Manuel Cánovas - Academia.edu (original) (raw)

Papers by Manuel Cánovas

Microbial Cell Factories, 2013

Background L(-)-carnitine production has been widely studied because of its beneficial properties... more Background L(-)-carnitine production has been widely studied because of its beneficial properties on various diseases and dysfunctions. Enterobacteria possess a specific biotransformation pathway which can be used for the enantioselective production of L(-)-carnitine. Although bioprocesses catalyzed by enzymes or whole cells can overcome the lack of enantioselectivity of chemical methods, current processes for L(−)-carnitine production still have severe disadvantages, such as the low yields, side reactions and the need of high catalyst concentrations and anaerobic conditions for proper expression of the biotransformation pathway. Additionally, genetically engineered strains so far constructed for L(-)-carnitine production are based on plasmids and, therefore, suffer from segregational unstability. Results In this work, a stable, high yielding strain for L(-)-carnitine production from low cost substrates was constructed. A metabolic engineering strategy was implemented in a multiple ...

Additional file 1: Table S1. Retention times and absorption spectra characteristics of carotenoid... more Additional file 1: Table S1. Retention times and absorption spectra characteristics of carotenoids. Figure S1. Biosynthetic pathway of lycopene in Escherichia coli from a native 2-C-methyl-D-erythritol 4-phosphate pathway (non-mevalonate pathway). Gene names and its encoded enzymes follow: dxr DXP reductoisomerase, dxs DXP synthase, idi IPP isomerase, ispA FPP synthase, crtE GGPP synthase, crtB phytoene synthase, crtI phytoene desaturase. Figure S2. Chromatogram of reference standards for 20 min measured at 472 and 285 nm, respectively. Figure S4. E. coli BL21 LF growth in the presence of octane during 24 h.

1 Depto. de Bioquímica y Biología Molecular B e Inmunología, Facultad de Química, Universidad de ... more 1 Depto. de Bioquímica y Biología Molecular B e Inmunología, Facultad de Química, Universidad de Murcia. Murcia. josempastor@um.es 2 Depto. de Ingeniería Química, Facultad de Química, Universidad de Murcia. Murcia. 3 Synbio lab, Faculty of Medical Sciences, University of Surrey, Reino Unido. 4 Instituto de Tecnologia Química e Biologica António Xavier. Universidade Nova de Lisboa. Oeiras. Portugal. 5 Dpt. of Biological Sciences, Purdue University. West Lafayette, EE.UU. 6 Depto. de Microbiología y Parasitología. Facultad de Farmacia. Universidad de Sevilla. Sevilla. 7 Área de Biología. Dirección de Nuevas Energías. Centro de Tecnología de Repsol. Repsol S.A. Móstoles. Madrid.

Overall, the reviewers refer to the need to perform additional experiments in order to better sup... more Overall, the reviewers refer to the need to perform additional experiments in order to better support the main conclusions. Without repeating all the comments listed below, among the more fundamental points are the following: Further experimentation is required to demonstrate that the effects of CobB on bacterial physiology are mediated by its deacetylase activity and that CobB regulates isocitrate lyase via deacetylation. Additional analyses providing further insights into the unexpected finding that loss of PatZ results in elevated acetylation levels in acetate media would significantly enhance the impact of the study. Referee #2 provides constructive comments regarding this point.

ABSTRACTTerpenoids are a family of compounds with high industrial interest and the development of... more ABSTRACTTerpenoids are a family of compounds with high industrial interest and the development of biotechnological production methods is essential to achieve more sustainable alternatives to traditional extraction and synthesis methods. The modification and engineering of the catalytic activity (kcat) have been shown to be a feasible strategy in the biotechnological realm. Accordingly, we introduce a novel optimization strategy based in the modification of the kcat of the enzymes and applied it to the maximization of the terpenoids synthesis in E. coli. This approach is fairly general and can be applied alone or in conjunction with classic optimization strategies such as the modification of enzymatic specific activities.For this purpose we first build up a reliable dynamic mathematical model of the alternative mevalonate pathway synthesis leading terpenoids biosynthesis in E. coli through the methyl-D-erythritol 4-phosphate (MEP) pathway. This model includes the 2-C-methyl-D-erythri...

Microbial Cell Factories, 2019

Background: The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively ... more Background: The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner-Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner-Doudoroff and Embden-Meyerhof-Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens. Results: Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner-Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden-Meyerhof-Parnas (EMP) route. Tracking isotopic label from [1-13 C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31 P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as fructose bisphosphatase, although it had been annotated previously as pyrophosphate-dependent phosphofructokinase. The gluconeogenic rather than glycolytic role of this enzyme in vivo is in agreement with the lack of 6-phosphofructokinase activity previously described.

Applied microbiology and biotechnology, 2016

Acetate is ubiquitously found in natural environments. Its availability in the gut is high as a r... more Acetate is ubiquitously found in natural environments. Its availability in the gut is high as a result of the fermentation of nutrients, and although it is rapidly absorbed by intestinal mucosa, it can also be used as carbon source by some members of gut microbiota. The metabolism of acetate in Escherichia coli has attracted the attention of the scientific community due to its role in central metabolism and its link to multiple physiological features. In this microorganism, acetate is involved directly or indirectly on the regulation of functional processes, such as motility, formation of biofilms, and responses to stress. Furthermore, it is a relevant nutrient in gut, where it serves additional roles, which regulate or, at least, modulate pathophysiological responses of E. coli and other bacteria. Acetate is one of the major by-products of anaerobic (fermenting) metabolism, and it is also produced under fully aerobic conditions. This acetate overflow is recognized as one of the maj...

BIO-PROTOCOL, 2012

Acetyl-coenzyme A synthethase (Acs, E.C.6.2.1.1) is an acetate activating enzyme widely represent... more Acetyl-coenzyme A synthethase (Acs, E.C.6.2.1.1) is an acetate activating enzyme widely represented in nature from bacteria to human. Its function is important for cellular catabolism, especially in order to support microbial growth at low concentrations of acetate (<10 mM) (Castano-Cerezo et al., 2011; Castano-Cerezo et al., 2009; Renilla et al., 2012). In this protocol, a continuous coupled enzymatic assay for Acs activity is described. Product formation is followed spectrophotometrically by the formation of NADH. The protocol is tailored for E. coli's Acs, but it can be adapted to assay Acs in any other organism.

Molecular Systems Biology, 2014

Although protein acetylation is widely observed, it has been associated with few specific regulat... more Although protein acetylation is widely observed, it has been associated with few specific regulatory functions making it poorly understood. To interrogate its functionality, we analyzed the acetylome in Escherichia coli knockout mutants of cobB, the only known sirtuin-like deacetylase, and patZ, the best-known protein acetyltransferase. For four growth conditions, more than 2,000 unique acetylated peptides, belonging to 809 proteins, were identified and differentially quantified. Nearly 65% of these proteins are related to metabolism. The global activity of CobB contributes to the deacetylation of a large number of substrates and has a major impact on physiology. Apart from the regulation of acetyl-CoA synthetase, we found that CobB-controlled acetylation of isocitrate lyase contributes to the fine-tuning of the glyoxylate shunt. Acetylation of the transcription factor RcsB prevents DNA binding, activating flagella biosynthesis and motility, and increases acid stress susceptibility. Surprisingly, deletion of patZ increased acetylation in acetate cultures, which suggests that it regulates the levels of acetylating agents. The results presented offer new insights into functional roles of protein acetylation in metabolic fitness and global cell regulation.

Process Biochemistry, 2007

Flow cytometry was used to monitor Escherichia coli cellular state during the biotransformation o... more Flow cytometry was used to monitor Escherichia coli cellular state during the biotransformation of crotonobetaine into L-carnitine using growing and resting cells in batch and high-cell-recycle continuous membrane reactors. The cell physiological state and the DNA, RNA and protein cell content were analyzed during the bioprocess. The cell growth cycle was followed by reference to cellular DNA concentration and the entry in the stationary phase resulted in an increase in intracellular protein. The biochemical activity of resting cells was assessed for the first time at the molecular level, protein synthesis being observed despite the absence of nutrients. Freely suspended growing, both in batch and continuous cultures, and, more importantly, resting E. coli cells were seen to be made up of subpopulations differing in reproductive ability, metabolic activity and membrane integrity. In the case of growing cells, biotransformation was mostly performed by fully viable cells (68-75%), while in a resting cell system, also dead cells (1-5%) and cells with doubtful viability (60-70%) appeared to be involved in the process; in later stages, a population made up of phantom cells, containing little or no cellular DNA, was detected. In cell-recycle continuous reactors, the recording of DNA (40 to 60 fg), RNA (50 to 120 fg) and protein (100 to 220 fg) levels per unit of cell, and the evolution of cell population heterogeneity (three different populations of cells) threw light on the stress conditions imposed by high cell densities. The use of FCM allowed to follow the recovery of cell catalytic activity for resting biotransformation batch processes, thus showing its potential for the optimization of bioprocesses.

Molecular Microbiology, 2011

Lysine acetylation is a well-established posttranslational modification widely conserved and dist... more Lysine acetylation is a well-established posttranslational modification widely conserved and distributed in bacteria. Although multiple regulatory roles have been proved, little is known about its regulation. Here, we present evidence that the transcription of the Gcn5-like acetyltransferase YfiQ of Escherichia coli (proposed name: PatZ) is regulated by cAMP-CRP and its implications on acetate metabolism regulation. The acetate scavenging acetyl-CoA synthetase (Acs) is regulated at the transcriptional and post-translational levels. Post-translational regulation depends on a protein acetyltransferase (yfiQ) and an NAD +-dependent deacetylase (cobB). We have studied their expression under different environmental conditions. cobB is constitutively expressed from a promoter located upstream nagK. The expression of yfiQ occurs from its own promoter; it is upregulated in the stationary phase and in the presence of non-PTS carbon sources and is positively regulated by cAMP-CRP. Two putative CRP binding sites are necessary for its full activity. Gene deletion revealed that cobB is essential for growth on acetate, yfiQ deletion restoring growth of the cobB mutant. The fine tuning of metabolic enzymes results from the integration of multiple mechanisms, and redundant systems may exist. Despite the existence of divergent catabolite repression systems, this may be a conserved strategy common to both Gram-positive and-negative bacteria.

Journal of Biotechnology, 2007

Biotechnology Letters, 2007

The effect of immobilization on cell physiology and how this determines cell metabolic performanc... more The effect of immobilization on cell physiology and how this determines cell metabolic performance is an important concern for developing bioprocess. This is particularly true for genetically modified microorganisms and their genetic stability. For this reason the stability and physiological state of plasmid-bearing E. coli cells were ascertained by flow cytometry. Differences in the cellular DNA and protein content (15-20%) permit discrimination of control and plasmid-bearing cells, as well as adaptation to continuous cultivation conditions in both freely suspended and immobilized states to be monitored. Moreover, the observed metabolic burden due to maintenance and over-expression of plasmid-coded genetic material and slow cell growth in poorly-viable immobilized cells were found to be the main factors contributing to strain stabilization.

Biotechnology Letters, 1992

13-Glucosidase from sweet almond was immobilized onto a nylon support and used to hydrolyze picro... more 13-Glucosidase from sweet almond was immobilized onto a nylon support and used to hydrolyze picrocrocin, the glycoside precursor of the saffron essential volatile oil, safranal. The nylon support was derivatized as hydrazide and the enzyme attached through Schiff base to bonds. The coupling efficiency was 46.8%, the immobiliTation yield 29.5%, and the derivative showed 24.2 and 4.0 U/rag activity for p-nitrophenylglucoside and picrocrocin, respectively, as substrates. Kinetic parameters of the immobilized derivative were determined, with picrocrocin as substrate, showing K M =7.2 mM and Vmax=4,0 U/rag. Glucose behaved as a competitive inhibitor (Ki-95.0 raM). The immobilized derivative was thermally stable up to 45"C; from that temperature onwards thermoinactivation occured. The operational deactivation showed a biphasic pattern, tl/2 being 4.2 days for the first four days of continuous operation, and 20.1 days from that point on. The immobilized enzyme lost ~ 50 % of its initial activity after 30.7 days of storage at 4~

Biotechnology and Bioengineering, 2009

Pulse experiments in continuous-culture are a valuable tool in microbial physiology research. How... more Pulse experiments in continuous-culture are a valuable tool in microbial physiology research. However, inferences become difficult when the cell response is followed by monitoring many biochemical variables or when several types of perturbations are compared. Moreover, there is no objective criterion to delimit the time-window, so that the recorded responses will render valid inferences. Hence, we have investigated the capability of a multivariate approach to deal with complex data from a previously described series of pulse experiments. Data are concerned with 12 biochemical variables that were monitored when an anaerobic, steady-continuous culture of E. coli O74K74 was disturbed by six types of pulses (glycerol, fumarate, acetate, crotonobetaine, hypersaline plus high-glycerol basal medium and crotonobetaine plus hypersaline basal medium). Our analysis determined the instantaneous uptake rate for the pulsed metabolite (Dynamical Chemical-Balances), reduced the multivariate observations to one response curve (Principal Component Analysis) and determined the optimal time-window (Cluster Analysis). Finally, input-output data were filtered (Orthogonal Signal Correction) while both blocks were mathematically connected (Partial Least-squares Regression). This systematic approach allowed us to detect several relevant patterns not previously revealed: (i) Glycerol uptake rate did not follow a Michaelian kinetics but showed a biphasic dependence on glycerol concentration; noticeably, net uptake decreased 136-fold despite the high availability of glycerol in the milieu. (ii) The structure of the bacterial response changed during time the glycerol-disturbance lasted (2 h), hence analyses had to be limited to the early response (time from 0 to 5 min). (iii) By mathematically relating the input (glycerol uptake rate) with the output (12 biochemical responses) it was possible to identify which of the monitored variables were primary targets of the glycerol disturbance (namely: ATP, formate, acetyl-CoA synthase, isocitrate dehydrogenase, and isocitrate lyase), which were secondarily responsive (ethanol) and those that were independent (acetate, carnitine, lactate, and NADH/NAD ratio). Identification was achieved even though all the analyzed variables were affected by the pulse. (iv) Some variables exhibited uncorrelated dynamics despite their close functional relationship (ATP and NADH/NAD ratio, ethanol and lactate; carnitine and the crotonobetaine hydratase complex; acetate and the enzymes phosphotransacetylase, acetyl-CoA synthase and isocitrate lyase). The results are discussed in terms of E. coli transcriptional control, and it is concluded that glycerol pulse produces a stressing effect. The consequent activation of the polyamine-dependent mechanisms involved in such stressing effect provides a unified explanation for how glycerol uptake is down-regulated in the presence of high glycerol availability and how acetate can be produced without de novo biosynthesis.

Biotechnology and Bioengineering, 2007

The aim was to understand how interaction of the central carbon and the secondary carnitine metab... more The aim was to understand how interaction of the central carbon and the secondary carnitine metabolisms is affected under salt stress and its effect on the production of L-carnitine by Escherichia coli. The biotransformation of crotonobetaine into L-carnitine by resting cells of E. coli O44 K74 was improved by salt stress, a yield of nearly twofold that for the control being obtained with 0.5 M NaCl. Crotonobetaine and the L-carnitine formed acted as an osmoprotectant during cell growth and biotransformation in the presence of NaCl. The enzyme activities involved in the biotransformation process (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA/acetate (pyruvate dehydrogenase, acetyl-CoA synthetase [ACS] and ATP/acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid cycle (isocitrate dehydrogenase [ICDH]) and glyoxylate shunt (isocitrate lyase [ICL]) were followed in batch with resting cells both in the presence and absence of NaCl and in perturbation experiments performed on growing cells in a high density cell recycle membrane reactor. Further, the levels of carnitine, crotonobetaine, gamma-butyrobetaine and ATP and the NADH/NAD(+) ratio were measured in order to know how the metabolic state was modified and coenzyme pools redistributed as a result of NaCl's effect on the energy content of the cell. The results provided the first experimental evidence of the important role played by salt stress during resting and growing cell biotransformation (0.5 M NaCl increased the L-carnitine production in nearly 85%), and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the main metabolic pathways and carbon flow operating during cell biotransformation was that controlled by the ICDH/ICL ratio, which decreased from 8.0 to 2.5, and the phosphotransferase/ACS ratio, which increased from 2.1 to 5.2, after a NaCl pulse fivefold the steady-state level. Resting E. coli cells were seen to be made up of heterogeneous populations consisting of several types of subpopulation (intact, depolarized, and permeabilized cells) differing in viability and metabolic activity as biotransformation run-time and the NaCl concentration increased. The results are discussed in relation with the general stress response of E. coli, which alters the NADH/NAD(+) ratio, ATP content, and central carbon enzyme activities.