Prebiotic Pathway from Ribose to RNA Formation.

At the focus of abiotic chemical reactions is the synthesis of ribose. No satisfactory explanation was provided as to the missing link between the prebiotic synthesis of ribose and prebiotic RNA (preRNA). Hydrogen cyanide (HCN) is assumed to have been the principal precursor in the prebiotic formation of aldopentoses in the formose reaction and in the synthesis of ribose. Ribose as the best fitting aldopentose became the exclusive sugar component of RNA. The elevated yield of ribose synthesis at higher temperatures and its protection from decomposition could have driven the polymerization of the ribose-phosphate backbone and the coupling of nucleobases to the backbone. RNA could have come into being without the involvement of nucleotide precursors. The first nucleoside monophosphate is likely to have appeared upon the hydrolysis of preRNA contributed by the presence of reactive 2'-OH moieties in the preRNA chain. As a result of phosphorylation, nucleoside monophosphates became nucleoside triphosphates, substrates for the selective synthesis of genRNA.

Conversion of L-arabinose to L-ribose by genetically engineered Candida tropicalis.

L-Ribose, a starting material for the synthesis of L-nucleoside, has attracted lots of attention since L-nucleoside is responsible for the antiviral activities of the racemic mixtures of nucleoside enantiomers. In this study, the L-ribulose-producing Candida tropicalis strain was engineered for the conversion of L-arabinose to L-ribose. For the construction of a uracil auxotroph, the URA3 gene was excised by homologous recombination. The expression cassette of codon-optimized L-ribose isomerase gene from Acinetobacter calcoaceticus DL-28 under the control of the GAPDH promoter was integrated to the uracil auxotroph. The resulting strain, K1 CoSTP2 LsaAraA AcLRI, was cultivated with the glucose/L-arabinose mixture. At 45.5 h of fermentation, 6.0 g/L of L-ribose and 3.2 g/L of L-ribulose were produced from 30 g/L of L-arabinose. The proportion between L-ribose and L-ribulose was approximately 2:1 and the conversion yield of L-arabinose to L-ribose was about 20% (w/w). The L-ribose-producing yeast strain was successfully constructed for the first time and could convert L-arabinose to L-ribose in one-pot fermentation using the mixture of glucose and L-arabinose.

Characterization of an L-Arabinose Isomerase from Bacillus velezensis and Its Application for L-Ribulose and L-Ribose Biosynthesis.

L-Ribulose and L-ribose are two high-value unnatural sugars that can be biosynthesized by sugar isomerases. In this paper, an L-arabinose isomerase (BvAI) from Bacillus velezensis CICC 24777 was cloned and overexpressed in Escherichia coli BL21 (DE3) strain. The maximum activity of recombinant BvAI was observed at 45 °C and pH 8.0, in the presence of 1.0 mM Mn2+. Approximately 207.2 g/L L-ribulose was obtained from 300 g/L L-arabinose in 1.5 h by E. coli harboring BvAI. In addition, approximately 74.25 g/L L-ribose was produced from 300 g/L L-arabinose in 7 h by E. coli co-expressing BvAI and L-RI from Actinotalea fermentans ATCC 43279 (AfRI). This study provides a feasible approach for producing L-ribose from L-arabinose using a co-expression system harboring L-Al and L-RI.

Phosphate sugar isomerases and their potential for rare sugar bioconversion.

Phosphate sugar isomerases, catalyzing the isomerization between ketopentose/ketohexose phosphate and aldopentose/aldohexose phosphate, play an important role in microbial sugar metabolism. They are present in a wide range of microorganisms. They have attracted increasing research interest because of their broad substrate specificity and great potential in the enzymatic production of various rare sugars. Here, the enzymatic properties of various phosphate sugar isomerases are reviewed in terms of their substrate specificities and their applications in the production of valuable rare sugars because of their functions such as low-calorie sweeteners, bulking agents, and pharmaceutical precursor. Specifically, we focused on the industrial applications of D-ribose-5-phosphate isomerase and D-mannose-6-phosphate isomerase to produce D-allose and L-ribose, respectively.

Encapsulation of Mannose-6-phosphate Isomerase in Yeast Spores and Its Application in l-Ribose Production.

A mannose-6-phosphate isomerase (MPI) from Geobacillus thermodenitrificans was expressed and successfully encapsulated into the Saccharomyces cerevisiae spores. Our results demonstrated that compared to the free enzyme, the MPI triple mutant encapsulated in osw2Δ spores exhibited much preferred enzymatic properties, such as enhanced catalytic activity, excellent reusability, thermostability, and tolerance to various harsh conditions. In combination with an l-arabinose isomerase (AI) also from G. thermodenitrificans, this technique of spore encapsulation was applied for producing a high-value rare sugar l-ribose from biomass-derived l-arabinose. Using a 10 mL reaction system, 350 mg of l-ribose was produced from 1 g of l-arabinose with a conversion yield of 35% by repeatedly reacting with 200 mg of AI-encapsulated spores and 300 mg of MPI-encapsulated spores. This study provides a very useful and concise approach for the synthesis of rare sugars and other useful compounds.

Dual metabolomic profiling uncovers Toxoplasma manipulation of the host metabolome and the discovery of a novel parasite metabolic capability.

The obligate intracellular parasite Toxoplasma gondii is auxotrophic for several key metabolites and must scavenge these from the host. It is unclear how T. gondii manipulates host metabolism to support its overall growth rate and non-essential metabolites. To investigate this question, we measured changes in the joint host-parasite metabolome over a time course of infection. Host and parasite transcriptomes were simultaneously generated to determine potential changes in expression of metabolic enzymes. T. gondii infection changed metabolite abundance in multiple metabolic pathways, including the tricarboxylic acid cycle, the pentose phosphate pathway, glycolysis, amino acid synthesis, and nucleotide metabolism. Our analysis indicated that changes in some pathways, such as the tricarboxylic acid cycle, were mirrored by changes in parasite transcription, while changes in others, like the pentose phosphate pathway, were paired with changes in both the host and parasite transcriptomes. Further experiments led to the discovery of a T. gondii enzyme, sedoheptulose bisphosphatase, which funnels carbon from glycolysis into the pentose phosphate pathway through an energetically driven dephosphorylation reaction. This additional route for ribose synthesis appears to resolve the conflict between the T. gondii tricarboxylic acid cycle and pentose phosphate pathway, which are both NADP+ dependent. Sedoheptulose bisphosphatase represents a novel step in T. gondii central carbon metabolism that allows T. gondii to energetically-drive ribose synthesis without using NADP+.

Microbial and enzymatic strategies for the production of L-ribose.

L-Ribose is a non-naturally occurring pentose that recently has become known for its potential application in the pharmaceutical industry, as it is an ideal starting material for use in synthesizing L-nucleosides analogues, an important class of antiviral drugs. In the past few decades, the synthesis of L-ribose has been mainly undertaken through the chemical route. However, chemical synthesis of L-ribose is difficult to achieve on an industrial scale. Therefore, the biotechnological production of L-ribose has gained considerable attention, as it exhibits many merits over the chemical approaches. The present review focuses on various biotechnological strategies for the production of L-ribose through microbial biotransformation and enzymatic catalysis, and in particular on an analysis and comparison of the synthetic methods and different enzymes. The physiological functions and applications of L-ribose are also elucidated. In addition, different sugar isomerases involved in the production of L-ribose from a number of sources are discussed in detail with regard to their biochemical properties. Furthermore, analysis of the separation issues of L-ribose from the reaction solution and different purification methods is presented.Key points • l -Arabinose, l -ribulose and ribitol can be used to produce l -ribose by enzymes. • Five enzymes are systematically introduced for production of l -ribose. • Microbial transformation and enzymatic methods are promising for yielding l -ribose.

The Biosynthetic Gene Cluster of Pyrazomycin-A C-Nucleoside Antibiotic with a Rare Pyrazole Moiety.

Pyrazomycin is a rare C-nucleoside antibiotic containing a naturally occurring pyrazole ring, the biosynthetic origin of which has remained obscure for decades. In this study we report the identification of the gene cluster responsible for pyrazomycin biosynthesis in Streptomyces candidus NRRL 3601, revealing that the StrR-family regulator PyrR is the cluster-situated transcriptional activator governing pyrazomycin biosynthesis. Furthermore, our results from in vivo reconstitution and stable-isotope feeding experiments provide support for the hypothesis that PyrN is a new nitrogen-nitrogen bond-forming enzyme that catalyzes the linkage of the ϵ-NH2 nitrogen atom of l-N6 -OH-lysine and the α-NH2 nitrogen atom of l-glutamic acid. This study lays the foundation for further genetic and biochemical characterization of pyrazomycin pathway enzymes involved in constructing the characteristic pyrazole ring.

The pentose phosphate pathway of cellulolytic clostridia relies on 6-phosphofructokinase instead of transaldolase.

The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C5 precursors (such as ribose) during growth on other (non-C5) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PPi-PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PPi-PFKs of Clostridium thermosuccinogenes and Clostridium thermocellum indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of Escherichia coli, which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PPi-PFK of cellulolytic clostridia has evolved the use of S7P. We further show that C. thermosuccinogenes contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of C. thermosuccinogenes that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its K½ value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia.

Production of l-ribose from l-arabinose by co-expression of l-arabinose isomerase and d-lyxose isomerase in Escherichia coli.

l-Ribose is an important pharmaceutical intermediate that is used in the synthesis of numerous antiviral and anticancer drugs. However, it is a non-natural and expensive rare sugar. Recently, the enzymatic synthesis of l-ribose has attracted considerable attention owing to its considerable advantages over chemical approaches. In this work, a new strategy was developed for the production of l-ribose from the inexpensive starting material l-arabinose. The l-arabinose isomerase (l-AIase) gene from Alicyclobacillus hesperidum and the d-lyxose isomerase (d-LIase) gene from Thermoflavimicrobium dichotomicum were cloned and co-expressed in Escherichia coli, resulting in recombinant cells harboring the vector pCDFDuet-Alhe-LAI/Thdi-DLI. The co-expression system exhibited optimal activity at a temperature of 70 °C and pH 6.0, and the addition of Co2+ enhanced the catalytic activity by 27.8-fold. The system containing 50 g L-1 of recombinant cells were relatively stable up to 55 °C. The co-expression system (50 g L-1 of recombinant cells) afforded 20.9, 39.7, and 50.3 g L-1 of l-ribose from initial l-arabinose concentrations of 100, 300, and 500 g L-1, corresponding to conversion rate of 20.9%, 13.2%, and 10.0%, respectively. Overall, this study provides a viable approach for producing l-ribose from l-arabinose under slightly acidic conditions using a co-expression system harboring l-AIase and d-LIase genes.

A plant/fungal-type phosphoenolpyruvate carboxykinase located in the parasite mitochondrion ensures glucose-independent survival of Toxoplasma gondii.

Toxoplasma gondii is considered to be one of the most successful intracellular pathogens, because it can reproduce in varied nutritional milieus, encountered in diverse host cell types of essentially any warm-blooded organism. Our earlier work demonstrated that the acute (tachyzoite) stage of T. gondii depends on cooperativity of glucose and glutamine catabolism to meet biosynthetic demands. Either of these two nutrients can sustain the parasite survival; however, what determines the metabolic plasticity has not yet been resolved. Here, we reveal two discrete phosphoenolpyruvate carboxykinase (PEPCK) enzymes in the parasite, one of which resides in the mitochondrion (TgPEPCKmt), whereas the other protein is not expressed in tachyzoites (TgPEPCKnet). Parasites with an intact glycolysis can tolerate genetic deletions of TgPEPCKmt as well as of TgPEPCKnet, indicating their nonessential roles for tachyzoite survival. TgPEPCKnet can also be ablated in a glycolysis-deficient mutant, while TgPEPCKmt is refractory to deletion. Consistent with this, the lytic cycle of a conditional mutant of TgPEPCKmt in the glycolysis-impaired strain was aborted upon induced repression of the mitochondrial isoform, demonstrating its essential role for the glucose-independent survival of parasites. Isotope-resolved metabolomics of the conditional mutant revealed defective flux of glutamine-derived carbon into RNA-bound ribose sugar as well as metabolites associated with gluconeogenesis, entailing a critical nodal role of PEPCKmt in linking catabolism of glucose and glutamine with anabolic pathways. Our data also suggest a homeostatic function ofTgPEPCKmt in cohesive operation of glycolysis and the tricarboxylic acid cycle in a normal glucose-replete milieu. Conversely, we found that the otherwise integrative enzyme pyruvate carboxylase (TgPyC) is dispensable not only in glycolysis-competent but also in glycolysis-deficient tachyzoites despite a mitochondrial localization. Last but not least, the observed physiology of T. gondii tachyzoites appears to phenocopy cancer cells, which holds promise for developing common therapeutics against both threats.

Ribose operon repressor (RbsR) contributes to the adhesion of Aeromonas hydrophila to Anguilla japonica mucus.

The characterization of adhesion between pathogenic bacteria and the host is critical. Pathogenic Aeromonas hydrophila was shown to adhere in vitro to the mucus of Anguilla japonica. To further investigate the adhesion mechanisms of A. hydrophila, a mini-Tn10 transposon mutagenesis system was used to generate an insertion mutant library by cell conjugation. Seven mutants that were impaired in adhesion to mucus were selected out of 332 individual colonies, and mutant M196 was the most severely impaired strain. National Center for Biotechnology Information (NCBI) blast analysis showed that mutant M196 was inserted by mini-Tn10 with an ORF of approximately 1,005 bp (GenBank accession numbers KP280172). This ORF is predicted to encode a protein consist of 334 amino acid, which displays the highest identity (98%) with the RbsR of A. hydrophila ATCC 7966. Random inactivation of rbsR gene affected the pleiotropic phenotypes of A. hydrophila. The adhesion ability and the survival level of the rbsR gene mutant (M196) were attenuated compared with the wild-type and rbsR complementary type. The findings of this study indicated that RbsR plays roles in the bacterial adhesion and intracellular survival of A. hydrophila.

A single and two step isomerization process for d-tagatose and l-ribose bioproduction using l-arabinose isomerase and d-lyxose isomerase.

l-ribose and d-tagatose are biochemically synthesized using sugar isomerases. The l-arabinose isomerase gene from Shigella flexneri (Sf-AI) was cloned and expressed in Escherichia coli BL-21. Sf-AI was applied for the bioproduction of d-tagatose from d-galactose. l-ribose synthesis was performed by two step isomerization using Sf-AI and d-lyxose/ribose isomerase from Cohnella laevoribosii. The overall 22.3% and 25% conversion rate were observed for d-tagatose and l-ribose production from d-galactose and l-arabinose respectively. In the present manuscript, synthesis of rare sugars from naturally available sugars is discussed along with the biochemical characterization of Sf-AI and its efficiency.

Efficient biosynthesis of d-ribose using a novel co-feeding strategy in Bacillus subtilis without acid formation.

Normally, low d-ribose production was identified as responsible for plenty of acid formation by Bacillus subtilis due to its carbon overflow. An approach of co-feeding glucose and sodium citrate is developed here and had been proved to be useful in d-ribose production. This strategy is critical because it affects the cell concentration, the productivity of d-ribose and, especially, the formation of by-products such as acetoin, lactate and acetate. d-ribose production was increased by 59·6% from 71·06 to 113·41 g l-1 without acid formation by co-feeding 2·22 g l-1  h-1 glucose and 0·036 g l-1  h-1 sodium citrate to a 60 g l-1 glucose reaction system. Actually, the cell density was also enhanced from 11·51 to 13·84 g l-1 . These parameters revealed the importance of optimization and modelling of the d-ribose production process. Not only could zero acid formation was achieved over a wide range of co-feeding rate by reducing glycolytic flux drastically but also the cell density and d-ribose yield were elevated by increasing the hexose monophosphate pathway flux. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus subtilis usually produce d-ribose accompanied by plenty of organic acids when glucose is used as a carbon source, which is considered to be a consequence of mismatched glycolytic and tricarboxylic acid cycle capacities. This is the first study to provide high-efficiency biosynthesis of d-ribose without organic acid formation in B. subtilis, which would be lower than the cost of separation and purification. The strain transketolase-deficient B. subtilis CGMCC 3720 can be potentially applied to the production of d-ribose in industry.

Improvement of D-Ribose Production from Corn Starch Hydrolysate by a Transketolase-Deficient Strain Bacillus subtilis UJS0717.

D-Ribose is a five-carbon sugar and generally used as an energy source to improve athletic performance and the ability. The culture conditions for maximum D-ribose production performance from cheap raw material corn starch hydrolysate were improved by using one-factor-at-a-time experiments and a three-level Box-Behnken factorial design. The optimal fermentation parameters were obtained as 36°C culture temperature, 10% inoculum volume, and 7.0 initial pH. The mathematical model was then developed to show the effect of each medium composition and their interactions on the production of D-ribose and estimated that the optimized D-ribose production performance with the concentration of 62.13 g/L, yield of 0.40 g/g, and volumetric productivity of 0.86 g/L·h could be obtained when the medium compositions were set as 157 g/L glucose, 21 g/L corn steep liquor, 3.2 g/L (NH4)2SO4, 1 g/L yeast extract, 0.05 g/L MnSO4·H2O, and 20 g/L CaCO3. These findings indicated the D-ribose production performance was significantly improved compared to that under original conditions.

Anaplerotic metabolism of alloreactive T cells provides a metabolic approach to treat graft-versus-host disease.

T-cell activation requires increased ATP and biosynthesis to support proliferation and effector function. Most models of T-cell activation are based on in vitro culture systems and posit that aerobic glycolysis is employed to meet increased energetic and biosynthetic demands. By contrast, T cells activated in vivo by alloantigens in graft-versus-host disease (GVHD) increase mitochondrial oxygen consumption, fatty acid uptake, and oxidation, with small increases of glucose uptake and aerobic glycolysis. Here we show that these differences are not a consequence of alloactivation, because T cells activated in vitro either in a mixed lymphocyte reaction to the same alloantigens used in vivo or with agonistic anti-CD3/anti-CD28 antibodies increased aerobic glycolysis. Using targeted metabolic (13)C tracer fate associations, we elucidated the metabolic pathway(s) employed by alloreactive T cells in vivo that support this phenotype. We find that glutamine (Gln)-dependent tricarboxylic acid cycle anaplerosis is increased in alloreactive T cells and that Gln carbon contributes to ribose biosynthesis. Pharmacological modulation of oxidative phosphorylation rapidly reduces anaplerosis in alloreactive T cells and improves GVHD. On the basis of these data, we propose a model of T-cell metabolism that is relevant to activated lymphocytes in vivo, with implications for the discovery of new drugs for immune disorders.

L-Ribose production from L-arabinose by immobilized recombinant Escherichia coli co-expressing the L-arabinose isomerase and mannose-6-phosphate isomerase genes from Geobacillus thermodenitrificans.

L-Ribose is an important precursor for antiviral agents, and thus its high-level production is urgently demanded. For this aim, immobilized recombinant Escherichia coli cells expressing the L-arabinose isomerase and variant mannose-6-phosphate isomerase genes from Geobacillus thermodenitrificans were developed. The immobilized cells produced 99 g/l L-ribose from 300 g/l L-arabinose in 3 h at pH 7.5 and 60 °C in the presence of 1 mM Co(2+), with a conversion yield of 33 % (w/w) and a productivity of 33 g/l/h. The immobilized cells in the packed-bed bioreactor at a dilution rate of 0.2 h(-1) produced an average of 100 g/l L-ribose with a conversion yield of 33 % and a productivity of 5.0 g/l/h for the first 12 days, and the operational half-life in the bioreactor was 28 days. Our study is first verification for L-ribose production by long-term operation and feasible for cost-effective commercialization. The immobilized cells in the present study also showed the highest conversion yield among processes from L-arabinose as the substrate.

Effects of oxygen supply and mixed sugar concentration on D-ribose production by a transketolase-deficient Bacillus subtilis SPK1.

D-Ribose is a value-added five-carbon sugar used for riboflavin production. To investigate the effects of oxygen supply and mixed sugar concentration on microbial production of D-ribose, a transketolase-deficient Bacillus subtilis SPK1 was cultured batch-wise using xylose and glucose. A change of agitation speed from 300 rpm to 600 rpm at 1 vvm of air supply increased both the xylose consumption rate and D-ribose production rate. Because the sum of the specific consumption rates for xylose and glucose was similar at all agitation speeds, metabolic preferences between xylose and glucose might depend on oxygen supply. Although B. subtilis SPK1 can take up xylose and glucose by the active transport mechanism, a high initial concentration of xylose and glucose was not beneficial for high D-ribose production.

Metabolic flexibility of D-ribose producer strain of Bacillus pumilus under environmental perturbations.

The metabolic reaction rate vector is a bridge that links gene and protein expression alterations to the phenotypic endpoint. We present a simple approach for the estimation of flux distribution at key branch points in the metabolic network by using substrate uptake, metabolite secretion rate, and biomass growth rate for transketolase (tkt) deficient Bacillus pumilus ATCC 21951. We find that the glucose-6-phosphate (G6P) and pseudo catabolic/anabolic branch points are flexible in the D: -ribose-producing tkt deficient strain of B. pumilus. The normalized flux through the pentose phosphate pathway (PPP) varied from 1.5 to 86 % under different growth conditions, thereby enabling substantial extracellular accumulation of D: -ribose under certain conditions. Interestingly, the flux through PPP was affected by the extracellular phosphate concentration and dissolved oxygen concentration. This metabolic flexibility may have been the underlying reason for this strain being selected from thousands of others in a screening for D: -ribose producers conducted in the 1970s.

Production of L-ribose from L-ribulose by a triple-site variant of mannose-6-phosphate isomerase from Geobacillus thermodenitrificans.

A triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase from Geobacillus thermodenitrificans was obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (k(cat)/K(m)) for L-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co(2+). The triple-site variant produced 213 g/liter l-ribose from 300 g/liter L-ribulose for 60 min, with a volumetric productivity of 213 g liter(-1) h(-1), which was 4.5-fold higher than that of the wild-type enzyme. The k(cat)/K(m) and productivity of the triple-site variant were approximately 2-fold higher than those of the Thermus thermophilus R142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.