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 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 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.     

An Escherichia coli mutant defective in the NAD-dependent succinate semialdehyde dehydrogenase

Escherichia coli mutants, unable to grown on 4-hydroxyphenylacetate, have been isolated and found to be defective in the NAD-dependent succinate semialdehyde dehydrogenase. When the mutants are grown with 4-aminobutyrate as sole nitrogen source an NAD-dependent succinate semialdehyde dehydrogenase seen in the parental strain is absent but, as in the parental strain, an NADP-dependent enzyme is induced. Growth of the mutants is inhibited by 4-hydroxyphenylacetate due to the accumulation of succinate semialdehyde. The mutants are more sensitive to inhibition by exogenous succinate semialdehyde than is the parental strain. Secondary mutants able to grow in the presence of 4-hydroxyphenylacetate but still unable to use it as sole carbon source were defective in early steps of 4-hydroxyphenylacetate catabolism and so did not form succinate semialdehyde from 4-hydroxyphenylacetate. The gene encoding the NAD-dependent succinate semialdehyde dehydrogenase of Escherichia coli K-12 was located at min 34.1 on the genetic map.     

Close linkage of prd and rel genes in Escherichia coli K-12

Summary The prd gene, the mutant allele of which permits growth of E. coli on 1,2-propanediol as sole carbon source, has been located by transduction very close to the rel (RNA control) gene. This close linkage provides a convenient means for the inter-strain transfer of rel.This work was supported in part by the Australian Meat Research Committee.     

Point mutation of the xylose reductase (XR) gene reduces xylitol accumulation and increases citric acid production in Aspergillus carbonarius

Aspergillus carbonarius accumulates xylitol when it grows on d-xylose. In fungi, d-xylose is reduced to xylitol by the NAD(P)H-dependent xylose reductase (XR). Xylitol is then further oxidized by the NAD⁺-dependent xylitol dehydrogenase (XDH). The cofactor impairment between the XR and XDH can lead to the accumulation of xylitol under oxygen-limiting conditions. Most of the XRs are NADPH dependent and contain a conserved Ile-Pro-Lys-Ser motif. The only known naturally occurring NADH-dependent XR (from Candida parapsilosis) carries an arginine residue instead of the lysine in this motif. In order to overcome xylitol accumulation in A. carbonarius a Lys-274 to Arg point mutation was introduced into the XR with the aim of changing the specificity toward NADH. The effect of the genetic engineering was examined in fermentation for citric acid production and xylitol accumulation by using d-xylose as the sole carbon source. Fermentation with the mutant strain showed a 2.8-fold reduction in xylitol accumulation and 4.5-fold increase in citric acid production compared to the wild-type strain. The fact that the mutant strain shows decreased xylitol levels is assumed to be associated with the capability of the mutated XR to use the NADH generated by the XDH, thus preventing the inhibition of XDH by the high levels of NADH and ensuring the flux of xylose through the pathway. This work shows that enhanced production of citric acid can be achieved using xylose as the sole carbon source by reducing accumulation of other by-products, such as xylitol.     

Xylitol Production by an Enterobacter Species

A xylose-utilizing bacterial strain was isolated from soil.

The strain, No. 553, was identified as Enterobacter liquefaciens from the result of the taxonomical studies. This bacterium grew well on D-xylose as a sole carbon source and accumulated pentitol extracellularly in shaking culture.

Pentitol produced was isolated from the culture broth and identified as xylitol.

The xylitol production reached the maximum after the cessation of the cell growth with a yield of 33.3 mg per ml in a medium containing 10% D-xylose as a sole carbon source and no significant decline of the amount of xylitol was observed through the period of the cultivation.     

PanB is involved in nicotine metabolism in Pseudomonas putida

A nicotine-sensitive mutant was generated from the nicotine-degrading bacterium, Pseudomonas putida strain J5, by mini-Tn5 transposon mutagenesis. This mutant was unable to grow with nicotine as the sole carbon source but could grow with glucose. Sequence analysis showed that the Tn5 transposon inserted at the site of the ketopantoate hydroxymethyltransferase gene (panB), which had 54% identity to PanB in Escherichia coli K-12. In-frame deletion of the panB gene abolished the nicotine-degrading ability of strain J5, while complementation with panB from P. putida J5 and E. coli K-12 restored the degrading activity of the mutant to the wild-type level. These results suggest that ketopantoate hydroxymethyltransferase is a crucial enzyme in nicotine metabolism in P. putida J5.     

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.     

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.     

Production of xylitol by a Coniochaeta ligniaria strain tolerant of inhibitors and defective in growth on xylose

In conversion of biomass to fuels or chemicals, inhibitory compounds arising from physical–chemical pretreatment of the feedstock can interfere with fermentation of the sugars to product. Fungal strain Coniochaeta ligniaria NRRL30616 metabolizes the furan aldehydes furfural and 5‐hydroxymethylfurfural, as well as a number of aromatic and aliphatic acids and aldehydes. Use of NRRL30616 to condition biomass sugars by metabolizing the inhibitors improves their fermentability. Wild‐type C. ligniaria has the ability to grow on xylose as sole source of carbon and energy, with no accumulation of xylitol. Mutants of C. ligniaria unable to grow on xylose were constructed. Xylose reductase and xylitol dehydrogenase activities were reduced by approximately two thirds in mutant C8100. The mutant retained ability to metabolize inhibitors in biomass hydrolysates. Although C. ligniaria C8100 did not grow on xylose, the strain converted a portion of xylose to xylitol, producing 0.59 g xylitol/g xylose in rich medium and 0.48 g xylitol/g xylose in corn stover dilute acid hydrolysate. 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016 © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:606–612, 2016     

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.     

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.     

Production of xylitol from D-xylose by a xylitol dehydrogenase gene-disrupted mutant of Candida tropicalis

Xylitol dehydrogenase (XDH) is one of the key enzymes in d-xylose metabolism, catalyzing the oxidation of xylitol to d-xylulose. Two copies of the XYL2 gene encoding XDH in the diploid yeast Candida tropicalis were sequentially disrupted using the Ura-blasting method. The XYL2-disrupted mutant, BSXDH-3, did not grow on a minimal medium containing d-xylose as a sole carbon source. An enzyme assay experiment indicated that BSXDH-3 lost apparently all XDH activity. Xylitol production by BSXDH-3 was evaluated using a xylitol fermentation medium with glucose as a cosubstrate. As glucose was found to be an insufficient cosubstrate, various carbon sources were screened for efficient cofactor regeneration, and glycerol was found to be the best cosubstrate. BSXDH-3 produced xylitol with a volumetric productivity of 3.23 g liter(-1) h(-1), a specific productivity of 0.76 g g(-1) h(-1), and a xylitol yield of 98%. This is the first report of gene disruption of C. tropicalis for enhancing the efficiency of xylitol production.     

Glutamine assimilation pathways in Neurospora crassa growing on glutamine as sole nitrogen and carbon source

Neurospora crassa wild-type is almost unable to grow on glutamine as sole nitrogen and carbon source but a GDH-; GS +/- double mutant strain, lacking NADP-dependent glutamate dehydrogenase and partially lacking glutamine synthetase did grow. Under these conditions, the double mutant had a higher chemical energy content than the wild-type. Enzyme assays and labelling experiments with glutamine indicated that in the double mutant glutamine was degraded to ammonium and to carbon skeletons by glutamate synthase, the catabolic (NADH-dependent) glutamate dehydrogenase and the glutamine transaminase-omega-amidase pathway.     

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.     

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.     

Selection ofEscherichia coli K12 1EA mutants with increased synthesis of ribitol dehydrogenase

Selection of an interspecific hybridEscherichia coli K 12 1EA in a chemostat on xylitol yielded a stable mutant synthesizing a four-fold amount of ribitol dehydrogenase (EC 1.1.1.56). Subsequent cultivation of the mutant under increased selection pressure resulted in an accumulation of a mutant with 12-fold higher level of ribitol dehydrogenase relative to the parent strain 1EA. A selection during which a UV-mutagenized population of the 1EA mutant was cultivated in a chemostat on xylitol was accompanied by monitoring the activities of ribitol dehydrogenase andD-arabinitol dehydrogenase (EC 1.1.1.11) of two adjacent catabolite operons. A several-fold increase in the activity of the two enzymes was followed by further increase in the activity of ribitol dehydrogenase and a concomitant drop in the activity ofD-arabinitol dehydrogenase. The two hyperproducing strains are compared with the parent mutant as to the rate of synthesis of the two dehydrogenases and growth parameters under the conditions of batch cultivation.     

Transport of acetate in mutants of Saccharomyces cerevisiae defective in monocarboxylate permeases

The strain Saccharomyces cerevisiae W303-1a, able to grow in a medium containing acetic acid as the sole carbon and energy source, was subjected to mutagenesis in order to obtain mutants deficient in monocarboxylate permeases. Two mutant clones exhibiting growth in ethanol, but unable to grow in a medium with acetic acid as the sole carbon and energy source, were isolated (mutants Ace12 and Ace8). In both mutants, the activity for the acetate carrier was strongly affected. The mutant Ace8 revealed not to be affected in the transport of lactate, while the mutant Ace12 did not display activity for that carrier. These results reinforced those previously found in the strain IGC 4072, where two distinct transport systems for monocarboxylates have been described, depending on the growth carbon source. It is tempting to postulate that the Ace8 mutant seems to be affected in the gene coding for an acetate permease. In contrast, the absence of activity for both monocarboxylate permeases in mutant Ace12 could be attributed to a mutation in a gene coding for a regulatory protein not detected before.