A comparison of alternate metabolic strategies for the utilization of D-arabinose

Summary Mutants ofKlebsiella aerogenes W70 that metabolize the uncommon pentose D-arabinose were isolated. These mutants were found to be either constitutive or indicible by D-arabinose for the synthesis of enzymes in the L-fucose pathway. Such mutants could then utilize L-fucose isomerase to convert the structurally similar D-arabinose molecule to D-ribulose. D-Ribulose is an inter-mediate and the inducer of an existing ribitol pathway and could thus be metabolized. In those D-arabinose-positive mutants where the ribitol pathway was blocked by mutation, D-ribulose could alternatively be metabolized by using the remaining L-fucose pathway enzymes. When the two D-arabinose catabolic routes were compared, catabolism of D-arabinose via the ribitol pathway was found to be more efficient. Catabolism of D-arabinose using the L-fucose pathway per-mitted D-ribulose to escape into the media and produced an unmetabolizable end product, L-glycolic acid. A comparison of growth using constitutive versus inducible control of the borrowed L-fucose isomerase did not reveal an advantage for one control type over the other. Several differences were observed,however, when we determined the degree to which these control mutations perturbed the normal functioning of the L-fucose and associated pathways. Growth of the constitutive mutant was impaired with L-fucose as substrate. The inducible-control mutant had altered growth characteristics on ribitol and L-rhamnose.     

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.     

Construction of an improved D-arabinose pathway in Escherichia coli K-12.

A ribitol catabolic pathway was transduced into Escherichia coli K-12 in an effort to determine whether the ribitol pathway would confer an advantage to D-arabinose-positive mutants growing on D-arabinose as the sole carbon source. Competition studies in chemostats showed that ribitol-positive strains, with a selection coefficient of 9%/h, have a significant competitive advantage over ribitol-negative strains. Ribitol-positive strains grown in batch culture also exhibited a shorter lag period than did ribitol-negative strains when transferred from glucose to D-arabinose. Repeated transfer of a ribitol-positive strain of E. coli K-12 on D-arabinose yielded a strain with further improved growth on D-arabinose. This "evolved" strain was found to constitutively synthesize L-fucose permease, isomerase, and kinase but had lost the ability to grow on L-fucose, apparently owing to the loss of a functional aldolase. This constitutive mutation is not linked to the fucose gene cluster and may be similar to an unlinked constitutive mutation described by Chen et al. (J. Bacteriol. 159:725-729, 1984).     

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.     

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.     

L-核糖的生产研究进展

L-核糖是合成许多抗病毒药物的关键中间体,自然界和生物体中并不存在。L-核糖的制备方法有化学合成法和生物合成法。与化学合成法相比,生物合成法对环境更加有利。生物合成法是应用微生物及其酶以核糖醇或L-阿拉伯糖为原料生产L-核糖。综述了L-核糖的制备方法以及产品的分离纯化技术,并对L-核糖的研究现状和前景进行了展望。     

Construction of intergeneric hybrids using bacteriophage P1CM: transfer of the Klebsiella aerogenes ribitol dehydrogenase gene to Escherichia coli.

Study of many of the interesting properties of Klebsiella aerogenes is limited by the lack of a well-characterized genetic system for this organism. Our investigations of the evolution of the enzyme ribitol dehydrogenase (EC 1.1.1.56) in K. aerogenes would be greatly facilitated by the availability of such a system, and we here report two approaches to developing one. We have isolated mutants sensitive to the coliphage P1, which will efficiently tranduce genetic markers between such sensitive strains and which will thus make detailed mapping studies possible. Derivatives of K. aerogenes lysogenic for P1 can be readily isolated by using the specialized transducing particle P1CMclr100. Bacteria lysogenic for this phage are chloramphenicol resistant and temperature sensitive. Phage particles produced by temperature induction of such lysogens can be used to transfer K. aerogenes genes to the natural host of P1 phage. Escherichia coli. We have used this method to prepare derivatives of E. coli K-12 carrying the K. aerogenes genes conferring the ability to metabolize the pentitols ribitol and D-arabitol. We have shown that these E. coli-K. aerogenes hybrids synthesize a ribitol dehydrogenase with the properties of the K. aerogenes enzyme and have mapped the position of the transferred gene on the E. coli chromosome. The ramifications of this methodology are discussed.     

Metabolism of D-arabinose: a new pathway in Escherichia coli

Several growth characteristics of Escherichia coli K-12 suggest that growth on l-fucose results in the synthesis of all the enzymes necessary for growth on d-arabinose. Conversely, when a mutant of E. coli is grown on d-arabinose, all of the enzymes necessary for immediate growth on l-fucose are present. Three enzymes of the l-fucose pathway in E. coli, l-fucose isomerase, l-fuculokinase, and l-fuculose-l-phospháte aldolase possess activity on d-arabinose, d-ribulose, and d-ribulose-l-phosphate, respectively. The products of the aldolase, with d-ribulose-l-phosphate as substrate, are dihydroxyacetone phosphate and glycolaldehyde. l-Fucose, but not d-arabinose, is capable of inducing these activities in wild-type E. coli. In mutants capable of utilizing d-arabinose as sole source of carbon and energy, these activities are induced in the presence of d-arabinose and in the presence of l-fucose. Mutants unable to utilize l-fucose, selected from strains capable of growth on d-arabinose, are found to have lost the ability to grow on d-arabinose. Enzymatic analysis of cell-free extracts, prepared from cultures of these mutants, reveals that a deficiency in any of the l-fucose pathway enzymes results in the loss of ability to utilize d-arabinose. Thus, the pathway of d-arabinose catabolism in E. coli K-12 is believed to be: d-arabinose right harpoon over left harpoon d-ribulose --> d-ribulose-l-phosphate right harpoon over left harpoon dihydroxyacetone phosphate plus glycolaldehyde. Evidence is presented which suggests that the glycolaldehyde is further oxidized to glycolate.     

Selection of Escherichia coli mutants lacking glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase

Glucose is metabolized in Escherichia coli chiefly via the phosphoglucose isomerase reaction; mutants lacking that enzyme grow slowly on glucose by using the hexose monophosphate shunt. When such a strain is further mutated so as to yield strains unable to grow at all on glucose or on glucose-6-phosphate, the secondary strains are found to lack also activity of glucose-6-phosphate dehydrogenase. The double mutants can be transduced back to glucose positivity; one class of transductants has normal phosphoglucose isomerase activity but no glucose-6-phosphate dehydrogenase. An analogous scheme has been used to select mutants lacking gluconate-6-phosphate dehydrogenase. Here the primary mutant lacks gluconate-6-phosphate dehydrase (an enzyme of the Enter-Doudoroff pathway) and grows slowly on gluconate; gluconate-negative mutants are selected from it. These mutants, lacking the nicotinamide dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase, grow on glucose at rates similar to the wild type. Thus, these enzymes are not essential for glucose metabolism in E. coli.     

Experimental evolution of a metabolic pathway for ethylene glycol utilization by Escherichia coli.

Spontaneous mutants of Escherichia coli able to grow on ethylene glycol as a sole source of carbon and energy were obtained from mutants that could grow on propylene glycol. Attempts to obtain ethylene glycol-utilizing mutants from wild-type E. coli were unsuccessful. The two major characteristics of the ethylene glycol-utilizing mutants were (i) increased activities of propanediol oxidoreductase, an enzyme present in the parental strain (a propylene glycol-positive strain), which also converted ethylene glycol into glycolaldehyde; and (ii) constitutive synthesis of high activities of glycolaldehyde dehydrogenase, which converted glycolaldehyde to glycolate. Glycolate was metabolized via the glycolate pathway, which was present in the wild-type cells; this was indicated by the induction in ethylene glycol-grown cells of glycolate oxidase, the first enzyme in the pathway. Glycolaldehyde dehydrogenase was partially characterized as an enzyme of this new metabolic pathway in E. coli, and glycolate was identified as the product of the reaction. This enzyme used NAD and NADP as coenzymes, although the NADP-dependent activity was about 10 times lower than the NAD-dependent activity. Uptake of [14C]ethylene glycol was dependent on the presence of the enzymes capable of metabolism of ethylene glycol. Glycolaldehyde and glycolate were identified as intermediate metabolites in the pathway.     

Natural and altered induction of the L-fucose catabolic enzymes in Klebsiella aerogenes.

Mutants of Klebsiella aerogenes W70 were isolated that had gained the ability to utilize the uncommon pentose D-arabinose as their sole source of carbon and energy. In contrast to the D-arabinose-negative, parent strain, these mutants were found to be either constitutive for certain enzymes of the L-fucose catabolic pathway or inducible for such enzymes when incubated in the presence of D-arabinose. The mutants used L-fucose isomerase to convert D-arabinose to D-ribulose, which is an intermediate and inducer of the ribitol catabolic pathway. The D-ribulokinase of the ribitol pathway was then induced. This enzyme catalyzed the phosphorylation of D-ribulose at the 5-carbon position. Mutants that were negative for D-ribulokinase could still dissimilate D-arabinose slowly by using all three enzymes, the isomerase, kinase, and aldolase, of the L-fucose pathway. Using condition negative mutants, we were able to demonstrate that the natural induction of the L-fucose pathway enzymes by L-fucose required the activity of a functional L-fucose isomerase and a functional L-fuculokinase but not an L-fuculose-1-phosphate aldolase. A metabolic intermediate, L-fuculose-1-phosphate, was thereby shown to be a probable inducer of at least the isomerase and kinase of the L-fucose catabolic pathway. Similar experiments, with D-arabinose-positive mutants, which were induced for the L-fucose pathway enzymes upon incubation with D-arabinose, revealed that the activities of the L-fucose isomerase and the L-fuculokinase were also required for the induction of the L-fucose enzymes. These D-arabinose-positive mutants apparently produced an altered regulatory protein that accepted both L-fuculose-1-phosphate and D-ribulose-1-phosphate as inducers. Examination of constitutive mutants revealed that L-fucose isomerase and L-fuculokinase were both synthesized constitutively, with the aldolase apparently under separate control.     

Increasing the oxidative stress response allows Escherichia coli to overcome inhibitory effects of condensed tannins

Tannins are plant-derived polyphenols with antimicrobial effects. The mechanism of tannin toxicity towards Escherichia coli was determined by using an extract from Acacia mearnsii (Black wattle) as a source of condensed tannins (proanthocyanidins). E. coli growth was inhibited by tannins only when tannins were exposed to oxygen. Tannins auto-oxidize, and substantial hydrogen peroxide was generated when they were added to aerobic media. The addition of exogenous catalase permitted growth in tannin medium. E. coli mutants that lacked HPI, the major catalase, were especially sensitive to tannins, while oxyR mutants that constitutively overexpress antioxidant enzymes were resistant. A tannin-resistant mutant was isolated in which a promoter-region point mutation increased the level of HPI by 10-fold. Our results indicate that wattle condensed tannins are toxic to E. coli in aerobic medium primarily because they generate H(2)O(2). The oxidative stress response helps E. coli strains to overcome their inhibitory effect.     

Selection of AraB and AraC Mutants of Escherichia coli B/r by Resistance to Ribitol

The growth of strain araC(c)67, which produces the enzymes of the ara operon constitutively, is inhibited by the addition of ribitol. Isolation of strains resistant to ribitol yields mutants of either the araB or araC genes. A model to account for the inhibition by ribitol is discussed.     

Mutants of Aerobacter aerogenes capable of utilizing xylitol as a novel carbon

Wild-type Aerobacter aerogenes 1033 is unable to utilize xylitol. A succession of mutants was isolated capable of growth on this compound (0.2%) at progressively faster rates. Whereas the ability to utilize xylitol was achieved in the first-stage mutant (X1) by constitutive production of ribitol dehydrogenase (for which xylitol is a substrate but not an inducer), the basis for enhanced utilization of xylitol in the second-stage mutant (X2) was an alteration of ribitol dehydrogenase. This enzyme was purified from the various mutants. The apparent K(m) for xylitol was 0.12 m with X2 enzyme and 0.29 m with X1 enzyme. The X2 enzyme was also less heat stable and, at 0.05 m substrate concentration, had a higher ratio of activity with xylitol compared to ribitol than did the X1 enzyme. The third mutant (X3), with an even faster growth rate on xylitol, produced a ribitol dehydrogenase indistinguishable physically or kinetically from that of X2. However, X3 produced constitutively an active transport system which accepts xylitol. The usual function of this system is apparently for the transport of d-arabitol since the latter is not only a substrate but also an inducer of the transport system in parental strains of X3. The sequence of mutations described herein illustrates how genes belonging to different metabolic systems can be mobilized to serve a new biochemical pathway.     

Phenol and Benzoate Metabolism by Pseudomonas putida: Regulation of Tangential Pathways

Catechol occurs as an intermediate in the metabolism of both benzoate and phenol by strains of Pseudomonas putida. During growth at the expense of benzoate, catechol is cleaved ortho (1,2-oxygenase) and metabolized via the beta-ketoadipate pathway; during growth at the expense of phenol or cresols, the catechol or substituted catechols formed are metabolized by a separate pathway following meta (2,3-oxygenase) cleavage of the aromatic ring of catechol. It is possible to explain the mutually exclusive occurrence of the meta and ortho pathway enzymes in phenol- and benzoate-grown cells of P. putida on the basis of differences in the mode of regulation of these two pathways. By use of both nonmetabolizable inducers and blocked mutants, gratuitous synthesis of some of the meta pathway enzymes was obtained. All four enzymes of the meta pathway are induced by the primary substrate, cresol or phenol, or its analogue. Three enzymes of the ortho pathway that catalyze the conversion of catechol to beta-ketoadipate enol-lactone are induced by cis,cis-muconate, produced from catechol by 1,2-oxygenase-mediated cleavage. Observations on the differences in specificity of induction and function of the two pathways suggest that they are not really either tangential or redundant. The meta pathway serves as a general mechanism for catabolism of various alkyl derivatives of catechol derived from substituted phenolic compounds. The ortho pathway is more specific and serves primarily in the catabolism of precursors of catechol and catechol itself.     

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.     

Effect of the proline analogue baikiain on proline metabolism in Salmonella typhimurium

A proline analogue, 4,5-dehydro-l-pipecolic acid (baikiain) induces the formation in Salmonella typhimurium of the two enzymes catalyzing the degradation of proline, proline oxidase and Delta(1)-pyrroline-5-carboxylic acid (P5C) dehydrogenase. The level of induction by 20 mm baikiain is about 10% of the maximum level induced by proline. Since the analogue is a substrate of proline oxidase the first enzyme of the proline catabolic pathway, the oxidation derivative rather than baikiain itself might be the actual effector. Baikiain is also an inducer of proline oxidase in Escherichia coli K-12 and E. coli W. An additional effect of this analogue on proline degradation in S. typhimurium is inhibition of P5C dehydrogenase. At a concentration of 5 x 10(-4)m, baikiain inhibits completely the growth of strains constitutive for proline oxidase. This inhibition, which can be overcome by proline, occurs in the presence or absence of P5C dehydrogenase activity. Three spontaneously occurring mutants resistant to baikiain were isolated from constitutive strains. All are pleiotropic-negative for the proline-degrading enzymes. The sites of these mutations are linked to the put region. Although the mechanism of toxicity has not been determined, baikiain provides a simple and direct selection for obtaining mutants unable to degrade proline. In addition, it allows selection for strains with an inducible rather than constitutive phenotype.     

Ribitol catabolic pathway in Klebsiella aerogenes

In Klebsiella aerogenes W70, there is an inducible pathway for the catabolism of ribitol consisting of at least two enzymes, ribitol dehydrogenase (RDH) and d-ribulokinase (DRK). These two enzymes are coordinately controlled and induced in response to d-ribulose, an intermediate of the pathway. Whereas wild-type K. aerogenes W70 are unable to utilize xylitol as a carbon and energy source, mutants constitutive for the ribitol pathway are able to utilize RDH to oxidize the unusual pentitol, xylitol, to d-xylulose. These mutants are able to grow on xylitol, presumably by utilization of the d-xylulose produced. Mutants constitutive for l-fucose isomerase can utilize the isomerase to convert d-arabinose to d-ribulose. In the presence of d-ribulose, RDH and DRK are induced, and such mutants are thus able to phosphorylate the d-ribulose by using the DRK of the ribitol pathway. Derivatives of an l-fucose isomerase-constitutive mutant were plated on d-arabinose, ribitol, and xylitol to select and identify mutations in the ribitol pathway. Using the transducing phage PW52, we were able to demonstrate genetic linkage of the loci involved. Three-point crosses, using constitutive mutants as donors and RDH(-), DRK(-) double mutants as recipients and selecting for DRK(+) transductants on d-arabinose, resulted in DRK(+)RDH(+)-constitutive, DRK(+)RDH(+)-inducible, and DRK(+)RDH(-)-inducible transductants but no detectable DRK(+)RDH(-) constitutive transductants, data consistent with the order rbtC-rbtD-rbtK, where rbtC is a control site and rbtD and rbtK correspond to the sites for the sites for the enzymes RDH and DRK, respectively.     

Secretion processing and activation of Erwinia chrysanthemi proteases

E chrysanthemi, a phytopathogenic enterobacterium, secretes several enzymes into the medium such as pectinases cellulases and proteases. It also produces 3 distinct and antigenically related extracellular proteases. The proteases secretion pathway seems to be distinct from that of the other extracellular enzymes since pleiotropic mutants impaired in cellulase and pectinase secretion are unimpaired in protease secretion. E chrysanthemi proteases B and C secretion occurs without an N-terminal signal peptide and is dependent upon specific secretion functions which are encoded by genes adjacent to the protease structural genes. This secretion pathway might be analogous to the alpha-hemolysin secretion pathway in E coli. Protection against intracellular proteolytic activity is achieved by 2 distinct mechanisms: the proteases are synthesized as inactive precursors with an N-terminal extension of 15 aminoacids (protease B) and 17 aminoacids (protease C) absent in the mature active extracellular enzymes; an intracellular specific protease inhibitor is produced by some E chrysanthemi strains.