Red cell ektacytometry in two patients with chronic hemolytic anemia and three new α-spectrin variants.

Red blood cell (RBC) morphology is, in general, the key diagnostic feature for hereditary spherocytosis (HS) and hereditary elliptocytosis (HE). However, in hereditary pyropoikilocytosis (HPP), the severe clinical form of HE, the morphological diagnosis is difficult due to the presence of a RBC morphological picture characterized by a mixture of elliptocytes, spherocytes, tear-drop cells, and fragmented cells. This difficulty increases in new-borns and/or patients requiring frequent transfusions, making impossible the prediction of the disease course or its severity. Recently, it has been demonstrated that the measurement of osmotic gradient ektacytometry (OGE), using a laser-assisted optical rotational ektacytometer LoRRca (MaxSis, RR Mechatronics), allows a clear differentiation between HS and HE, where the truncated osmoscan curve reflects the inability of the already elliptical cells to deform further under shear stress in the face of hypotonicity. In HPP, however, the RBCs appear to have a significantly decreased ability to maintain deformability in these conditions, and the classical trapezoidal profile of HE is less evident or indistinguishable from HS. Here, two unrelated patients with hereditary hemolytic anemia (HHA) due to HPP and HS, respectively, are described with the joint inheritance of a complex set of five genetic defects. Two of these defects are novel alpha-spectrin gene (SPTA1) variants, one is a microdeletion that removes the entire SPTA1 gene, and two are well-known low-expression polymorphic alleles: α-LELY and α-LEPRA. In the HPP patient (ID1), with many circulating spherocytes, the interactions between the two SPTA1 gene variants may lead, in addition to an elongation defect (elliptocytes), to a loss of membrane stability and vesiculation (spherocytes), and RBCs appear to have a significantly decreased ability to maintain deformability in hypotonic conditions. Due to this, the classical trapezoidal profile of HE may become less evident or indistinguishable from HS. The second patient (ID2) was a classical severe form of HS with the presence of more than 20% of spherocytes and few pincered cells. The severity of clinical manifestation is due to the coinheritance of a microdeletion of chromosome 1 that removes the entire SPTA1 gene with a LEPRA SPTA1 variant in trans. The diagnostic interest of both observations is discussed.

Sickle cell nephropathy. Clinical manifestations and new mechanisms involved in kidney injury.

Kidney problems are among the most common complications in sickle cell disease (SCD). They occur early in childhood and are one of the main factors related to mortality in these patients. The main underlying pathogenic mechanisms are vaso-occlusion and haemolysis. The renal medulla has ideal conditions for the sickling of red cells due to its low partial pressure of oxygen, high osmolarity and acidic pH. Initially, sickle-cell formation in the vasa recta of the renal medulla causes hyposthenuria. This is universal and appears in early childhood. Microscopic and macroscopic haematuria also occur, in part related to renal papillary necrosis when the infarcts are extensive. Release of prostaglandins in the renal medulla due to ischaemia leads to an increase in the glomerular filtration rate (GFR). Adaptively, sodium reabsorption in the proximal tubule increases, accompanied by increased creatinine secretion. Therefore, the GFR estimated from creatinine may be overestimated. Focal segmental glomerulosclerosis is the most common glomerular disease. Albuminuria is very common and reduction has been found in 72.8% of subjects treated with ACE inhibitors or ARB. Recent evidence suggests that free haemoglobin has harmful effects on podocytes, and may be a mechanism involved in impaired kidney function in these patients. These effects need to be better studied in SCD, as they could provide a therapeutic alternative in sickle cell nephropathy.

Absolute Lymphocytes, Ferritin, C-Reactive Protein, and Lactate Dehydrogenase Predict Early Invasive Ventilation in Patients With COVID-19.

OBJECTIVE: Early detection of patients with COVID-19 who will need mechanical invasive ventilation (MIV) may aid in delivering proper care and optimizing the use of limited resources. METHODS: In this single-center retrospective observational study, we aimed to identify simple laboratory parameters that in combination with ferritin (a surrogate marker of severe inflammation) may help predict early (first 48 hours) MIV. A total of 160 patients with COVID-19 in whom serum ferritin, absolute lymphocyte count (ALC), platelet count, C-reactive protein (CRP), and lactate dehydrogenase (LDH) had been analyzed at admission were included. RESULTS: We found that ferritin, LDH, ALC, and CRP predicted with 88% accuracy the probability of early MIV. Results indicated that LDH showed the greater area under the curve (AUC), with a value of 89.1%. Using the AUC, we established cutoff values for clinical application. Finally, we developed a classification tree based on LDH for its clinical use. CONCLUSION: Ferritin, LDH, ALC, and CRP predict with 88% accuracy the probability of early MIV.

Mild Thalassemia Intermedia Due to Interaction of δß-Thalassemia with Triplicated α-Globin Genes.

Here we report a Spanish family in which two members, mother and daughter, present with a phenotype of mild non transfusion-dependent thalassemia (NTDT) due to compound heterozygosity for δß-thalassemia (δß-thal) and the α gene triplication ααα-3.7. They carry the most prevalent form of δß-thal in Spain, the so-called Spanish δß0-thal, which consists of a deletion of 114 kb that affects the δ and ß genes. A mild microcytic anemia [hemoglobin (Hb) 10.6 g/dL and mean corpuscular volume (MCV) 72.8 fL, and Hb 10.9 g/dL and MCV 70.0 fL, respectively], hypocromia [mean corpuscular Hb (MCH), 23.4 and 22.6 pg, respectively], increased red blood cell (RBC) distribution width (RDW) (20.0 and 21.9%, respectively), high fetal Hb (Hb F) (23.7 and 21.6%, respectively) with Hb A2 within the normal range, and splenomegaly, were present in the affected subjects. In areas were δß-thal is prevalent, the interaction with triplicated α-globin genes should be suspected in cases of mild NTDT if Hb F is high and Hb A2 is not increased.

Ameliorative Effect of Spinach on Non-Alcoholic Fatty Liver Disease Induced in Rats by a High-Fat Diet.

The purpose of this work was to evaluate the effect of dietary carotenoids from spinach on the inflammation and oxidative stress biomarkers, liver lipid profile, and liver transcriptomic and metabolomics profiles in Sprague-Dawley rats with steatosis induced by a high-fat diet. Two concentrations of spinach powder (2.5 and 5%) were used in two types of diet: high-fat (H) and standard (N). Although rats fed diet H showed an accumulation of fat in hepatocytes, they did not show differences in the values of adiponectin, tumor necrosis factor alpha (TNF-α), and oxygen radical absorption (ORAC) in plasma or of isoprostanes in urine compared with animals fed diet N. The consumption of spinach and the accumulation of α and ß carotenes and lutein in the liver was inversely correlated with serum total cholesterol and glucose and the content of hepatic cholesterol, increasing monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA) and reducing cholesterol in the livers of rats fed diet H and spinach. In addition, changes in the expression of genes related to the fatty liver condition occurred, and the expression of genes involved in the metabolism of fatty acids and cholesterol increased, mainly through the overexpression of peroxisome proliferator activated receptors (PPARs). Related to liver metabolites, animals fed with diet H showed hypoaminoacidemia, mainly for the glucogenic aminoacids. Although no changes were observed in inflammation and oxidative stress biomarkers, the consumption of spinach modulated the lipid metabolism in liver, which must be taken into consideration during the dietary treatment of steatosis.

Metabolomic responses of mussel Mytilus galloprovincialis to fluoranthene exposure under different nutritive conditions.

Biomarkers are useful tools to assess biological effects of pollutants that are extensively used in monitoring programs to assess ecosystem health. However, they are strongly affected by mussel physiological state, especially nutritive status, which has led to the search of new biological indicators of chemical pollutants exposition. Environmental metabolomics is an approach for examining the metabolic responses (measurement of low molecular weight endogenous metabolites) of an organism to both natural and anthropogenic stressors that can occur in its environment. The aim of the present work was to assess the effect of the polycyclic aromatic hydrocarbon fluoranthene (FLU) exposure on the metabolomic profiles of mussel digestive glands under different nutritive conditions. To achieve this objective, mussels were reared, for a period of 56 days, under three different food rations in order to obtain a gradient of nutritive status (negative, zero and positive energy balance), and after that, they were exposed, during 3 weeks, to a nominal concentration of 3 µg FLU L-1. A total of 43 metabolites, including aminoacids (Ala, Val, Leu, Ile, etc.), energy metabolism related metabolites (ATP, AMP, etc.), organic osmolytes (taurine, etc.), redox metabolism (GSH, NADP+) and nucleotides, were identified and quantified in the digestive glands of the mussels. Principal Component Analysis (PCA) defined two principal components (PC1 and PC2) that explained 55.6% of the total variance, although the first component explains more than 80% of this variance, this being related to the mussel nutritive condition. The effect of the toxicant, explained by the PC2, is similar to that produced under conditions of food restriction, which masks the effect of the toxicant under these conditions. As the feeding conditions are more favorable, the toxic effect becomes more apparent. Therefore, the great influence of nutritive condition on mussel metabolome implies a handicap for the use of metabolomic biomarkers, as previously demonstrated for biochemical and other molecular biomarkers, in large-scale monitoring programs in which several food conditions coexist with pollution levels.

The drought-tolerant Solanum pennellii regulates leaf water loss and induces genes involved in amino acid and ethylene/jasmonate metabolism under dehydration.

Breeding for drought-tolerant crops is a pressing issue due to the increasing frequency and duration of droughts caused by climate change. Although important sources of variation for drought tolerance exist in wild relatives, the mechanisms and the key genes controlling tolerance in tomato are little known. The aim of this study is to determine the drought response of the tomato wild relative Solanum pennellii (Sp) compared with the cultivated tomato Solanum lycopersicum (Sl). The paper investigates the physiological and molecular responses in leaves of Sp and Sl plants without stress and moderate drought stress. Significant physiological differences between species were found, with Sp leaves showing greater ability to avoid water loss and oxidative damage. Leaf transcriptomic analysis carried out when leaves did not as yet show visual dehydration symptoms revealed important constitutive expression differences between Sp and Sl species. Genes linked to different physiological and metabolic processes were induced by drought in Sp, especially those involved in N assimilation, GOGAT/GS cycle and GABA-shunt. Up-regulation in Sp of genes linked to JA/ET biosynthesis and signaling pathways was also observed. In sum, genes involved in the amino acid metabolism together with genes linked to ET/JA seem to be key actors in the drought tolerance of the wild tomato species.

Anaemia in the elderly. / Anemia del anciano.

Anaemia is common in the elderly and is associated with an increased risk of physical, functional, and cognitive impairment, hospitalisation and mortality. Although it is unknown whether anaemia is a causal factor or a subrogated marker of worse health status, its correction can improve the patients' physical and functional capacity. Detection, classification, and treatment of anaemia should be a priority for the health system. The main causes of anaemia in the elderly are nutritional deficiencies and chronic disease, with or without kidney failure, although some cases are of indeterminate origin. Medical history and physical examination help to clarify its aetiology. A diagnostic algorithm based on data from the lab allows anaemia classification with a therapeutic orientation. Supplements of iron and maturation factors, as well as erythropoiesis-stimulating agents, constitute the mainstay of treatment, along with that of the underlying disease, whereas red blood cell transfusion should be reserved for severe cases.

Metabolomic responses in caged clams, Ruditapes decussatus, exposed to agricultural and urban inputs in a Mediterranean coastal lagoon (Mar Menor, SE Spain).

The Mar Menor is a coastal lagoon affected by the growth of intensive agriculture and urban development in the surrounding area. Large amounts of chemical pollutants from these areas are discharged into El Albujón, a permanent water-course flowing into the lagoon. Biomarkers such as the activity of acetylcholinesterase or antioxidant enzymes have been previously tested in this lagoon demonstrating the presence of neurotoxicity and oxidative stress in clams transplanted in sites affected by the dispersion of the effluent from El Albujón. To complete this traditional toxicology work, a metabolomic profiling of these transplanted organisms has been carried out for the detection of metabolic biomarkers induced by agricultural/urban pollutants. More than 70 metabolites have been quantified using a targeting metabolomics platform based on HPLC-MS. The intracellular metabolic pattern was analyzed by PCA from the digestive gland of clams after 7 and 22 days of transplantation. Results showed a different profile of metabolite between organisms collected from control and exposed sites. At the shorter exposure time, there was an increase in several metabolites in the latter when compared with those from control sites, whereas metabolic profiling at 22 days showed that those metabolites were drastically diminished, with even lower levels than at control sites. These metabolites included: (i) 12 amino acids from the 21 proteogenic and HomoSer, (ii) osmotic protectants such as γ-butyrobetaine and taurine and (iii) nucleotides such as ITP. Regarding sulfur-containing molecules, taurine could be highlighted as a potential biomarker since its concentration was reduced by more than 30 times after 22 days of exposure, whereas the antioxidant glutathione remained constant in the organisms from both control and exposed sites. Although targeted metabolomics has been shown as an early technique of pollutant effect detection, the two-phase pattern could highlight a more complicated metabolite response to pollutants than classical biomarkers.

Systematic production of inactivating and non-inactivating suppressor mutations at the relA locus that compensate the detrimental effects of complete spot loss and affect glycogen content in Escherichia coli.

In Escherichia coli, ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of spoT null (ΔspoT) mutants obtained by transducing a ΔspoT allele from ΔrelAΔspoT double mutants into relA+ cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 ΔspoT clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in relA and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of ΔspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 ΔspoT clones displaying high glycogen phenotypes possessed relA mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45-85% of those of wild type cells. None of the ΔspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the ΔspoT clones revealed that suppressor mutations were restricted to the relA locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in relA, but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the in vivo regulation of E. coli RelA ppGpp synthetase.

Metabolic profiling of insect cell lines: Unveiling cell line determinants behind system's productivity.

Baculovirus infection boosts the host biosynthetic activity towards the production of viral components and the recombinant protein of interest, hyper-productive phenotypes being the result of a successful adaptation of the cellular network to that scenario. Spodoptera frugiperda derived Sf9 and Trichoplusia ni derived High Five cell lines have a major track record for the production of recombinant proteins, with High Five cells presenting higher productivities. A metabolic profiling of the two insect cell lines was pursued to underpin specific cellular traits behind productive phenotypes. Multivariate analysis identified cell-line dependent metabolic signatures linked to productivity. Pathway analysis highlighted cellular pathways of paramount importance in supporting infection and protein production. Moreover, better producer phenotypes proved to be correlated with the capacity of cells to shift their metabolism in favor of energy-generating pathways to fuel biosynthesis, a scenario observed in the High Five cell line. Metabolomic profiling allowed us to identify metabolic pathways involved in infection and recombinant protein production, which can be selected as targets for further improvement of the system.

Lipid biomarkers and metabolic effects of lycopene from tomato juice on liver of rats with induced hepatic steatosis.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver disorders, covering steatosis to nonalcoholic steatohepatitis (NASH). Dietary factors may modulate its evolution, and antioxidants have been proposed as therapeutic agents. Among them, lycopene has been demonstrated to prevent the development of steatohepatitis and even to inhibit NASH-promoted early hepatocarcinogenesis induced by a high-fat diet in rats. These conclusions have been related to its antioxidant activity; however, NAFLD is more complex than a simple redox imbalance state since it disturbs several metabolic systems in the liver. In consequence, there is a lack of information related to the action of lycopene beyond antioxidant biomarkers. In this work, NAFLD was induced in rats using a hypercholesterolemic and high-fat diet to evaluate the effect of lycopene consumption from tomato juice on liver metabolism. Several classical antioxidant biomarkers related to NAFLD were measured to check the state of this disease after 7 weeks of the controlled diet. Moreover, a metabolomics platform was applied to measure more than 70 metabolites. Results showed clear differences in the classical antioxidant biomarkers as well as in the metabolic pattern, attending not only to the diet but also to the intake of lycopene from tomato juice. Interestingly, tomato juice administration partially reverted the metabolic pattern from a high-fat diet to a normal diet even in metabolites not related to the redox state, which could lead to new targets for therapeutic agents against NAFLD and to achieving a better understanding of the role of lycopene in liver metabolism.

Role of central metabolism in the osmoadaptation of the halophilic bacterium Chromohalobacter salexigens.

Bacterial osmoadaptation involves the cytoplasmic accumulation of compatible solutes to counteract extracellular osmolarity. The halophilic and highly halotolerant bacterium Chromohalobacter salexigens is able to grow up to 3 m NaCl in a minimal medium due to the de novo synthesis of ectoines. This is an osmoregulated pathway that burdens central metabolic routes by quantitatively drawing off TCA cycle intermediaries. Consequently, metabolism in C. salexigens has adapted to support this biosynthetic route. Metabolism of C. salexigens is more efficient at high salinity than at low salinity, as reflected by lower glucose consumption, lower metabolite overflow, and higher biomass yield. At low salinity, by-products (mainly gluconate, pyruvate, and acetate) accumulate extracellularly. Using [1-(13)C]-, [2-(13)C]-, [6-(13)C]-, and [U-(13)C6]glucose as carbon sources, we were able to determine the main central metabolic pathways involved in ectoines biosynthesis from glucose. C. salexigens uses the Entner-Doudoroff pathway rather than the standard glycolytic pathway for glucose catabolism, and anaplerotic activity is high to replenish the TCA cycle with the intermediaries withdrawn for ectoines biosynthesis. Metabolic flux ratios at low and high salinity were similar, revealing a certain metabolic rigidity, probably due to its specialization to support high biosynthetic fluxes and partially explaining why metabolic yields are so highly affected by salinity. This work represents an important contribution to the elucidation of specific metabolic adaptations in compatible solute-accumulating halophilic bacteria.

GlgS, described previously as a glycogen synthesis control protein, negatively regulates motility and biofilm formation in Escherichia coli.

Escherichia coli glycogen metabolism involves the regulation of glgBXCAP operon expression and allosteric control of the GlgC [ADPG (ADP-glucose) pyrophosphorylase]-mediated catalysis of ATP and G1P (glucose-1-phosphate) to ADPG linked to glycogen biosynthesis. E. coli glycogen metabolism is also affected by glgS. Though the precise function of the protein it encodes is unknown, its deficiency causes both reduced glycogen content and enhanced levels of the GlgC-negative allosteric regulator AMP. The transcriptomic analyses carried out in the present study revealed that, compared with their isogenic BW25113 wild-type strain, glgS-null (ΔglgS) mutants have increased expression of the operons involved in the synthesis of type 1 fimbriae adhesins, flagella and nucleotides. In agreement, ΔglgS cells were hyperflagellated and hyperfimbriated, and displayed elevated swarming motility; these phenotypes all reverted to the wild-type by ectopic glgS expression. Also, ΔglgS cells accumulated high colanic acid content and displayed increased ability to form biofilms on polystyrene surfaces. F-driven conjugation based on large-scale interaction studies of glgS with all the non-essential genes of E. coli showed that deletion of purine biosynthesis genes complement the glycogen-deficient, high motility and high biofilm content phenotypes of ΔglgS cells. Overall the results of the present study indicate that glycogen deficiency in ΔglgS cells can be ascribed to high flagellar propulsion and high exopolysaccharide and purine nucleotides biosynthetic activities competing with GlgC for the same ATP and G1P pools. Supporting this proposal, glycogen-less ΔglgC cells displayed an elevated swarming motility, and accumulated high levels of colanic acid and biofilm. Furthermore, glgC overexpression reverted the glycogen-deficient, high swarming motility, high colanic acid and high biofilm content phenotypes of ΔglgS cells to the wild-type. As on the basis of the present study GlgS has emerged as a major determinant of E. coli surface composition and because its effect on glycogen metabolism appears to be only indirect, we propose to rename it as ScoR (surface composition regulator).

EasyLCMS: an asynchronous web application for the automated quantification of LC-MS data.

BACKGROUND: Downstream applications in metabolomics, as well as mathematical modelling, require data in a quantitative format, which may also necessitate the automated and simultaneous quantification of numerous metabolites. Although numerous applications have been previously developed for metabolomics data handling, automated calibration and calculation of the concentrations in terms of µmol have not been carried out. Moreover, most of the metabolomics applications are designed for GC-MS, and would not be suitable for LC-MS, since in LC, the deviation in the retention time is not linear, which is not taken into account in these applications. Moreover, only a few are web-based applications, which could improve stand-alone software in terms of compatibility, sharing capabilities and hardware requirements, even though a strong bandwidth is required. Furthermore, none of these incorporate asynchronous communication to allow real-time interaction with pre-processed results. FINDINGS: Here, we present EasyLCMS (http://www.easylcms.es/), a new application for automated quantification which was validated using more than 1000 concentration comparisons in real samples with manual operation. The results showed that only 1% of the quantifications presented a relative error higher than 15%. Using clustering analysis, the metabolites with the highest relative error distributions were identified and studied to solve recurrent mistakes. CONCLUSIONS: EasyLCMS is a new web application designed to quantify numerous metabolites, simultaneously integrating LC distortions and asynchronous web technology to present a visual interface with dynamic interaction which allows checking and correction of LC-MS raw data pre-processing results. Moreover, quantified data obtained with EasyLCMS are fully compatible with numerous downstream applications, as well as for mathematical modelling in the systems biology field.

Quantitative analysis of the dynamic signaling pathway involved in the cAMP mediated induction of l-carnitine biosynthesis in E. coli cultures.

L-(-)-carnitine can be synthesized from waste bioprecursors in the form of crotonobetaine. Such biotransformation is carried out in E. coli by the enzymes encoded by operons regulated by the cAMP receptor proteins. Non-phosphorylated sugars, such as glycerol are used as energy and carbon source since glucose inhibits cAMP synthesis. Until now little attention has been paid to the regulatory signaling structure that operates during the transition from a glucose-consuming, non-l-carnitine producing steady state, to a glycerol-consuming l-carnitine producing steady state. In this work we aim to elucidate and quantify the underlying regulatory mechanisms operating in the abolition of the glucose inhibiting effect. For this purpose we make use of the systemic approach by integrating the available information and our own experimentally generated data to construct a mathematical model. The model is built using power-law representation and is used as a platform to make predictive simulations and to assess the consistency of the regulatory structure of the overall process. The model is subsequently checked for quality through stability and a special, dynamic sensitivity analysis. The results show that the model is able to deal with the observed system transient phase. The model is multi-hierarchical, comprising the metabolic, gene expression, signaling and bioreactor levels. It involves variables and parameters of a very different nature that develop in different time scales and orders of magnitude. Some of the most relevant conclusions obtained are: (i) the regulatory interactions among glucose, glycerol and cAMP metabolism are far stronger than those present in the l-carnitine transport, production and degradation processes; (ii) carnitine biosynthesis is very sensitive to the cAMP signaling system since it reacts at very low cAMP receptor concentrations, and (iii) ATP is a critical factor in the transient dynamics. All these model-derived observations have been experimentally confirmed by separate studies. As a whole, the model contributes to our general understanding of the transient regulation through the signal regulatory structure, thus enabling more accurate optimization strategies to be used.

Production of L-carnitine by secondary metabolism of bacteria.

The increasing commercial demand for L-carnitine has led to a multiplication of efforts to improve its production with bacteria. The use of different cell environments, such as growing, resting, permeabilized, dried, osmotically stressed, freely suspended and immobilized cells, to maintain enzymes sufficiently active for L-carnitine production is discussed in the text. The different cell states of enterobacteria, such as Escherichia coli and Proteus sp., which can be used to produce L-carnitine from crotonobetaine or D-carnitine as substrate, are analyzed. Moreover, the combined application of both bioprocess and metabolic engineering has allowed a deeper understanding of the main factors controlling the production process, such as energy depletion and the alteration of the acetyl-CoA/CoA ratio which are coupled to the end of the biotransformation. Furthermore, the profiles of key central metabolic activities such as the TCA cycle, the glyoxylate shunt and the acetate metabolism are seen to be closely interrelated and affect the biotransformation efficiency. Although genetically modified strains have been obtained, new strain improvement strategies are still needed, especially in Escherichia coli as a model organism for molecular biology studies. This review aims to summarize and update the state of the art in L-carnitine production using E. coli and Proteus sp, emphasizing the importance of proper reactor design and operation strategies, together with metabolic engineering aspects and the need for feed-back between wet and in silico work to optimize this biotransformation.

Role of wet experiment design in data generation: from in vivo to in silico and back.

A thorough understanding of the in vivo kinetics of microorganisms requires the analysis of different data sets and therefore needs support from different sources of genome, transcriptome, proteome and metabolome data, as well as to generate new data in the laboratory to depict cell phenotypes in different scenarios. The value of dynamic metabolic data depends on the adequate design of wet experiments. In this paper a schematic representation of wet dynamic experiments to generate data is discussed. As a case study, the linking of the central metabolism with the carnitine secondary metabolism in E. coli is presented. The feed-back between the data generated and in silico modeling helps our understanding of the Escherichia coli expressed phenotype and permits new wet experiments to be designed to generate data concerning metabolic optimization.

Model of central and trimethylammonium metabolism for optimizing L-carnitine production by E. coli.

The application of metabolic engineering principles to the rational design of microbial production processes crucially depends on the ability to make quantitative descriptions of the systemic ability of the central carbon metabolism to redirect fluxes to the product-forming pathways. The aim of this work was to further our understanding of the steps controlling the biotransformation of trimethylammonium compounds into L-carnitine by Escherichia coli. Despite the importance of L-carnitine production processes, development of a model of the central carbon metabolism linked to the secondary carnitine metabolism of E. coli has been severely hampered by the lack of stoichiometric information on the metabolic reactions taking place in the carnitine metabolism. Here we present the design and experimental validation of a model which, for the first time, links the carnitine metabolism with the reactions of glycolysis, the tricarboxylic acid cycle and the pentose-phosphate pathway. The results demonstrate a need for a high production rate of ATP to be devoted to the biotransformation process. The results demonstrate that ATP is used up in a futile cycle, since both trimethylammonium compound carriers CaiT and ProU operate simultaneously. To improve the biotransformation process, resting processes as well as CaiT or ProU knock out mutants would yield a more efficient system for producing L-carnitine from crotonobetaine or D-carnitine.