Metabolism of L-fucose and L-rhamnose in Escherichia coli: aerobic-anaerobic regulation of L-lactaldehyde dissimilation.

L-Lactaldehyde is a branching point in the metabolic pathway of L-fucose and L-rhamnose utilization. Under aerobic conditions, L-lactaldehyde is oxidized to L-lactate by the enzyme lactaldehyde dehydrogenase, while under anaerobic conditions, L-lactaldehyde is reduced to L-1,2-propanediol by the enzyme propanediol oxidoreductase. Aerobic growth on either of the methyl pentoses induces a lactaldehyde dehydrogenase enzyme which is inhibited by NADH and is very stable under anaerobic conditions. In the absence of oxygen, the cell shifts from the oxidation of L-lactaldehyde to its reduction, owing to both the induction of propanediol oxidoreductase activity and the decrease in the NAD/NADH ratio. The oxidation of L-lactaldehyde to L-lactate is again restored upon a change to aerobic conditions. In this case, only the NAD/NADH ratio may be invoked as a regulatory mechanism, since both enzymes remain active after this change. Experimental evidence in the presence of rhamnose with mutants unable to produce L-lactaldehyde and mutants capable of producing but not further metabolizing it points toward L-lactaldehyde as the effector molecule in the induction of lactaldehyde dehydrogenase. Analysis of a temperature-sensitive mutation affecting the synthesis of lactaldehyde dehydrogenase permitted us to locate an apparently single regulator gene linked to the ald locus at 31 min and probably acting as a positive control element on the expression of the structural gene.     

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.     

Cross-induction of the L-fucose system by L-rhamnose in Escherichia coli.

Dissimilation of L-fucose as a carbon and energy source by Escherichia coli involves a permease, an isomerase, a kinase, and an aldolase encoded by the fuc regulon at minute 60.2. Utilization of L-rhamnose involves a similar set of proteins encoded by the rha operon at minute 87.7. Both pathways lead to the formation of L-lactaldehyde and dihydroxyacetone phosphate. A common NAD-linked oxidoreductase encoded by fucO serves to reduce L-lactaldehyde to L-1,2-propanediol under anaerobic growth conditions, irrespective of whether the aldehyde is derived from fucose or rhamnose. In this study it was shown that anaerobic growth on rhamnose induces expression of not only the fucO gene but also the entire fuc regulon. Rhamnose is unable to induce the fuc genes in mutants defective in rhaA (encoding L-rhamnose isomerase), rhaB (encoding L-rhamnulose kinase), rhaD (encoding L-rhamnulose 1-phosphate aldolase), rhaR (encoding the positive regulator for the rha structural genes), or fucR (encoding the positive for the fuc regulon). Thus, cross-induction of the L-fucose enzymes by rhamnose requires formation of L-lactaldehyde; either the aldehyde itself or the L-fuculose 1-phosphate (known to be an effector) formed from it then interacts with the fucR-encoded protein to induce the fuc regulon.     

Constitutive activation of L-fucose genes by an unlinked mutation in Escherichia coli.

Wild-type Escherichia coli cannot grow on L-1,2-propanediol; mutants that can do so have increased basal activity of an NAD-linked L-1,2-propanediol oxidoreductase. This enzyme belongs to the L-fucose system and functions normally as L-lactaldehyde reductase during fermentation of the methylpentose. In wild-type cells, the activity of this enzyme is fully induced only anaerobically. Continued aerobic selection for mutants with an improved growth rate on L-1,2-propanediol inevitably leads to full constitutive expression of the oxidoreductase activity. When this occurs, L-fuculose 1-phosphate aldolase concomitantly becomes constitutive, whereas L-fucose permease, L-fucose isomerase, and L-fuculose kinase become noninducible. It is shown in this study that the noninducibility of the three proteins can be changed by two different kinds of suppressor mutations: one mapping external to and the other within the fuc gene cluster. Both mutations result in constitutive synthesis of the permease, the isomerase, and the kinase, without affecting synthesis of the oxidoreductase and the aldolase. Since expression of the fuc structural genes is activated by a protein specified by the regulator gene fucR, and since all the known genes of the fuc system are clustered at minute 60.2 of the chromosome, the external gene in which the suppressor mutation can occur probably has an unrelated function in the wild-type strain. The internal suppressor mutation might be either in fucR or in the promoter region of the genes encoding the permease, the isomerase, and the kinase, if these genes belong to the same operon.     

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.     

L-1,2-propanediol exits more rapidly than L-lactaldehyde from Escherichia coli.

Catabolism of the six-carbon compound L-fucose results in formation of dihydroxyacetone phosphate (C-1-to-C-3 fragment) and L-lactaldehyde (C-4-to-C-6 fragment) as intermediates. The fate of lactaldehyde depends on the respiratory growth conditions. Aerobically, lactaldehyde is oxidized to L-lactate by an NAD-linked dehydrogenase (ald product). L-Lactate, in turn, is converted to pyruvate, which enters the general metabolic pool. Anaerobically, lactaldehyde is reduced to L-1,2-propanediol by an NADH-linked oxidoreductase (fucO product). L-1,2-Propanediol is excreted as a terminal fermentation product. In a previous study, we showed that retention of the C-4-to-C-6 fragment of fucose depended on the competition for lactaldehyde by aldehyde dehydrogenase and propanediol oxidoreductase (Y. Zhu and E.C.C. Lin, J. Bacteriol. 169:785-789, 1987). In this study, we compared the wild-type strain and isogenic mutant strains defective in ald, fucO, or both for ability to accumulate radioactivity when incubated with fucose labeled at either the C-1 or the C-6 position. The results showed that although blocking the oxidation of lactaldehyde prevented its assimilation, rapid exit of the 3-carbon unit occurred only when the compound was reduced to propanediol. Moreover, growth experiments on fucose indicated that a double ald fucO mutant accumulated inhibiting concentrations of lactaldehyde. The inner cell membrane therefore appears to be much more permeable to the 3-carbon alcohol than to the 3-carbon aldehyde. The almost instantaneous exit of propanediol appears to be a facilitated process.     

Fermentation mechanism of fucose and rhamnose in Salmonella typhimurium and Klebsiella pneumoniae.

An equimolar amount of 1,2-propanediol was detected in the medium when Salmonella typhimurium or Klebsiella pneumoniae fermented L-fucose or L-rhamnose. These metabolic conditions induced a propanediol oxidoreductase that converted the lactaldehyde formed in the dissimilation of either sugar into the diol. The enzyme was further identified by cross-reaction with antibodies against Escherichia coli propanediol oxidoreductase. This indicates that L-fucose and L-rhamnose fermentation takes place in these species by 1,2-propanediol production and excretion.     

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.     

Aldehyde dehydrogenase induction by glutamate in Escherichia coli. Role of 2-oxoglutarate.

In Escherichia coli, an aldehyde dehydrogenase that catalyzes the oxidation of L-lactaldehyde to L-lactate is induced not only by L-fucose, L-rhamnose or D-arabinose, but also by growth in the presence of glutamate or amino acids yielding glutamate, with the exception of proline. Induction by these amino acids requires glutamate accumulation. 4-Aminobutyric acid also induces this aldehyde dehydrogenase through its transamination to glutamate. Growth on 2-oxoglutarate, the tricarboxylic acid cycle intermediate with which glutamate is in equilibrium, also induces this aldehyde dehydrogenase. Conditions in which the conversion of 2-oxoglutarate into glutamate is highly restricted displayed unchanged rates of induction by 2-oxoglutarate, indicating that glutamate induces the aldehyde dehydrogenase through 2-oxoglutarate formation. Evidence is presented showing that L-fucose- and 2-oxoglutarate-inducing systems share the same regulatory protein. Induction by growth on either of these two compounds is repressed both by glucose and by glycerol. Addition of cAMP to these cultures partially recovers the glucose-repressed aldehyde dehydrogenase activity, while this nucleotide has no effect on the glycerol-mediated repression. These results indicate that ald is under carbon regulation mediated by at least two different mechanisms.     

Metabolic fate of L-lactaldehyde derived from an alternative L-rhamnose pathway

Fungal Pichia stipitis and bacterial Azotobacter vinelandii possess an alternative pathway of L-rhamnose metabolism, which is different from the known bacterial pathway. In a previous study (Watanabe S, Saimura M & Makino K (2008) Eukaryotic and bacterial gene clusters related to an alternative pathway of non-phosphorylated L-rhamnose metabolism. J Biol Chem283, 20372-20382), we identified and characterized the gene clusters encoding the four metabolic enzymes [L-rhamnose 1-dehydrogenase (LRA1), L-rhamnono-gamma-lactonase (LRA2), L-rhamnonate dehydratase (LRA3) and l-2-keto-3-deoxyrhamnonate aldolase (LRA4)]. In the known and alternative L-rhamnose pathways, L-lactaldehyde is commonly produced from l-2-keto-3-deoxyrhamnonate and L-rhamnulose 1-phosphate by each specific aldolase, respectively. To estimate the metabolic fate of L-lactaldehyde in fungi, we purified L-lactaldehyde dehydrogenase (LADH) from P. stipitis cells L-rhamnose-grown to homogeneity, and identified the gene encoding this enzyme (PsLADH) by matrix-assisted laser desorption ionization-quadruple ion trap-time of flight mass spectrometry. In contrast, LADH of A. vinelandii (AvLADH) was clustered with the LRA1-4 gene on the genome. Physiological characterization using recombinant enzymes revealed that, of the tested aldehyde substrates, L-lactaldehyde is the best substrate for both PsLADH and AvLADH, and that PsLADH shows broad substrate specificity and relaxed coenzyme specificity compared with AvLADH. In the phylogenetic tree of the aldehyde dehydrogenase superfamily, PsLADH is poorly related to the known bacterial LADHs, including that of Escherichia coli (EcLADH). However, despite its involvement in different L-rhamnose metabolism, AvLADH belongs to the same subfamily as EcLADH. This suggests that the substrate specificities for L-lactaldehyde between fungal and bacterial LADHs have been acquired independently.     

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 1,2-propanediol from glycerol in Saccharomyces cerevisiae

Glycerol has become an attractive carbon source in the biotechnology industry owing to its low price and reduced state. However, glycerol is rarely used as a carbon source in Saccharomyces cerevisiae because of its low utilization rate. In this study, we used glycerol as a main carbon source in S. cerevisiae to produce 1,2-propanediol. Metabolically engineered S. cerevisiae strains with overexpression of glycerol dissimilation pathway genes, including glycerol kinase (GUT1), glycerol 3-phosphate dehydrogenase (GUT2), glycerol dehydrogenase (gdh), and a glycerol transporter gene (GUP1), showed increased glycerol utilization and growth rate. More significant improvement of glycerol utilization and growth rate was accomplished by introducing 1,2-propanediol pathway genes, mgs (methylglyoxal synthase) and gldA (glycerol dehydrogenase) from Escherichia coli. By engineering both glycerol dissimilation and 1,2-propanediol pathways, the glycerol utilization and growth rate were improved 141% and 77%, respectively, and a 2.19 g 1,2- propanediol/l titer was achieved in 1% (v/v) glycerolcontaining YEPD medium in engineered S. cerevisiae.     

Eukaryotic and bacterial gene clusters related to an alternative pathway of nonphosphorylated L-rhamnose metabolism

The Entner-Doudoroff (ED) pathway is a classic central pathway of d-glucose metabolism in all three phylogenetic domains. On the other hand, Archaea and/or bacteria possess several modified versions of the ED pathway, in which nonphosphorylated intermediates are involved. Several fungi, including Pichia stipitis and Debaryomyces hansenii, possess an alternative pathway of L-rhamnose metabolism, which is different from the known bacterial pathway. Gene cluster related to this hypothetical pathway was identified by bioinformatic analysis using the metabolic enzymes involved in analogous sugar pathways to the ED pathway. Furthermore, the homologous gene cluster was found not only in many other fungi but also several bacteria, including Azotobacter vinelandii. Four putative metabolic genes, LRA1-4, were cloned, overexpressed in Escherichia coli, and purified. Substrate specificity and kinetic analysis revealed that nonphosphorylated intermediates related to L-rhamnose are significant active substrates for the purified LRA1-4 proteins. Furthermore, L-2-keto-3-deoxyrhamnonate was structurally identified as both reaction products of dehydration by LRA3 and aldol condensation by LRA4. These results suggested that the LRA1-4 genes encode L-rhamnose 1-dehydrogenase, L-rhamnono-gamma-lactonase, L-rhamnonate dehydratase, and L-KDR aldolase, respectively, by which L-rhamnose is converted into pyruvate and L-lactaldehyde through analogous reaction steps to the ED pathway. There was no evolutionary relationship between L-KDR aldolases from fungi and bacteria.     

A mutant crp allele that differentially activates the operons of the fuc regulon in Escherichia coli.

L-Fucose is used by Escherichia coli through an inducible pathway mediated by a fucP-encoded permease, a fucI-encoded isomerase, a fucK-encoded kinase, and a fucA-encoded aldolase. The adolase catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde. Anaerobically, lactaldehyde is converted by a fucO-encoded oxidoreductase to L-1,2-propanediol, which is excreted. The fuc genes belong to a regulon comprising four linked operons: fucO, fucA, fucPIK, and fucR. The positive regulator encoded by fucR responds to fuculose 1-phosphate as the effector. Mutants serially selected for aerobic growth on propanediol became constitutive in fucO and fucA [fucO(Con) fucA(Con)], but noninducible in fucPIK [fucPIK(Non)]. An external suppressor mutation that restored growth on fucose caused constitutive expression of fucPIK. Results from this study indicate that this suppressor mutation occurred in crp, which encodes the cyclic AMP-binding (or receptor) protein. When the suppressor allele (crp-201) was transduced into wild-type strains, the recipient became fucose negative and fucose sensitive (with glycerol as the carbon and energy source) because of impaired expression of fucA. The fucPIK operon became hyperinducible. The growth rate on maltose was significantly reduced, but growth on L-rhamnose, D-galactose, L-arabinose, glycerol, or glycerol 3-phosphate was close to normal. Lysogenization of fuc+ crp-201 cells by a lambda bacteriophage bearing crp+ restored normal growth ability on fucose. In contrast, lysogenization of [fucO(Con)fucA(Con)fucPIK(Non)crp-201] cells by the same phage retarded their growth on fucose.     

L-rhamnose and L-fucose suppress cancer growth in mice

It is documented that deficient fucosylation may play an important role in the pathogenesis of cancer. Since the supplementation of L-fucose could restore fucosylation in both in vitro and in vivo conditions, our intent was to examine the effect of intraperitoneal administration of L-fucose and L-rhamnose (a similar deoxysaccharide) on tumour growth, mitotic activity and metastatic setting of a solid form of Ehrlich carcinoma as well as on the survival rate of tumour bearing mice. Both L-fucose and L-rhamnose exerted a significant suppressive effect on tumour growth (P<0.05). After 10 days of therapy, the greatest inhibition of tumour growth expressed as a percentage of controls was observed in L-rhamnose at a dose of 3 g/kg/day (by 62%) and L-fucose at a dose of 5 g/kg/day (by 47%). Moreover, the mitotic index decreased with increasing doses of L-fucose and L-rhamnose. Prolonged survival of tumour bearing mice was observed after 14 consecutive days of daily administering L-rhamnose. Its optimal dose was estimated to be 3.64 g/kg/day. L-Fucose, however, displayed only a slight effect on the survival of the mice. Our results suggest that L-fucose and especially L-rhamnose have anticancer potential. This study is the first to demonstrate the tumour-inhibitory effect of L-rhamnose.     

Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene

Salmonella enterica degrades 1,2-propanediol by a pathway dependent on coenzyme B12 (adenosylcobalamin [AdoCb1]). Previous studies showed that 1,2-propanediol utilization (pdu) genes include those for the conversion of inactive cobalamins, such as vitamin B12, to AdoCbl. However, the specific genes involved were not identified. Here we show that the pduO gene encodes a protein with ATP:cob(I)alamin adenosyltransferase activity. The main role of this protein is apparently the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation. Genetic tests showed that the function of the pduO gene was partially replaced by the cobA gene (a known ATP:corrinoid adenosyltransferase) but that optimal growth of S. enterica on 1,2-propanediol required a functional pduO gene. Growth studies showed that cobA pduO double mutants were unable to grow on 1,2-propanediol minimal medium supplemented with vitamin B(12) but were capable of growth on similar medium supplemented with AdoCbl. The pduO gene was cloned into a T7 expression vector. The PduO protein was overexpressed, partially purified, and, using an improved assay procedure, shown to have cob(I)alamin adenosyltransferase activity. Analysis of the genomic context of genes encoding PduO and related proteins indicated that particular adenosyltransferases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of AdoCbl. Such analyses also indicated that PduO is a bifunctional enzyme. The possibility that genes of unknown function proximal to adenosyltransferase homologues represent previously unidentified AdoCbl-dependent enzymes is discussed.     

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.