is suggested that utilization of exogenous D-arabinose in mycobacteria involves two dehydrogenases that catalyze the reactions D-arabinose NADPH----D-arabitol NAD+----D-xylulose, by virtue of which an aldopentose is converted into a ketopentose. The alditol: NADP oxidoreductase was isolated from homogenates of D-arabinose-adapted mycobacteria, and purified by DEAE-cellulose chromatography. The enzymatic activity was restricted to a single band which, under denaturing conditions, comigrated with albumin (approximately 46 kDa). It was insensitive to 2-mercaptoethanol, EDTA and NaF, and was inactivated at 70 degrees C.     

Levels of enzymes of the pentose phosphate pathway in Pachysolen tannophilus Y-2460 and selected mutants

The compositions of intracellular pentose phosphate pathway enzymes have been examined in mutants of Pachysolen tannophilus NRRL Y-2460 which possessed enhanced D-xylose fermentation rates. The levels of oxidoreductive enzymes involved in converting D-xylose to D-xylulose via xylitol were 1.5–14.7-fold higher in mutants than in the parent. These enzymes were still under inductive control by D-xylose in the mutants. The D-xylose reductase activity (EC 1.1.1.21) which catalyses the conversion of D-xylose to xylitol was supported with either NADPH or NADH as coenzyme in all the mutant strains. Other enzyme specific activities that generally increased were: xylitol dehydrogenase (EC 1.1.1.9), 1.2–1.6-fold; glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 1.9–2.6-fold; D-xylulose-5-phosphate phosphoketolase (EC 4.1.2.9), 1.2–2.6-fold; and alcohol dehydrogenase (EC 1.1.1.1), 1.5–2.7-fold. The increase of enzymatic activities, 5.3–10.3-fold, occurring in D-xylulokinase (EC 2.7.1.17), suggested a pivotal role for this enzyme in utilization of D-xylose by these mutants. The best ethanol-producing mutant showed the highest ratio of NADH- to NADPH-linked D-xylose reductase activity and high levels of all other pentose phosphate pathway enzymes assayed.     

Characterization of xylitol-utilizing mutants of Erwinia uredovora.

Of the four pentitols ribitol, xylitol, D-arabitol, and L-arabitol, Erwinia uredovora was able to utilize only D-arabitol as a carbon and energy source. Although attempts to isolate ribitol- or L-arabitol-utilizing mutants were unsuccessful, mutants able to grow on xylitol were isolated at a frequency of 9 X 10(-8). Xylitol-positive mutants constitutively synthesized both a novel NAD-dependent xylitol-4-dehydrogenase, which oxidized xylitol to L-xylulose, and an L-xylulokinase. The xylitol dehydrogenase had a Km for xylitol of 48 mM and showed best activity with xylitol and D-threitol as substrates. However, D-threitol was not a growth substrate for E. uredovora, and its presence did not induce either dehydrogenase or kinase activity. Attempts to determine the origin of the xylitol catabolic enzymes were unsuccessful; neither enzyme was induced on any growth substrate or in the presence of any polyol tested. Analysis of xylitol-negative mutants isolated after Tn5 mutagenesis suggested that the xylitol dehydrogenase and the L-xylulokinase structural genes were components of two separate operons but were under common regulatory control.     

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

Summary All of ourEscherichia coli C mutants blocked in the first step of D-arabitol catabolism (D-arabitol dehydrogenase) became unable to grow in the presense 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.     

Growth of a mutant of Escherichia coli K-12 on xylitol by recruiting enzymes for D-xylose and L1,2-propanediol metabolism.

Wild type Escherichia coli K-12 cannot grow on xylitol and we have been unsuccessful in isolating a mutant directly which had acquired this new growth ability. However, a mutant had been selected previously for growth on L-1,2-propanediol as the sole source of carbon and energy. This mutant constitutively synthesized a propanediol dehydrogenase. Recently, we have found that this dehydrogenase fortuitously converted xylitol to D-xylose which could normally be metabolized by E. coli K-12. In addition, it was also discovered that the D-xylose permease fortuitously transported xylitol into the cell. A second mutant was thus isolated from the L-1,2-propanediol-growing mutant that was constitutive for the enzymes of the D-xylose pathway. This mutant could indeed grow on xylitol as the sole source of carbon and energy, by utilizing the enzymes normally involved in D-xylose and L-1,2-propanediol metabolism.     

Growth of a mutant of Escherichia coli K-12 on xylitol by recruiting enzymes for D-xylose and L1,2-propanedol metabolism

Wild type Escherichia coli K-12 cannot grow on xylitol and we have been unsuccessful in isolating a mutant directly which had acquired this new growth ability. However, a mutant had been selected previously for growth on L-1,2-propanediol as the sole source of carbon and energy. This mutant constitutively synthesized a propanediol dehydrogenase. Recently, we have found that this dehydrogenase fortuitously converted xylitol to D-xylose which could normally be metabolized by E. coli K-12. In addition, it was also discovered that the D-xylose permease fortuitously transported xylitol into the cell. A second mutant was thus isolated from the L-1,2-propanediol-growing mutant that was constitutive for enzymes of the D-xylose pathway. This mutant could indeed grow on xylitol as the sole source of carbon and energy, by utilizing the enzymes normally involved in D-xylose and L-1,2-propanediol metabolism.     

Biosynthesis and catabolism of 6-deoxy L-talitol by Klebsiella aerogenes mutants.

A mutant strain of Klebsiella aerogenes was constructed and, when incubated anaerobically with L-fucose and glycerol, synthesized and excreted a novel methyl pentitol, 6-deoxy L-talitol. The mutant was constitutive for the synthesis of L-fucose isomerase but unable to synthesize L-fuculokinase activity. Thus, it could convert the L-fucose to L-fuculose but was incapable of phosphorylating L-fuculose to L-fuculose 1-phosphate. The mutant was also constitutive for the synthesis of ribitol dehydrogenase, and in the presence of sufficient reducing power this latter enzyme catalyzed the reduction of the L-fuculose to 6-deoxy L-talitol. The reducing equivalents required for this reaction were generated by the oxidation of glycerol to dihydroxyacetone with an anaerobic glycerol dehydrogenase. The parent strain of K. aerogenes was unable to utilize the purified 6-deoxy L-talitol as a sole source of carbon and energy for growth; however, mutant could be isolated which had gained this ability. Such mutants were found to be constitutive for the synthesis of ribitol dehydrogenase and were thus capable of oxidizing 6-deoxy L-talitol to L-fuculose. Further metabolism of L-fuculose was shown by mutant analysis to be mediated by the enzymes of the L-fucose catabolic pathway.     

Phylogenetic Relationships between different D-Xylose Biogroups in Wild-type Salmonella typhimurium strains and a suggested Evolutionary Pathway

Sixty-two wild-type Salmonella typhimurium strains were characterized for their D-xylose enzyme activities. Strains from the xylose strong biogroup synthesized high levels of D-xylose isomerase and D-xylulokinase and transported D-xylose. Strains from the xylose weak biogroup synthesized only low levels of D-xylulokinase and low, or no, levels of D-xylose isomerase and were deficient in the ability to transport D-xylose. These findings are discussed in the light of known phylogenetic relationships among the biotypes of Salm. typhimurium.     

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.     

Genetics and regulation of D-xylose utilization in Salmonella typhimurium LT2.

Twenty-one Xyl- mutants of Salmonella typhimurium were selected: all had lost one or more of the activities for D-xylose isomerase, C-xylulokinase, or D-xylose transport. The mutants were classified into five functional groups: xylR, pleiotropic negative (12 mutants); xylA, D-xylose isomerase defective (3 mutants); xylB, D-xylulokinase defective (2 mutants); xylT, D-xylose transport defective (1 mutant); and 3 mutants with defective D-xylose isomerase and D-xylulokinase. Some nonsense mutations were identified among the xylR mutants. Two F'xyl plasmids were isolated by selection for early transfer of xyl+ by an Hfr which transfers xyl as a terminal gene; a plasmid with a mutation in the xyl genes, F'xylR1, was also isolated. Complementation tests using F'xyl plasmids indicate that expression of the xylA, xylB, and xylT genes is under the positive control of the xylR regulatory gene. Conjugation crosses and P22-mediated transduction data indicate that all the xyl mutations tested are in a cluster of genes at 78 units on the linkage map, and that the gene order is xylT--xylR--xylB--xylA--glyS--mtlA,D.     

Metabolic control analysis of Aspergillus niger L-arabinose catabolism

A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography, and their kinetic properties were characterized. For the other enzymes of the pathway the kinetic data were available from the literature. The metabolic model was used to analyze flux and metabolite concentration control of the L-arabinose catabolic pathway. The model demonstrated that flux control does not reside at the enzyme following the intermediate with the highest concentration, L-arabitol, but is distributed over the first three steps in the pathway, preceding and following L-arabitol. Flux control appeared to be strongly dependent on the intracellular L-arabinose concentration. At 5 mM intracellular L-arabinose, a level that resulted in realistic intermediate concentrations in the model, flux control coefficients for L-arabinose reductase, L-arabitol dehydrogenase and L-xylulose reductase were 0.68, 0.17 and 0.14, respectively. The analysis can be used as a guide to identify targets for metabolic engineering aiming at either flux or metabolite level optimization of the L-arabinose catabolic pathway of A. niger. Faster L-arabinose utilization may enhance utilization of readily available organic waste containing hemicelluloses to be converted into industrially interesting metabolites or valuable enzymes or proteins.     

Isolation and characterization of the Salmonella typhimurium LT2 xylose regulon

Salmonella DNA was partially digested with EcoRI, and the digest was fractionated to obtain fragments larger than 8 kilobases (kb). These were ligated into EcoRI-cut pBR322, and the mixture was used to transform Salmonella Xyl- cells selecting for ampR xyl+ transformants. A 21- and a 27-kb plasmid were isolated, both of which contained the entire xylose regulon. The xylose regulon was localized to a 6.3-kb segment of a 13.5-kb EcoRI fragment. Subclones were constructed which contained either the genes for D-xylose isomerase and D-xylulokinase or the genes for the D-xylose transport and the D-xylose regulatory factors. The gene order determined by the subcloning experiments is consistent with that determined by genetic mapping. The spots corresponding to D-xylose isomerase and D-xylulokinase subunits were identified in two-dimensional gels of several xylose-induced strains. Each of them had a molecular weight of 45,000 and an isoelectric point of 6.2 +/- 0.1.     

Uptake and catabolism of D-xylose in Salmonella typhimurium LT2.

Salmonella typhimurium LT2 grows on D-xylose as sole carbon source with a generation time of 105 to 110 min. The following activities are induced at the indicated time after the addition of the inducer, D-xylose: D-xylulokinase (5 min), D-xylose isomerase (7 to 8 min), and D-xylose transport (10 min). All other pentoses and pentitols tested failed to induce isomerase or kinase. Synthesis of D-xylose isomerase was subject to catabolite repression, which was reversed by the addition of cyclic adenosine monophosphate. Most of the radioactive counts from D-[14C]xylose were initially accumulated in the cell in the form of D-xylose or D-xylulose. D-Xylose uptake in a mutant which was deficient in D-xylose isomerase was equal to that of the wild type. The apparent Km for D-xylose uptake was 0.41 mM. Some L-arabinose was accumulated in D-xylose-induced cells, and some D-xylose was accumulated in L-arabinose-induced cells. D-Xylitol and L-arabinose competed against C-xylose uptake, but D-arabinose, D-lyxose, and L-lyxose did not. Osmotic shock reduced the uptake of D-xylose by about 50%; by equilibrium dialysis, a D-xylose-binding protein was detected in the supernatant fluid after spheroplasts were formed from D-xylose-induced cells.     

The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis

Recent research has significantly advanced our understanding of the phenylpropanoid pathway but has left in doubt the pathway by which sinapic acid is synthesized in plants. The reduced epidermal fluorescence1 (ref1) mutant of Arabidopsis thaliana accumulates only 10 to 30% of the sinapate esters found in wild-type plants. Positional cloning of the REF1 gene revealed that it encodes an aldehyde dehydrogenase, a member of a large class of NADP(+)-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. Consistent with this finding, extracts of ref1 leaves exhibit low sinapaldehyde dehydrogenase activity. These data indicate that REF1 encodes a sinapaldehyde dehydrogenase required for sinapic acid and sinapate ester biosynthesis. When expressed in Escherichia coli, REF1 was found to exhibit both sinapaldehyde and coniferaldehyde dehydrogenase activity, and further phenotypic analysis of ref1 mutant plants showed that they contain less cell wall-esterified ferulic acid. These findings suggest that both ferulic acid and sinapic acid are derived, at least in part, through oxidation of coniferaldehyde and sinapaldehyde. This route is directly opposite to the traditional representation of phenylpropanoid metabolism in which hydroxycinnamic acids are instead precursors of their corresponding aldehydes.