Propionate Kinase Research Articles

258 Investigating the Ruminal Metagenome of Neonatal Holstein Dairy Calves fed an Essential oil-Based Feed Additive

Abstract Antibiotics remain the most effective tool for treating and preventing animal diseases in livestock production. However, the widespread use of antibiotics in food animals, as well as concerns about antibiotic resistance among bacterial pathogens, have raised awareness of the need to limit their use. As neonatal dairy calves are very susceptible to gastrointestinal infections that can result in high mortality and morbidity, the need to find alternatives to antibiotics has become a priority. Although essential oils (EO) have emerged as promising anti-microbial alternatives, their potential effects on the rumen microbiome are still poorly understood due to the complexity of their chemical components and limited understanding of their mechanisms of action. To gain further insight into the effects of EO on the bacterial communities of pre-weaned calves, we investigated the rumen metagenome of neonatal Holstein dairy calves that had been supplemented with 3.75g/feeding of a blend of EO (carvacrol, caryophyllene, p-cymene, cineole, terpinene, and thymol) as part of a previous study. Genomic DNA extracted from rumen contents collected from one of the EO-supplemented animals was used for high throughput sequencing using an Illumina Miseq (2x250) platform. Raw sequence reads were assembled into genomic contigs using custom-written Perl scripts. Coding sequences (CDS) were identified and annotated using RAST, with assignment of open reading frames (ORFs) to metabolic pathways using KEGG pathways. After assembly, a total of 3,052 contigs between 279 and 259,890 nt in length were obtained. From this set, 571 contigs affiliated to Prevotella ruminicola, representing 18.7% of total contigs built, were further analyzed. Data mining for specific functions within categories such as ‘protein metabolism’, ‘carbohydrates metabolism’, and ‘amino acids and derivatives’ was performed. This analysis resulted in the identification of all the enzymes involved in glycolysis. Two outcomes were predicted for pyruvate: conversion to lactate through lactate dehydrogenases (EC 1.1.1.27, EC 1.1.1.28), and the production of formate and acetyl-CoA through the activity of pyruvate formate-lyase (EC 2.3.1.54). Acetate (acetate kinase, EC 2.7.2.1) and propionate (Propionate kinase, EC 2.7.2.15) were other predicted end products. In addition to glucose fermentation pathways, enzymes potentially involved in the utilization of plant-based secondary metabolites were also identified, such as enzymes involved in Quinate-Chorismate metabolism: 3-dehydroquinate dehydratase (EC 4.2.1.10), Shikimate 5-dehydrogenase I alpha (EC 1.1.1.25), Shikimate kinase (EC 2.7.1.71), 5-Enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19), and Chorismate synthase (EC 4.2.3.5). Identification of such metabolic pathways suggests that perhaps the supplemented EO additive could be used as a precursor for the synthesis of aromatic amino acids. Together, these findings indicate that EO supplementation may benefit animal health and performance by promoting the growth of bacterial species that can metabolize compounds in EO additives.

Neisseria meningitidis Sibling Small Regulatory RNAs Connect Metabolism with Colonization by Controlling Propionate Use.

Neisseria meningitidis (meningococcus) colonizes the human nasopharynx, primarily as a commensal, but sporadically causing septicemia and meningitis. During colonization and invasion, it encounters different niches with specific nutrient compositions. Small noncoding RNAs (sRNAs) are used to fine-tune expression of genes, allowing adaptation to their physiological differences. We have previously characterized sRNAs (Neisseria metabolic switch regulators [NmsRs]) controlling switches between cataplerotic and anaplerotic metabolism. Here, we extend the NmsR regulon by studying methylcitrate lyase (PrpF) and propionate kinase (AckA-1) involved in the methylcitrate cycle and serine hydroxymethyltransferase (GlyA) and 3-hydroxyacid dehydrogenase (MmsB) involved in protein degradation. These proteins were previously shown to be dysregulated in a ΔnmsRs strain. Levels of transcription of target genes and NmsRs were assessed by reverse transcriptase quantitative PCR (RT-qPCR). We also used a novel gene reporter system in which the 5' untranslated region (5' UTR) of the target gene is fused to mcherry to study NmsRs-target gene interaction in the meningococcus. Under nutrient-rich conditions, NmsRs downregulate expression of PrpF and AckA-1 by direct interaction with the 5' UTR of their mRNA. Overexpression of NmsRs impaired growth under nutrient-limiting growth conditions with pyruvate and propionic acid as the only carbon sources. Our data strongly suggest that NmsRs downregulate propionate metabolism by lowering methylcitrate enzyme activity under nutrient-rich conditions. Under nutrient-poor conditions, NmsRs are downregulated, increasing propionate metabolism, resulting in higher tricarboxylic acid (TCA) activities. IMPORTANCE Neisseria meningitidis colonizes the human nasopharynx, forming a reservoir for the sporadic occurrence of epidemic invasive meningococcal disease like septicemia and meningitis. Propionic acid generated by other bacteria that coinhabit the human nasopharynx can be utilized by meningococci for replication in this environment. Here, we showed that sibling small RNAs, designated NmsRs, riboregulate propionic acid utilization by meningococci and, thus, colonization. Under conditions mimicking the nasopharyngeal environment, NmsRs are downregulated. This leads to the conversion of propionic acid to pyruvate and succinate, resulting in higher tricarboxylic acid cycle activity, allowing colonization of the nasopharynx. NmsRs link metabolic state with colonization, which is a crucial step on the trajectory to invasive meningococcal disease.

Propionate metabolism in a human pathogenic fungus: proteomic and biochemical analyses

Fungi of the complex Paracoccidioides spp. are thermodimorphic organisms that cause Paracoccidioidomycosis, one of the most prevalent mycoses in Latin America. These fungi present metabolic mechanisms that contribute to the fungal survival in host tissues. Paracoccidioides lutzii activates glycolysis and fermentation while inactivates aerobic metabolism in iron deprivation, a condition found during infection. In lungs Paracoccidioides brasiliensis face a glucose poor environment and relies on the beta-oxidation to support energy requirement. During mycelium to yeast transition P. lutzii cells up-regulate transcripts related to lipid metabolism and cell wall remodeling in order to cope with the host body temperature. Paracoccidioides spp. cells also induce transcripts/enzymes of the methylcitrate cycle (MCC), a pathway responsible for propionyl-CoA metabolism. Propionyl-CoA is a toxic compound formed during the degradation of odd-chain fatty acids, branched chain amino acids and cholesterol. Therefore, fungi require a functional MCC for full virulence and the ability to metabolize propionyl-CoA is related to the virulence traits in Paracoccidioides spp. On this way we sought to characterize the propionate metabolism in Paracoccidioides spp. The data collected showed that P. lutzii grows in propionate and activates the MCC by accumulating transcripts and proteins of methylcitrate synthase (MCS), methylcitrate dehydratase (MCD) and methylisocitrate lyase (MCL). Biochemical characterization of MCS showed that the enzyme is regulated by phosphorylation, an event not yet described. Proteomic analyses further indicate that P. lutzii yeast cells degrades lipids and amino acids to support the carbon requirement for propionate metabolism. The induction of a putative propionate kinase suggests that fungal cells use propionyl-phosphate as an intermediate in the production of toxic propionyl-CoA. Concluding, the metabolism of propionate in P. lutzii is under regulation at transcriptional and phosphorylation levels and that survival on this carbon source requires additional mechanisms other than activation of MCC.

Lactobacillus reuteri NAD(P)H oxidase: Properties and coexpression with propanediol-utilization enzymes for enhancing 3-hydroxypropionic acid production from 3-hydroxypropionaldehyde

Lactobacillus reuteri metabolizes glycerol through propanediol-utilization (Pdu) pathway to 1,3-propanediol (1,3-PD) via 3-hydroxypropionaldehyde (3-HPA) as intermediate. In the resting cells, the oxidized co-factor obtained in the reaction is regenerated by simultaneous oxidation of 3-HPA to 3-hydroxypropionic acid (3-HP) using propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL) and propionate kinase (PduW). We have earlier shown that the use of resting cells of recombinant Escherichia coli expressing the oxidative pathway gives the highest theoretical yield of 1 mol 3-HP per mol 3-HPA but is limited by cofactor depletion. In the present study, the gene encoding the enzyme NAD(P)H oxidase (LreuNox) that utilizes molecular oxygen as substrate, was isolated from L. reuteri and heterologously overexpressed in E. coli. LreuNox has a pH optimum of 6 and exhibits Vmax of 101.1 ± 2.2 U/mg with NADH, which is 30% higher than that for NADPH. Co-expression of LreuNox with PduP, PduL and PduW in E. coli enhances the biocatalytic lifetime as well as productivity at least two-fold compared to that achieved without co-factor regeneration.

Crosslinked, cryostructured Lactobacillus reuteri monoliths for production of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1,3-propanediol from glycerol

Crosslinked, cryostructured monoliths prepared from Lactobacillus reuteri cells were evaluated as potential immobilized whole-cell biocatalyst for conversion of glycerol, to potentially important chemicals for the biobased industry, i.e. 3-hydroxypropionaldehyde (3HPA), 3-hydroxypropionic acid (3HP) and 1,3-propanediol (1,3PDO). Glutaraldehyde, oxidized dextran and activated polyethyleneimine/modified polyvinyl alcohol (PEI/PVA) were evaluated as crosslinkers; the latter gave highly stable preparations with maintained viability and biocatalytic activity. Scanning electron microscopy of the PEI/PVA monoliths showed high density of crosslinked cells with wide channels allowing liquid flow through. Flux analysis of the propanediol-utilization pathway, incorporating glycerol/diol dehydratase, propionaldehyde dehydrogenase, 1,3PDO oxidoreductase, phosphotransacylase, and propionate kinase, for conversion of glycerol to the three chemicals showed that the maximum specific reaction rates were −562.6, 281.4, 62.4 and 50.5mg/gCDWh for glycerol consumption, and 3HPA (extracellular), 3HP and 1,3PDO production, respectively. Under optimal conditions using monolith operated as continuous plug flow reactor, 19.7g/L 3HPA was produced as complex with carbohydrazide at a rate of 9.1g/Lh and a yield of 77mol%. Using fed-batch operation, 1,3PDO and 3HP were co-produced in equimolar amounts with a yield of 91mol%. The monoliths embedded in plastic carriers showed high mechanical stability under different modes in a miniaturized plug flow reactor.

Structures of substrate- and nucleotide-bound propionate kinase from Salmonella typhimurium: substrate specificity and phosphate-transfer mechanism.

Kinases are ubiquitous enzymes that are pivotal to many biochemical processes. There are contrasting views on the phosphoryl-transfer mechanism in propionate kinase, an enzyme that reversibly transfers a phosphoryl group from propionyl phosphate to ADP in the final step of non-oxidative catabolism of L-threonine to propionate. Here, X-ray crystal structures of propionate- and nucleotide-bound Salmonella typhimurium propionate kinase are reported at 1.8-2.0 Å resolution. Although the mode of nucleotide binding is comparable to those of other members of the ASKHA superfamily, propionate is bound at a distinct site deeper in the hydrophobic pocket defining the active site. The propionate carboxyl is at a distance of ∼ 5 Å from the γ-phosphate of the nucleotide, supporting a direct in-line transfer mechanism. The phosphoryl-transfer reaction is likely to occur via an associative SN2-like transition state that involves a pentagonal bipyramidal structure with the axial positions occupied by the nucleophile of the substrate and the O atom between the β- and the γ-phosphates, respectively. The proximity of the strictly conserved His175 and Arg236 to the carboxyl group of the propionate and the γ-phosphate of ATP suggests their involvement in catalysis. Moreover, ligand binding does not induce global domain movement as reported in some other members of the ASKHA superfamily. Instead, residues Arg86, Asp143 and Pro116-Leu117-His118 that define the active-site pocket move towards the substrate and expel water molecules from the active site. The role of Ala88, previously proposed to be the residue determining substrate specificity, was examined by determining the crystal structures of the propionate-bound Ala88 mutants A88V and A88G. Kinetic analysis and structural data are consistent with a significant role of Ala88 in substrate-specificity determination. The active-site pocket-defining residues Arg86, Asp143 and the Pro116-Leu117-His118 segment are also likely to contribute to substrate specificity.

Production of 3-hydroxypropionic acid from 3-hydroxypropionaldehyde by recombinant Escherichia coli co-expressing Lactobacillus reuteri propanediol utilization enzymes

3-Hydroxypropionic acid (3-HP) is an important platform chemical for the biobased chemical industry. Lactobacillus reuteri produces 3-HP from glycerol via 3-hydroxypropionaldehyde (3-HPA) through a CoA-dependent propanediol utilization (Pdu) pathway. This study was performed to verify and evaluate the pathway comprising propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL), and propionate kinase (PduW) for formation of 3-HP from 3-HPA. The pathway was confirmed using recombinant Escherichia coli co-expressing PduP, PduL and PduW of L. reuteri DSM 20016 and mutants lacking expression of either enzyme. Growing and resting cells of the recombinant strain produced 3-HP with a yield of 0.3mol/mol and 1mol/mol, respectively, from 3-HPA. 3-HP was the sole product with resting cells, while growing cells produced 1,3-propanediol as co-product. 3-HP production from glycerol was achieved with a yield of 0.68mol/mol by feeding recombinant E. coli with 3-HPA produced by L. reuteri and recovered using bisulfite-functionalized resin.

A large genomic island allows Neisseria meningitidis to utilize propionic acid, with implications for colonization of the human nasopharynx.

Neisseria meningitidis is an important human pathogen that is capable of killing within hours of infection. Its normal habitat is the nasopharynx of adult humans. Here we identify a genomic island (the prp gene cluster) in N. meningitidis that enables this species to utilize propionic acid as a supplementary carbon source during growth, particularly under nutrient poor growth conditions. The prp gene cluster encodes enzymes for a methylcitrate cycle. Novel aspects of the methylcitrate cycle in N. meningitidis include a propionate kinase which was purified and characterized, and a putative propionate transporter. This genomic island is absent from the close relative of N. meningitidis, the commensal Neisseria lactamica, which chiefly colonizes infants not adults. We reason that the possession of the prp genes provides a metabolic advantage to N. meningitidis in the adult oral cavity, which is rich in propionic acid-generating bacteria. Data from classical microbiological and sequence-based microbiome studies provide several lines of supporting evidence that N. meningitidis colonization is correlated with propionic acid generating bacteria, with a strong correlation between prp-containing Neisseria and propionic acid generating bacteria from the genus Porphyromonas, and that this may explain adolescent/adult colonization by N. meningitidis.

Mechanistic features of Salmonella typhimurium propionate kinase (TdcD): Insights from kinetic and crystallographic studies

Short-chain fatty acids (SCFAs) play a major role in carbon cycle and can be utilized as a source of carbon and energy by bacteria. Salmonella typhimurium propionate kinase (StTdcD) catalyzes reversible transfer of the γ-phosphate of ATP to propionate during l-threonine degradation to propionate. Kinetic analysis revealed that StTdcD possesses broad ligand specificity and could be activated by various SCFAs (propionate>acetate≈butyrate), nucleotides (ATP≈GTP>CTP≈TTP; dATP>dGTP>dCTP) and metal ions (Mg2+≈Mn2+>Co2+). Inhibition of StTdcD by tricarboxylic acid (TCA) cycle intermediates such as citrate, succinate, α-ketoglutarate and malate suggests that the enzyme could be under plausible feedback regulation. Crystal structures of StTdcD bound to PO4 (phosphate), AMP, ATP, Ap4 (adenosine tetraphosphate), GMP, GDP, GTP, CMP and CTP revealed that binding of nucleotide mainly involves hydrophobic interactions with the base moiety and could account for the broad biochemical specificity observed between the enzyme and nucleotides. Modeling and site-directed mutagenesis studies suggest Ala88 to be an important residue involved in determining the rate of catalysis with SCFA substrates. Molecular dynamics simulations on monomeric and dimeric forms of StTdcD revealed plausible open and closed states, and also suggested role for dimerization in stabilizing segment 235–290 involved in interfacial interactions and ligand binding. Observation of an ethylene glycol molecule bound sufficiently close to the γ-phosphate in StTdcD complexes with triphosphate nucleotides supports direct in-line phosphoryl transfer.

Crystal structures of Salmonella typhimurium propionate kinase and its complex with Ap4A: Evidence for a novel Ap4A synthetic activity

Propionate kinase catalyses the last step in the anaerobic breakdown of L-threonine to propionate in which propionyl phosphate and ADP are converted to propionate and ATP. Here we report the structures of propionate kinase (TdcD) in the native form as well as in complex with diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) by X-ray crystallography. Structure of TdcD obtained after cocrystallization with ATP showed Ap4A bound to the active site pocket suggesting the presence of Ap4A synthetic activity in TdcD. Binding of Ap4A to the enzyme was confirmed by the structure determination of a TdcD-Ap4A complex obtained after cocrystallization of TdcD with commercially available Ap4A. Mass spectroscopic studies provided further evidence for the formation of Ap4A by propionate kinase in the presence of ATP. In the TdcD-Ap4A complex structure, Ap4A is present in an extended conformation with one adenosine moiety present in the nucleotide binding site and other in the proposed propionate binding site. These observations tend to support direct in-line transfer of phosphoryl group during the kinase reaction.

Structure and function of enzymes involved in the anaerobic degradation of L-threonine to propionate

In Escherichia coli and Salmonella typhimurium, L-threonine is cleaved non-oxidatively to propionate via 2-ketobutyrate by biodegradative threonine deaminase, 2-ketobutyrate formate-lyase (or pyruvate formate-lyase), phosphotransacetylase and propionate kinase. In the anaerobic condition, L-threonine is converted to the energy-rich keto acid and this is subsequently catabolised to produce ATP via substrate-level phosphorylation, providing a source of energy to the cells. Most of the enzymes involved in the degradation of L-threonine to propionate are encoded by the anaerobically regulated tdc operon. In the recent past, extensive structural and biochemical studies have been carried out on these enzymes by various groups. Besides detailed structural and functional insights, these studies have also shown the similarities and differences between the other related enzymes present in the metabolic network. In this paper, we review the structural and biochemical studies carried out on these enzymes.

Crystal Structures of ADP and AMPPNP-bound Propionate Kinase (TdcD) from Salmonella typhimurium: Comparison with Members of Acetate and Sugar Kinase/Heat Shock Cognate 70/Actin Superfamily

Recently, it has been shown that l-threonine can be catabolized non-oxidatively to propionate via 2-ketobutyrate. Propionate kinase (TdcD; EC 2.7.2.-) catalyses the last step of this metabolic process by enabling the conversion of propionyl phosphate and ADP to propionate and ATP. To provide insights into the substrate-binding pocket and catalytic mechanism of TdcD, the crystal structures of the enzyme from Salmonella typhimurium in complex with ADP and AMPPNP have been determined to resolutions of 2.2A and 2.3A, respectively, by molecular replacement using Methanosarcina thermophila acetate kinase (MAK; EC 2.7.2.1). Propionate kinase, like acetate kinase, contains a fold with the topology betabetabetaalphabetaalphabetaalpha, identical with that of glycerol kinase, hexokinase, heat shock cognaten 70 (Hsc70) and actin, the superfamily of phosphotransferases. The structure consists of two domains with the active site contained in a cleft at the domain interface. Examination of the active site pocket revealed a plausible structural rationale for the greater specificity of the enzyme towards propionate than acetate. This was further confirmed by kinetic studies with the purified enzyme, which showed about ten times lower K(m) for propionate (2.3 mM) than for acetate (26.9 mM). Comparison of TdcD complex structures with those of acetate and sugar kinase/Hsc70/actin obtained with different ligands has permitted the identification of catalytically essential residues involved in substrate binding and catalysis, and points to both structural and mechanistic similarities. In the well-characterized members of this superfamily, ATP phosphoryl transfer or hydrolysis is coupled to a large conformational change in which the two domains close around the active site cleft. The significant amino acid sequence similarity between TdcD and MAK has facilitated study of domain movement, which indicates that the conformation assumed by the two domains in the nucleotide-bound structure of TdcD may represent an intermediate point in the pathway of domain closure.

Characterization of the Acetate Binding Pocket in theMethanosarcina thermophilaAcetate Kinase

Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP gamma-phosphoryl group to acetate. Inspection of the crystal structure of the Methanosarcina thermophila enzyme containing only ADP revealed a solvent-accessible hydrophobic pocket formed by residues Val(93), Leu(122), Phe(179), and Pro(232) in the active site cleft, which identified a potential acetate binding site. The hypothesis that this was a binding site was further supported by alignment of all acetate kinase sequences available from databases, which showed strict conservation of all four residues, and the recent crystal structure of the M. thermophila enzyme with acetate bound in this pocket. Replacement of each residue in the pocket produced variants with K(m) values for acetate that were 7- to 26-fold greater than that of the wild type, and perturbations of this binding pocket also altered the specificity for longer-chain carboxylic acids and acetyl phosphate. The kinetic analyses of variants combined with structural modeling indicated that the pocket has roles in binding the methyl group of acetate, influencing substrate specificity, and orienting the carboxyl group. The kinetic analyses also indicated that binding of acetyl phosphate is more dependent on interactions of the phosphate group with an unidentified residue than on interactions between the methyl group and the hydrophobic pocket. The analyses also indicated that Phe(179) is essential for catalysis, possibly for domain closure. Alignments of acetate kinase, propionate kinase, and butyrate kinase sequences obtained from databases suggested that these enzymes have similar catalytic mechanisms and carboxylic acid substrate binding sites.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of propionate kinase (TdcD) from Salmonella typhimurium.

In the cell, propionate is mainly formed during beta-oxidation of odd-numbered carbon-chain fatty acids, fermentation of carbohydrates and degradation of the amino acids threonine, valine, isoleucine and methionine. Recently, it has been shown that L-threonine is non-oxidatively cleaved to propionate via 2-ketobutyrate. The last step in this process, conversion of propionyl phosphate and ADP to propionate and ATP, is catalysed by propionate kinase (EC 2.7.1.-). Here, the cloning of propionate kinase (molecular weight 44 kDa) from Salmonella typhimurium with an N-terminal hexahistidine affinity tag and its overexpression in Escherichia coli are reported. Purified propionate kinase was found to cocrystallize with ADP in the hanging-drop vapour-diffusion and microbatch methods. Crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 111.47, c = 66.52 A. A complete data set to 2.2 A resolution has been collected using an image-plate detector system mounted on a rotating-anode X-ray generator.

Propanol as an end product of threonine fermentation.

Clostridium sp. strain 17cr1 was able to ferment L-threonine to propionate and propanol. Electrons arising in the oxidation of 2-oxobutyrate to propionyl-CoA were apparently used in reductive pathway leading to propanol formation. Part of the propionyl-CoA was used to form propionate in an ATP-forming pathway via a propionate kinase, so that the final ATP yield was 0.5 mol per mol of L-threonine metabolised. Other growth substrates were fermented mainly to acetate and butyrate, and the reductive formation of butyrate, from 2 mol of acetyl-CoA or from crotonate or 3-hydroxybutyrate, was the main route for recycling reduced electron carriers arising during oxidative pathways for most substrates.

Propionyl coenzyme A is a common intermediate in the 1,2-propanediol and propionate catabolic pathways needed for expression of the prpBCDE operon during growth of Salmonella enterica on 1,2-propanediol.

The studies reported here identify propionyl coenzyme A (propionyl-CoA) as the common intermediate in the 1,2-propanediol and propionate catabolic pathways of Salmonella enterica serovar Typhimurium LT2. Growth on 1,2-propanediol as a carbon and energy source led to the formation and excretion of propionate, whose activation to propionyl-CoA relied on the activities of the propionate kinase (PduW)/phosphotransacetylase (Pta) enzyme system and the CobB sirtuin-controlled acetyl-CoA and propionyl-CoA (Acs, PrpE) synthetases. The different affinities of these systems for propionate ensure sufficient synthesis of propionyl-CoA to support wild-type growth of S. enterica under low or high concentrations of propionate in the environment. These redundant systems of propionyl-CoA synthesis are needed because the prpE gene encoding the propionyl-CoA synthetase enzyme is part of the prpBCDE operon under the control of the PrpR regulatory protein, which needs 2-methylcitrate as a coactivator. Because the synthesis of 2-methylcitrate by PrpC (i.e., the 2-methylcitrate synthase enzyme) requires propionyl-CoA as a substrate, the level of propionyl-CoA needs to be raised by the Acs or PduW-Pta system before 2-methylcitrate can be synthesized and prpBCDE transcription can be activated.

DOE awards chemical makers funding for biomass conversion

Crosslinked, cryostructured monoliths prepared from Lactobacillus reuteri cells were evaluated as potential immobilized whole-cell biocatalyst for conversion of glycerol, to potentially important chemicals for the biobased industry, i.e. 3-hydroxypropionaldehyde (3HPA), 3-hydroxypropionic acid (3HP) and 1,3-propanediol (1,3PDO). Glutaraldehyde, oxidized dextran and activated polyethyleneimine/modified polyvinyl alcohol (PEI/PVA) were evaluated as crosslinkers; the latter gave highly stable preparations with maintained viability and biocatalytic activity. Scanning electron microscopy of the PEI/PVA monoliths showed high density of crosslinked cells with wide channels allowing liquid flow through. Flux analysis of the propanediol-utilization pathway, incorporating glycerol/diol dehydratase, propionaldehyde dehydrogenase, 1,3PDO oxidoreductase, phosphotransacylase, and propionate kinase, for conversion of glycerol to the three chemicals showed that the maximum specific reaction rates were −562.6, 281.4, 62.4 and 50.5 mg/gCDW h for glycerol consumption, and 3HPA (extracellular), 3HP and 1,3PDO production, respectively. Under optimal conditions using monolith operated as continuous plug flow reactor, 19.7 g/L 3HPA was produced as complex with carbohydrazide at a rate of 9.1 g/Lh and a yield of 77 mol%. Using fed-batch operation, 1,3PDO and 3HP were co-produced in equimolar amounts with a yield of 91 mol%. The monoliths embedded in plastic carriers showed high mechanical stability under different modes in a miniaturized plug flow reactor.

Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate.

An immunological analysis of an Escherichia coli strain unable to synthesize the main pyruvate formate-lyase enzyme Pfl revealed the existence of a weak, cross-reacting 85 kDa polypeptide that exhibited the characteristic oxygen-dependent fragmentation typical of a glycyl radical enzyme. Polypeptide fragmentation of this cross-reacting species was shown to be dependent on Pfl activase. Cloning and sequence analysis of the gene encoding this protein revealed that it coded for a new enzyme, termed TdcE, which has 82% identity with Pfl. On the basis of RNA analyses, the tdcE gene was shown to be part of a large operon that included the tdcABC genes, encoding an anaerobic threonine dehydratase, tdcD, coding for a propionate kinase, tdcF, the function of which is unknown, and the tdcG gene, which encodes a L-serine dehydratase. Expression of the tdcABCDEFG operon was strongly catabolite repressed. Enzyme studies showed that TdcE has both pyruvate formate-lyase and 2-ketobutyrate formate-lyase activity, whereas the TdcD protein is a new propionate/acetate kinase. By monitoring culture supernatants from various mutants using 1H nuclear magnetic resonance (NMR), we followed the anaerobic conversion of L-threonine to propionate. These studies confirmed that 2-ketobutyrate, the product of threonine deamination, is converted in vivo by TdcE to propionyl-CoA. These studies also revealed that Pfl and an as yet unidentified thiamine pyrophosphate-dependent enzyme(s) can perform this reaction. Double null mutants deficient in phosphotransacetylase (Pta) and acetate kinase (AckA) or AckA and TdcD were unable to metabolize threonine to propionate, indicating that propionyl-CoA and propionyl-phosphate are intermediates in the pathway and that ATP is generated during the conversion of propionyl-P to propionate by AckA or TdcD.

Precursorbildung und Biosynthese des Makrolidantibioticums A 6599 (Turimycin) durchStreptomyces hygroscopicus JA 6599

The possible role of some metabolic systems producing acetyl-CoA, and methylmalonyl-CoA as initial precursors in the biosynthesis of the macrolide antibiotic A 6599 by Streptomyces hygroscopicus JA 6599 was studied. The activities of pyruvate decarboxylase exceeded in two higher producing strains about twofold those found in the mycelium of a lower producing one suggesting that in this organism an enhanced production of acetyl-CoA should be one of the prerequisites necessary for an improved antibiotic biosynthesis. No clear interrelationship was established, however, between the biosynthesis of the secondary metabolite A 6599 on the one hand and the acetate and propionate kinase content on the other hand. In S. hygroscopicus JA 6599 the carboxylation of acetyl-CoA or propionyl-CoA seems to be the major pathway giving malonyl-CoA or methylmalonyl-CoA, respectively. Thus, the activities of acetyl-CoA and propionyl-CoA carboxylases corresponded with both the levels of antibiotic production in several strains and with variations observed in the specific antibiotic production rate during the cultivation. Some other pathways synthesizing these precursors, e.g. via oxaloacetate, are assumed to be negligible since even in the mycelium of the lower producing strain increased activities of phosphoenolpyruvate carboxylase were present.

Precursorbildung und Biosynthese des Makrolidantibioticums A 6599 (Turimycin) durch Streptomyces hygroscopicus JA 6599

AbstractThe possible role of some metabolic systems producing acetyl‐CoA, malonyl‐CoA, and methylmalonyl‐CoA as initial precursors in the biosynthesis of the macrolide antibiotic A 6599 by Streptomyces hygroscopicus JA 6599 was studied.The activities of pyruvate decarboxylase exceeded in two higher producing strains about twofold those found in the mycelium of a lower producing one suggesting that in this organism an enhanced production of acetyl‐CoA should be one of the prerequisites necessary for an improved antibiotic biosynthesis. No clear interrelationship was established, however, between the biosynthesis of the secondary metabolite A 6599 on the one hand and the acetate and propionate kinase content on the other hand.In S. hygroscopicus JA 6599 the carboxylation of acetyl‐CoA or propionyl‐CoA seems to be the major pathway giving malonyl‐CoA or methylmalonyl‐CoA, respectively. Thus, the activities of acetyl‐CoA and propionyl‐CoA carboxylases corresponded with both the levels of antibiotic production in several strains and with variations observed in the specific antibiotic production rate during the cultivation. Some other pathways synthesizing these precursors, e.g. via oxaloacetate, are assumed to be negligible since even in the mycelium of the lower producing strain increased activities of phosphoenolpyruvate carboxylase were present.