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.     

Growth of a mutant of Escherichia coli K-12 on xylitol by recruiting enzymes for -xylose and 1,2-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 -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 -xylose which could normally be metabolized by E. coli K-12. In addition, it was also discovered that the -xylose permease fortuitously transported xylitol into the cell. A second mutant was thus isolated from the -1,2-propanediol-growing mutant that was constitutive for enzymes of the -xylose pathway. This mutant could indeed grow on xylitol as the sole source of carbon and energy, by utilizing the enzymes normally involved in -xylose and -1,2-propanediol metabolism.     

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.     

Ferrous-activated nicotinamide adenine dinucleotide-linked dehydrogenase from a mutant of Escherichia coli capable of growth on 1, 2-propanediol

A nicotinamide adenine dinucleotide-linked dehydrogenase has been partially purified from a mutant of Escherichia coli K-12 able to grow on l-1,2-propanediol as carbon and energy source. This enzyme catalyzes the dehydrogenation at carbon 1 of l-1,2-propanediol, glycerol, 1,3-propanediol, ethylene glycol, and ethyl alcohol. The purified protein requires added ferrous or managanous ions. The V(max) and the apparent K(m) for a given substrate vary with the particular metal used.     

Evolution of propanediol utilization in Escherichia coli: mutant with improved substrate-scavenging power.

Wild-type strains of Escherichia coli are unable to use L-1,2-propanediol as a carbon and energy source. A series of mutants, able to grow on this compound at progressively faster rates, had been isolated by repeated transfers to a medium containing 20 mM L-1,2-propanediol. These strains synthesize at high constitutive levels a propanediolmicotinamide adenine dinucleotide oxidoreductase, an enzyme serving as a lactaldehyde during L-fucose fermentation by wild type cells. In this study, a mutant that can grow rapidly on the novel carbon source was subjected to further selection in a medium containing L-1,2-propanediol never exceeding 0.5 mM to obtain a derivative that has an increased power to extract the substrate from the medium. The emerging mutant exhibited four changes at the enzymatic level: (i) fuculose 1-phosphate aldolase activity is lost; (ii) the constitutive propanediol oxidoreductase activity is increased in its level; (iii) lactaldehyde dehydrogenase becomes constitutive and shows an elevated specific activity in crude extracts; and (iv) at low concentrations of propanediol, the facilitated diffusion across the cell membrane is enhanced. Changes two to four seem to act in concert in the trapping of propanediol by hastening its rate of entry and conversion to an ionized metabolite, lactate.     

Rhamnose-induced propanediol oxidoreductase in Escherichia coli: purification, properties, and comparison with the fucose-induced enzyme.

Escherichia coli are capable of growing anaerobically on L-rhamnose as a sole source of carbon and energy and without any exogenous hydrogen acceptor. When grown under such condition, synthesis of a nicotinamide adenine dinucleotide-linked L-lactaldehydepropanediol oxidoreductase is induced. The functioning of this enzyme results in the regeneration of nicotinamide adenine dinucleotide. The enzyme was purified to electrophoretic homogeneity. It has a molecular weight of 76,000, with two subunits that are indistinguishable by electrophoretic mobility. The enzyme reduces L-lactaldehyde to L-1,2-propanediol with reduced nicotinamide adenine dinucleotide as a cofactor. The Km were 0.035 mM L-lactaldehyde and 1.25 mM L-1,2-propanediol, at pH 7.0 and 9.5, respectively. The enzyme acts only on the L-isomers. Strong substrate inhibition was observed with L-1,2-propanediol (above 25 mM) in the dehydrogenase reaction. The enzyme has a pH optimum of 6.5 for the reduction of L-lactaldehyde and of 9.5 for the dehydrogenation of L-1,2-propanediol. The enzyme is, according to the parameters presented in this report, indistinguishable from the propanediol oxidoreductase induced by anaerobic growth on fucose.     

Utilization of γ-Aminobutyric Acid as the Sole Carbon and Nitrogen Source by Escherichia coli K-12 Mutants

Wild-type strains of Escherichia coli K-12 cannot grow in media with gamma-aminobutyrate (GABA) as the sole source of carbon or nitrogen. Mutants were isolated which could utilize GABA as the sole source of nitrogen. These mutants were found to have six- to ninefold higher activities of gamma-aminobutyrate-alpha-ketoglutarate transaminase (EC 2.6.1.19) and succinate semialdehyde dehydrogenase (EC 1.2.1.16) than those of the wild-type parent strains. Secondary mutants derived from these GABA-nitrogen-utilizing strains were able to grow on GABA as the sole source of carbon and nitrogen. They also grew faster on a variety of other carbon and nitrogen sources, and their growth was more strongly inhibited by different metabolic inhibitors than was that of the parent strains. The nature of the two mutations and the possible genes involved are discussed. A scheme of the pathway for GABA breakdown in E. coli K-12 is presented.     

Loss of aldehyde dehydrogenase in an Escherichia coli mutant selected for growth on the rare sugar L-galactose.

Escherichia coli K-12 converts L-fucose to dihydroxyacetone phosphate (C-1 to C-3) and L-lactaldehyde (C-4 to C-6) by a pathway specified by the fuc regulon. Aerobically, L-lactaldehyde serves as a carbon and energy source by the action of an aldehyde dehydrogenase of broad specificity; the product, L-lactate, is then converted to pyruvate. Anaerobically, L-lactaldehyde serves as an electron acceptor to regenerate NAD from NADH by the action of an oxidoreductase; the reduced product, L-12-propanediol, is excreted. A strain selected for growth on L-galactose (a structural analog of L-fucose) acquired a broadened inducer specificity because of an altered fucR gene encoding the activator protein for the fuc regulon (Y. Zhu and E. C. C. Lin, J. Mol. Evol. 23:259-266, 1986). In this study, a second mutation that abolished aldehyde dehydrogenase activity was discovered. The L-fucose pathway converts L-galactose to dihydroxyacetone phosphate and L-glyceraldehyde. Aldehyde dehydrogenase then converts L-glyceraldehyde to L-glycerate, which is toxic. Loss of the dehydrogenase averts the toxicity during growth on L-galactose, but reduces by one-half the aerobic growth yield on L-fucose. When mutant cells induced in the L-fucose system were incubated with radioactive L-fucose, accumulation of radioactivity occurred if the substrate was labeled at C-1 but not if it was labeled C-6. Complete aerobic utilization of carbons 4 through 6 of L-fucose depends not only on an adequate activity of aldehyde dehydrogenase to trap L-lactaldehyde as its anionic acid but also on the lack of L-1,2-propanediol oxidoreductase activity, which converts L-lactaldehyde to a readily excreted alcohol.     

NAD-linked aldehyde dehydrogenase for aerobic utilization of L-fucose and L-rhamnose by Escherichia coli.

Mutant analysis revealed that complete utilization of L-fucose and L-rhamnose by Escherichia coli requires the activity of a common NAD-linked aldehyde dehydrogenase which converts L-lactaldehyde to L-lactate. Mutations affecting this activity mapped to the ald locus at min 31, well apart from the fuc genes (min 60) encoding the trunk pathway for L-fucose dissimilation (as well as L-1,2-propanediol oxidoreductase) and the rha genes (min 88) encoding the trunk pathway for L-rhamnose dissimilation. Mutants that grow on L-1,2-propanediol as a carbon and energy source also depend on the ald gene product for the conversion of L-lactaldehyde to L-lactate.     

Regulatory changes in the fucose system associated with the evolution of a catabolic pathway for propanediol in Escherichia coli.

Wild-type strains of Escherichia coli are unable to use L-1,2-propanediol as a carbon and energy source. Strain 3, a mutant selected for the ability to grow on this compound at progressively more rapid rates, synthesizes constitutively a nicotinamide adenine dinucleotide-linked propanediol oxidoreductase. This enzyme is normally synthesized during anaerobic growth on L-fucose when it functions as a lactaldehyde reductase. Propanediol, the end product of this fermentation process, escapes irretrievably into the medium. The propanediol-utilizing mutant can no longer grow on fucose in either the presence or absence of molecular oxygen. In the present study nine independent lines of propanediol-positive mutants were characterized. One mutant, strain 418, attained a propanediol growth rate close to that of strain 3 without loss of the ability to grow on fucose. In all cases examined, however, prolonged selection on propanediol did result in the emergence of fucose-negative mutants. All of these mutants had enzyme patterns similar to that of strain 3; namely, fucose permease, fucose isomerase, and fuculose kinase were noninducible, whereas fuculose 1-phosphate aldolase was constitutive. In strain 418 and in the fucose-positive predecessors of the other mutants, the first four enzymes in the pathway remained inducible, as in the wild-type strain. Improvements in the growth rate on propanediol appeared to reflect principally the increased activity level of the oxidoreductase during the early stages of evolution. According to transductional analysis, the mutations affecting the ability to grow on propanediol and those that affect the expression of the first enzymes in the fucose pathway were very closely linked. The loss of the ability to grow on fucose is thought to be a mechanistic consequence incidental to the remodeling of the regulatory system in favor of the utilization of the novel carbon source.     

Multienzyme complexes of fatty acid oxidation from Escherichia coli K12 and from a mutant with a defective L-3-hydroxyacyl coenzyme A dehydrogenase

An Escherichia coli mutant (fadB64), with a defective L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) which is unable to grow on long-chain fatty acids as the sole carbon source, was shown to possess a fatty acid oxidation complex that contains five beta-oxidation enzymes, including L-3-hydroxyacyl-CoA dehydrogenase. A comparative study of the complexes from the mutant, from its parental strain and from wild-type E. coli B demonstrated the immunological and gross structural identity of all three fatty acid oxidation complexes. A kinetic evaluation of the complexes led to the suggestion that the mutation may have affected the active site of L-3-hydroxyacyl-CoA dehydrogenase so that it is inactive with acetoacetyl-CoA as a substrate, but exhibits an increasing percentage of the parental dehydrogenase activity with increasing chain length of the substrate.     

Fermentation of 1,2-Propanediol and 1,2-Ethanediol by Some Genera of Enterobacteriaceae, Involving Coenzyme B12-Dependent Diol Dehydratase

Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 was able to grow anaerobically on 1,2-propanediol and 1,2-ethanediol as carbon and energy sources. Whole cells of the bacterium grown anaerobically on 1,2-propanediol or on glycerol catalyzed conversion of 1,2-diols and aldehydes to the corresponding acids and alcohols. Glucose-grown cells also converted aldehydes, but not 1,2-diols, to acids and alcohols. The presence of activities of coenzyme B(12)-dependent diol dehydratase, alcohol dehydrogenase, coenzyme-A-dependent aldehyde dehydrogenase, phosphotransacetylase, and acetate kinase was demonstrated with crude extracts of 1,2-propanediol-grown cells. The dependence of the levels of these enzymes on growth substrates, together with cofactor requirements in in vitro conversion of these substrates, indicates that 1,2-diols are fermented to the corresponding acids and alcohols via aldehydes, acyl-coenzyme A, and acyl phosphates. This metabolic pathway for 1,2-diol fermentation was also suggested in some other genera of Enterobacteriaceae which were able to grow anaerobically on 1,2-propanediol. When the bacteria were cultivated in a 1,2-propanediol medium not supplemented with cobalt ion, the coenzyme B(12)-dependent conversion of 1,2-diols to aldehydes was the rate-limiting step in this fermentation. This was because the intracellular concentration of coenzyme B(12) was very low in the cells grown in cobalt-deficient medium, since the apoprotein of diol dehydratase was markedly induced in the cells grown in the 1,2-propanediol medium. Better cell yields were obtained when the bacteria were grown anaerobically on 1,2-propanediol. Evidence is presented that aerobically grown cells have a different metabolic pathway for utilizing 1,2-propanediol.     

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.     

Malate Dehydrogenase Mutants in Escherichia coli K-12

Mutants devoid of malate dehydrogenase activity have been isolated in Escherichia coli K-12. They do not possess detectable malate dehydrogenase when grown aerobically or anaerobically on glucose as sole carbon source. All mutants revert spontaneously; a few partial revertants have been found with a malate dehydrogenase exhibiting altered electrophoretic mobility. Therefore, only one such enzyme appears to exist in the strains examined. No evidence could be obtained for the presence of a malate dehydrogenase not linked to nicotinamide adenine dinucleotide. Mutants deficient in both malate dehydrogenase and phosphoenol pyruvate carboxylase activities will grow anaerobically on minimal glucose plus succinate medium; also, malate dehydrogenase mutants do not require succinate for anaerobic growth on glucose. The anaerobic pathway oxaloacetate to succinate or succinate to aspartate appears to be accomplished by aspartase. Malate dehydrogenase is coded for by a locus somewhere relatively near the histidine operon, i.e., a different chromosomal location than that known for other citric acid cycle enzymes.     

Disruption of the fucose pathway as a consequence of genetic adaptation to propanediol as a carbon source in Escherichia coli.

In Escherichia coli, L-fucose is dissimilated via an inducible pathway mediated by L-fucose permease, L-fucose isomerase, L-fucose kinase, and L-fuculose 1-phosphate aldolase. The last enzyme cleaves the six-carbon substrate into dihydroxyacetone phosphate and L-lactaldehyde. Aerobically, lactaldehyde is oxidized to L-lactate by a nicotinamide adenine dinucleotide (NAD)-linked dehydrogenase. Anaerobically, lactaldehyde is reduced by an NADH-COUPLED REDUCTASE TO L-1,2-propanediol, which is lost into the medium irretrievably, even when oxygen is subsequently introduced. Propanediol excretion is thus the end result of a dismutation that permits further anaerobic metabolism of dihydroxy-acetone phosphate. A mutant selected for its ability to grow aerobically on propanediol as a carbon and energy source was reported to produce lactaldehyde reductase constitutively and at high levels, even aerobically. Under the new situation, this enzyme serves as a propanediol dehydrogenase. It was also reported that the mutant had lost the ability to grow on fucose. In the present study, it is shown that in wild-type cells the full synthesis of lactaldehyde dehydrogenase requires the presence of both molecular oxygen and a small molecule effector, and the full synthesis of lactaldehyde reductase requires anaerobiosis and the presence of a small molecule effector. The failure of mutant cells to grow on fucose reflects the impairment of a regulatory element in the fucose system that prevents the induction of the permease, the isomerase, and the kinase. The aldolase, on the other hand, is constitutively synthesized. Three independent fucose-utilizing revertants of the mutant all produce the permease, the isomerase, the kinase, as well as the aldolase, constitutively. These strains grow less well than the parental mutant on propanediol.     

Amino sugar assimilation by Escherichia coli

The carbon skeleton of glucose is extensively randomized during conversion to cell wall glucosamine by Escherichia coli K-12. Exogenous glucosamine-1-(14)C is selectively oxidized, and isotope incorporation into cellular glucosamine is greatly diluted during assimilation. A mutant unable to grow with N-acetylglucosamine as a carbon and energy source was isolated from E. coli K-12. This mutant was found to be defective in glucosamine-6-phosphate deaminase. Glucosamine-1-(14)C and N-acetylglucosamine-1-(14)C were assimilated during the growth of mutant cultures without degradation or carbon randomization. Assimilated isotopic carbon resided entirely in cell wall glucosamine and muramic acid. Some isotope dilution occurred from biosynthesis, but at high concentrations (0.2 mm) of added N-acetylglucosamine nearly all cellular amino sugar was derived from the exogenous source. Growth of the mutant was inhibited with 1 mmN-acetylglucosamine.     

Chromosomal mutation for citrate utilization by Escherichia coli K-12.

A mutant strain of Escherichia coli K-12 that utilizes citrate as a sole source of carbon and energy was isolated. Citrate utilization arose as the consequence of two mutations in genes citA and citB, which are linked to the gal operon. The mutant strain expresses a semiconstitutive citrate transport system, and it utilizes both citrate and isocitrate as carbon and energy sources. It is capable of utilizing cis- and trans-aconitate, but only if it is preinduced by growth on citrate.     

Endogenous xylose pathway in Saccharomyces cerevisiae

The baker's yeast Saccharomyces cerevisiae is generally classified as a non-xylose-utilizing organism. We found that S. cerevisiae can grow on D-xylose when only the endogenous genes GRE3 (YHR104w), coding for a nonspecific aldose reductase, and XYL2 (YLR070c, ScXYL2), coding for a xylitol dehydrogenase (XDH), are overexpressed under endogenous promoters. In nontransformed S. cerevisiae strains, XDH activity was significantly higher in the presence of xylose, but xylose reductase (XR) activity was not affected by the choice of carbon source. The expression of SOR1, encoding a sorbitol dehydrogenase, was elevated in the presence of xylose as were the genes encoding transketolase and transaldolase. An S. cerevisiae strain carrying the XR and XDH enzymes from the xylose-utilizing yeast Pichia stipitis grew more quickly and accumulated less xylitol than did the strain overexpressing the endogenous enzymes. Overexpression of the GRE3 and ScXYL2 genes in the S. cerevisiae CEN.PK2 strain resulted in a growth rate of 0.01 g of cell dry mass liter(-1) h(-1) and a xylitol yield of 55% when xylose was the main carbon source.     

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.