Isotopologue Profiling of Legionella pneumophila: ROLE OF SERINE AND GLUCOSE AS CARBON SUBSTRATES*

Legionella pneumophila (Lp) is commonly found in freshwater habitats but is also the causative agent of Legionnaires'' disease when infecting humans. Although various virulence factors have been reported, little is known about the nutrition and the metabolism of the bacterium. Here, we report the application of isotopologue profiling for analyzing the metabolism of L. pneumophila. Cultures of Lp were supplied with [U-¹³C₃]serine, [U-¹³C₆]glucose, or [1,2-¹³C₂]glucose. After growth, ¹³C enrichments and isotopologue patterns of protein-derived amino acids and poly-3-hydroxybutyrate were determined by mass spectrometry and/or NMR spectroscopy. The labeling patterns detected in the experiment with [U-¹³C₃]serine showed major carbon flux from serine to pyruvate and from pyruvate to acetyl-CoA, which serves as a precursor of poly-3-hydroxybutyrate or as a substrate of a complete citrate cycle with Si specificity of the citrate synthase. Minor carbon flux was observed between pyruvate and oxaloacetate/malate by carboxylation and decarboxylation, respectively. The apparent lack of label in Val, Ile, Leu, Pro, Phe, Met, Arg, and Tyr confirmed that L. pneumophila is auxotrophic for these amino acids. Experiments with [¹³C]glucose showed that the carbohydrate is also used as a substrate to feed the central metabolism. The specific labeling patterns due to [1,2-¹³C₂]glucose identified the Entner-Doudoroff pathway as the predominant route for glucose utilization. In line with these observations, a mutant lacking glucose-6-phosphate dehydrogenase (Δzwf) did not incorporate label from glucose at significant levels and was slowly outcompeted by the wild type strain in successive rounds of infection in Acanthamoeba castellanii, indicating the importance of this enzyme and of carbohydrate usage in general for the life cycle of Lp.     

Purification and characterization of glycerate kinase from the thermoacidophilic archaeonThermoplasma acidophilum: An enzyme belonging to the second glycerate kinase family

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at 59°C and pH 2. Along with another thermoacidophilic archaeon,Sulfolobus solfataricus, it is known to metabolize glucose by the non-phosphorylated Entner-Doudoroff (nED) pathway. In the course of these studies, the specific activities of glyceraldehyde dehydrogenase and glycerate kinase, two enzymes that are involved in the downstream part of the nED pathway, were found to be much higher inT. acidophilum than inS. solfataricus. To characterize glycerate kinase, the enzyme was purified to homogeneity fromT. acidophilum cell extracts. TheN-terminal sequence of the purified enzyme was in exact agreement with that of Ta0453m in the genome database, with the removal of the initiator methionine. Furthermore, the enzyme was a monomer with a molecular weight of 49 kDa and followed Michaelis-Menten kinetics withK _m values of 0.56 and 0.32 mM forDL-glycerate and ATP, respectively. The enzyme also exhibited excellent thermal stability at 70°C. Of the seven sugars and four phosphate donors tested, onlyDL-glycerate and ATP were utilized by glycerate kinase as substrates. In addition, a coupled enzyme assay indicated that 2-phosphoglycerate was produced as a product. When divalent metal ions, such as Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, Ca²⁺, and Sr²⁺, were substituted for Mg²⁺, the enzyme activities were less than 10% of that obtained in the presence of Mg²⁺. The amino acid sequence ofT. acidophilum glycerate kinase showed no similarity withE. coli glycerate kinases, which belong to the first glycerate kinase family. This is the first report on the biochemical characterization of an enzyme which belongs to a member of the second glycerate kinase family.     

Carbohydrate metabolism in Zymomonas mobilis: a catabolic highway with some scenic routes

Abstract Sucrose, glucose and fructose are degraded in the Gram-negative bacterium Zymomonas mobilis via an anaerobic version of the Entner-Doudoroff pathway, to an equimolar mixture of ethanol and carbon dioxide. Sucrose is split extracellularly into glucose and fructose (or levan). The two sugars are transported into the cell via facilitated diffusion (uniport). A periplasmic enzyme, glucose-fructose oxidoreductase, provides the novel compatible solute, sorbitol, to counteract detrimental osmotic stress. Carbon flux and its regulation, and branches into anabolic pathways are discussed together with recent approaches to broaden the substrate range of the bacterium.     

Strain engineering for microbial production of value-added chemicals and fuels from glycerol

While the widespread reliance on fossil fuels is driven by their low cost and relative abundance, this fossil-based economy has been deemed unsustainable and, therefore, the adoption of sustainable and environmentally compatible energy sources is on the horizon. Biorefinery is an emerging approach that integrates metabolic engineering, synthetic biology, and systems biology principles for the development of whole-cell catalytic platforms for biomanufacturing. Due to the high degree of reduction and low cost, glycerol, either refined or crude, has been recognized as an ideal feedstock for the production of value-added biologicals, though microbial dissimilation of glycerol sometimes can be difficult particularly under anaerobic conditions. While strain development for glycerol biorefinery is widely reported in the literature, few, if any, commercialized bioprocesses have been developed as a result, such that engineering of glycerol metabolism in microbial hosts remains an untapped opportunity in biomanufacturing. Here we review the recent progress made in engineering microbial hosts for the production of biofuels, diols, organic acids, biopolymers, and specialty chemicals from glycerol. We begin with a broad outline of the major pathways for fermentative and respiratory glycerol dissimilation and key end metabolites, and then focus our analysis on four key genera of bacteria known to naturally dissimilate glycerol, i.e. Klebsiella, Citrobacter, Clostridium, and Lactobacillus, in addition to Escherichia coli, and systematically review the progress made toward engineering these microorganisms for glycerol biorefinery. We also identify the major biotechnological and bioprocessing advantages and disadvantages of each genus, and bottlenecks limiting the production of target metabolites from glycerol in engineered strains. Our analysis culminates in the development of potential strategies to overcome the current technical limitations identified for commonly employed strains, with an outlook on the suitability of different hosts for the production of key metabolites and avenues for their future development into biomanufacturing platforms.     

Glyceraldehyde dehydrogenases from the thermoacidophilic euryarchaeota Picrophilus torridus and Thermoplasma acidophilum, key enzymes of the non-phosphorylative Entner-Doudoroff pathway, constitute a novel enzyme family within the aldehyde dehydrogenase superfamily

Cells of Picrophilus torridus, grown on glucose, contained all enzyme activities of a non-phosphorylative Entner-Doudoroff pathway, including glucose dehydrogenase, gluconate dehydratase, 2-keto-3-deoxygluconate aldolase, glyceraldehyde dehydrogenase (GADH), glycerate kinase (2-phosphoglycerate forming), enolase and pyruvate kinase. GADH was purified to homogeneity. The 115-kDa homodimeric protein catalyzed the oxidation of glyceraldehyde with NADP+ at highest catalytic efficiency. NAD+ was not used. By MALDI-TOF analysis, open reading frame (ORF) Pto0332 was identified in the genome of P. torridus as the encoding gene, designated gadh, and the recombinant GADH was characterized. In Thermoplasma acidophilum ORF Ta0809 represents a gadh homolog with highest sequence identity; the gene was expressed and the recombinant protein was characterized as functional GADH with properties very similar to the P. torridus enzyme. Sequence comparison and phylogenetic analysis define both GADHs as members of novel enzyme family within the aldehyde dehydrogenase superfamily.     

Glucose fermentation to acetate and alanine in resting cell suspensions of Pyrococcus furiosus: Proposal of a novel glycolytic pathway based on 13C labelling data and enzyme activities

Abstract Suspensions of maltose-grown cells of the hyperthermophilic archaeon Pyrococcus furiosus , when incubated at 90°C with 35 mM [1-¹³C]glucose or [3-¹³C]glucose, consumed glucose at a rate of about 10 nmol min⁻¹ (mg protein)⁻¹. Acetate (10 mM), alanine (3 mM), CO₂ and H₂ were the fermentation products. The ¹³C-labelling pattern in alamine and acetate were analyzed. With [1-¹³C]glucose the methyl group of both alanine and acetate was labelled; with [3-¹³C]glucose only the carboxyl group of alanine was labelled whereas acetate was unlabelled. Extracts of maltose-grown cells contained glucose isomerase (12.8 U mg⁻¹, 100°C), ketohexokinase (0.23 U mg⁻¹, 100°C), and fructose 1-phosphate aldolase (0.06 U mg^{−1, 100°C). Enzymes catalyzing the formation of fructose 1,6-bisphosphate from fructose 1-phosphate or fructose 6-phosphate could not be detected. As publihed previously by our group and other authors P. furiosus also contains enzymes of glyceraldehyde conversion to 2-phosphoglycerate according to a non-phosphorylated Entner-Doudoroff pathway, of dihydroxyacetone phosphate conversion to 2-phosphoglycerate according to the Embden-Meyerhof pathway, and of 2-phosphoglycerate conversion - via pyruvate - to acetate and alanine. Based on the enzyme activities in P. furiosus , the following pathway for glucose degradation to alanine and acetate in cell suspensions is proposed which can explain the [13}C]glucose labelling data: glucose→ fructose → fructose 1- phosphate → dihydroxyacetone phosphate + glyceraldehyde and further conversion of both trioses to alanine and acetate via pyruvate.     

Isobutanol production in Corynebacterium glutamicum: Suppressed succinate by-production by pckA inactivation and enhanced productivity via the Entner–Doudoroff pathway

On the basis of our previous studies of microbial L-valine production under oxygen deprivation, we developed isobutanol-producing Corynebacterium glutamicum strains. The artificial isobutanol synthesis pathway was composed of the first three steps of the L-valine synthesis pathway; and the subsequent Ehrlich Pathway: pyruvate was converted to 2-ketoisovalerate in the former reactions; and the 2-keto acid was decarboxylated into isobutyraldehyde, and subsequently reduced into isobutanol in the latter reactions. Although there exists redox cofactor imbalance in the overall reactions, i.e., NADH is generated via glycolysis whereas NADPH is required to synthesize isobutanol, it was resolved by taking advantage of the NAD-preferring mutant acetohydroxy acid isomeroreductase encoded by ilvC^TM and the NAD-specific alcohol dehydrogenase encoded by adhA. Each enzyme activity to synthesize isobutanol was finely tuned by using two kinds of lac promoter derivatives. Efficient suppression of succinate by-production and improvement of isobutanol yield resulted from inactivation of pckA, which encodes phosphoenolpyruvate carboxykinase, whereas glucose consumption and isobutanol production rates decreased because of the elevated intracellular NADH/NAD⁺ ratio. On the other hand, introduction of the exogenous Entner–Doudoroff pathway effectively enhanced glucose consumption and productivity. Overexpression of phosphoenolpyruvate:carbohydrate phosphotransferase system specific to glucose and deletion of ilvE, which encodes branched-chain amino acid transaminase, further suppressed by-products and improved isobutanol productivity. Finally, the produced isobutanol concentration reached 280 mM at a yield of 84% (mol/mol glucose) in 24 h.